Microbial transformation of 7-epi-10-deacetylbaccatin III to 10-deacetylbaccatin III

Article abstract of DOI:10.1016/j.molcatb.2010.01.030

The microbial transformation of 7-epi-10-deacetylbaccatin III (7-epi-10-DAB III) to 10-deacetylbaccatin III (10-DAB III) was studied. In this report, seven microorganisms were found to be able to realize the transformation at yields from 20.0% to as high as 70.8%. The optimized conditions such as the solvent, pH value of the medium, the microorganisms, transformation time, and substrate concentration were investigated.

Full text of DOI:10.1016/j.molcatb.2010.01.030

Journal of Molecular Catalysis B: Enzymatic 64 (2010) 45–47
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Microbial transformation of 7-epi-10-deacetylbaccatin III to
1
0-deacetylbaccatin III
∗
Xu Feng, Lingzhi Sun, Shaobing Fu, Zhongmei Zou, Di-An Sun
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151 Malianwa North Road, Beijing 100193, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The microbial transformation of 7-epi-10-deacetylbaccatin III (7-epi-10-DAB III) to 10-deacetylbaccatin
III (10-DAB III) was studied. In this report, seven microorganisms were found to be able to realize the
transformation at yields from 20.0% to as high as 70.8%. The optimized conditions such as the solvent,
pH value of the medium, the microorganisms, transformation time, and substrate concentration were
investigated.
Received 20 October 2009
Received in revised form 28 January 2010
Accepted 28 January 2010
Available online 4 February 2010
©
2010 Elsevier B.V. All rights reserved.
Keywords:
7
1
-epi-10-Deacetylbaccatin III
0-Deacetylbaccatin III
Microorganism
Biotransformation
1
. Introduction
7-epi-10-Deacetylbaccatin III (7-epi-10-DAB III will be used
thereafter) is one of the taxanes that can be produced by actino-
mycetes [11] or recovered from conifers [12] and Taxus species
albeit in a very low yield. In our previous work on taxanes, we
have made great effort to increase the production of 10-DAB III,
including the chemical modification of the taxanes with the core
structure of 10-DAB III. Although most of the 10-DAB III (about
80%) remained intact, small part (about 20%) of it was transformed
into 7-epi-10-DAB III during the chemical reaction and purification
process (unpublished results). It is very difficult to transform 7-epi-
10-DAB III to 10-DAB III by chemical method and 7-epi-10-DAB III
is usually considered as a by-product. In this report, to make use of
7-epi-10-DAB III, 7-epi-10-DAB III was converted to 10-DAB III by
microbial transformations.
Paclitaxel (Taxol) was first isolated from the bark of Taxus bre-
vifolia in 0.02% yield [1], however, the harvest of the yew bark
is devastating to yew trees and the supply issue of Taxol was a
tremendous obstacle that slowed the development of Paclitaxel
into market. A lot of efforts have been made to increase the supply
of Paclitaxel including isolation from other natural sources, total
synthesis, semi-synthesis from 10-deacetylbaccatin III (10-DAB III
will be used thereafter), etc. The potential of semi-synthesis was
first recognized by Potier and co-workers, who found that 10-DAB
III was a relatively abundant taxane in the needles of Taxus bac-
cata L., the European yew, with a yield of 0.02% [2]. The isolated
yield could be improved to 0.1% [3]. More significantly, 10-DAB III
is easier to isolate from yew needles than Taxol and the needles
are easily regenerated. 10-DAB III seems to be an easily and perma-
nently accessible Taxol precursor. The first semi-synthesis of Taxol
from 10-deacetylbaccatin III was achieved by Denis et al. in 1988
2. Experimental
2.1. General
[
3].
Because of the highly regio- and stereo-selectivity of bio-
NMR spectra were measured on a Bruker Avance DRX-500 spec-
transformation, various microorganisms were used for the
transformation of taxanes in an attempt to search for new potent
anticancer agents. The reactions usually happened to taxanes were
hydroxylation [4–7], oxidation [5], acetylation [8], epimerization
trometer operating at 500 MHz with Me Si as internal standard.
4
TM
The analytical HPLC was carried out on a Waters 600 Controller
TM
and Pump, with Waters
996 Photodiode Array Detector and a
GraceSmart RP 18 column (5 ␮m, 4.6 mm × 250 mm). The mobile
[
9] as well as the impressive rearrangement of taxanes [6,10].
phase consisted of 55% methanol and 45% water. The flow rate
◦
was 1 ml/min. The column temperature was set at 25 C. The detec-
tion wave-length was 230 nm. The injection volume was 20 ␮l. The
retention times for 10-DAB III and 7-epi-10-DAB III were 7.2 min
and 11.4 min, respectively. Analytical TLC was carried out on Sil-
ica gel GF254 plates (Qingdao Oceanic Chemicals, China), and the
∗ Corresponding author. Tel.: +86 10 62890759.
E-mail addresses: dasun@implad.ac.cn, diansun@sina.com (D.-A. Sun).
1
381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.01.030

4
6
X. Feng et al. / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 45–47
visualization of TLC plates was performed by spraying with 10%
H SO in methanol followed by heating.
2
4
2.2. Substrate
The substrate 7-epi-10-DAB III was obtained from Beijing Fei-
SiDe(First) Bio-pharmaceutical Development Co. Ltd. 7-epi-10-DAB
III is the by-product during the production of 10-DAB III from other
taxanes by chemical method and was purified to obtain the purity
of more than 99% [17].
Scheme 1. Microbial transformation of 7-epi-10-DAB III.
cultures were harvested and extracted three times with equal vol-
umes of CH Cl and evaporated to dryness under reduced pressure
2
2
2.3. Microorganisms, culture and screening procedures
to give a brown oil.
The crude residue (250 mg) was purified by column chro-
matography on silica gel with a stepwise elution with petroleum
ether/EtOAc (from 3:1 to 1:2). The fractions containing the product
were further purified by recrystallization in acetonitrile to afford
a white solid metabolite 2 (117.8 mg, 58.9%). 63 mg (31.5%) of the
substrate (7-epi-10-DAB III) was recycled.
Compound 2 was obtained as a solid. The purity of the product
was checked by TLC and HPLC. The structure of the isolated prod-
uct was determined by comparing its NMR spectra with authentic
samples of 10-DAB III.
Seven strains: Pseudomonas oleovorans AS1.1641, Pseudomonas
putida AS1.1003, Pseudomonas fluorescens AS1.33, Nocardia coral-
lina AS4.1037, Bacillus cereus AS1.126, Bacillus mycoides AS1.182,
Bacillus megaterium AS1.127 were used in the experiment. All the
microbes were purchased from Institute of Microbiology, Chinese
Academy of Sciences (IM/CAS), Beijing, China.
Cultures were grown according to the standard two-stage fer-
mentation protocol. Screening experiments were performed in
conical Erlenmeyer flasks (250 ml) containing 100 ml of sterile
beef-protein medium (beef-protein medium, Peptone 10.0 g/l, beef
extract 3.0 g/l, NaCl 5.0 g/l, the pH value was adjusted to 7.0 before
3. Results and discussion
◦
autoclaving for 20 min at 121 C).
Stage I cultures were inoculated with microorganism obtained
from freshly grown agar slants. 3–5% inoculums from stage I cul-
tures were used to initiate stage II cultures. The stage II cultures
were incubated for 2 days before receiving 1 mg of substrate in
One of the main drawbacks in biotransformation of 7-epi-10-
DAB III is the low solubility of the substrate in water, which
diminishes reaction rates and overall productivity. Different sol-
vents were investigated to improve the solubility of the substrate
-epi-10-DAB III. Acetone was found to be the best solvent and was
used in all of the experiments.
Seven strains: P. oleovorans AS1.1641, P. putida AS1.1003, P. fluo-
rescens AS1.33, N. corallina AS4.1037, B. cereus AS1.126, B. mycoides
AS1.182, B. megaterium AS1.127 were able to directly convert 7-epi-
0
.1 ml of acetone. The cultures were further incubated for 7 days
7
◦
on a rotary shaker at 170 rpm at 27 C. The cultures were then har-
vested and extracted three times with equal volumes (50 ml) of
CH Cl and evaporated to dryness under reduced pressure. Two
2
2
controls of culture with microorganism but without substrate and
culture with substrate but without microorganism were run syn-
chronously with the fermentation and work-up in the same way.
The fermentations were analyzed by TLC and HPLC analysis with
pure samples as reference.
1
0-DAB III into 10-DAB III as the single product (Scheme 1). This is
the first successful direct conversion of 7-epi-10-DAB III to 10-DAB
III by microorganism. This finding may open a new way to make
use of the 7-epi-10-DAB III and thus to increase the yield of the
important intermediate 10-DAB III. From the data of screening (as
shown in Fig. 1), B. mycoides AS1.182 has the highest transforma-
tion rate (as high as 70.8% in HPLC checking) and was used for the
next optimization experiment.
A series of experiments to optimize the conditions of microbial
transformation were investigated, including the pH value of the
medium, culture time and concentration of substrate, etc.
Since the kinetics of reaction during incubation is greatly influ-
enced by pH value, the pH effects on the reaction were also
2.4. pH value kinetics of the B. mycoides AS1.182 growth period
To each of the seven Erlenmeyer flasks (250 ml) containing
1
00 ml of sterile beef-protein medium was added 3–5% inoculums
◦
from stage I cultures. Flasks were incubated at 27 C on a rotary
shaker at 170 rpm. The pH value of one flask was measured at 24 h
interval.
2.5. The influence of medium pH value on biotransformation rate
To each of the three Erlenmeyer flasks (250 ml) containing
00 ml of sterile beef-protein medium (with pH values 6.5, 7.5, and
1
8
.5, respectively) was added 1 mg of substrate in 0.1 ml of acetone.
◦
The flasks were incubated at 27 C on a rotary shaker at 170 rpm for
7
days. The extraction and HPLC checking procedure was the same
as above.
2.6. Preparative biotransformation of 7-epi-10-DAB III to 10-DAB
III
B. mycoides AS1.182 was grown in four 1000 ml Erlenmeyer
flasks each containing 500 ml of liquid beef-protein medium. The
◦
flasks were incubated at 27 C on an orbital shaker (170 rpm). After
4
8 h incubation, a total of 200 mg of 7-epi-10-DAB III 1, dissolved
in acetone (2 ml), was evenly distributed among 4 flasks contain-
ing stage II cultures (final concentration 0.1 g/l). After 10 days, the
◦
Fig. 1. Effect of microorganisms on the biotransformation rate (27 C, 170 rpm, 7
days).

X. Feng et al. / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 45–47
47
significantly when the substrate concentration was above 1 g/l (as
shown in Fig. 3).
The C-7 hydroxyl group was one of the most accessible
functional groups on the taxane ring, and was subjected to epimer-
ization. The 7-epitaxol and other R-epimers were determined as the
more stable form than the S-epimers in alcohol and other mixed
solvents [13]. Some biotransformations of 10-DAB III have been
reported in the literatures [8,14,15]. 10-DAB III could be trans-
formed to 7-epi-10-DAB III by Curvularia lunata CBS 215.54 [15].
However, the transformation of 7-epi-10-DAB III to 10-DAB III by
microbes has never been reported previously.
In this report, the more stable epimer 7-epi-10-DAB III was con-
verted to its less stable isomer 10-DAB III by microorganisms which
seems contradictive to the results from the chemical transforma-
tions [16]. This is a very interesting finding. It was indicated that
the enzymatic system of the microbes has the ability to stabilize
the 7-S-epimers. This enzymatic system may also exist in the Taxus
species, because there is much high yield of 10-DAB III than that of
Fig. 2. Effect of culturing time on the biotransformation rate of 7-epi-10-DAB III by
Bacillus mycoides AS1.182 (27 C, 170 rpm).
◦
7
-epi-10-DAB III in the natural yew trees. More investigations are
needed to understand the mechanism of this new discovery.
4. Conclusion
We reported for the first time the microbial transformation of
7
-epi-10-DAB III to 10-DAB III in high yield. The optimization of
Fig. 3. Effect of substrate concentration on the biotransformation rate of 7-epi-10-
DAB III by Bacillus mycoides AS1.182 (27 C, 170 rpm, 7 days).
◦
conditions for this transformation was investigated. The substrate
-epi-10-DAB III was dissolved in acetone at a concentration of 1 g/l
7
and was transformed in neutral medium for 5 days by B. mycoides
AS1.182. The yield of 10-DAB III by this biotransformation is 70.8%
by HPLC checking and 58.9% after isolation.
investigated systematically with B. mycoides AS1.182 as the exam-
ple.
The pH value of the medium was adjusted to 7.0 before incu-
bation. After the addition of the B. mycoides AS1.182 seeds, the pH
value increased from 7.03 to 8.26 during the first day and stayed at
Acknowledgements
8
.26 to 8.50 for the next 6 days during fermentation. The result indi-
This work is supported by the National Nature Science Foun-
dation of China (project no. 20772158 and no. 20972193) and the
Nature Science Foundation of Beijing (project no. 7092066).
cated that the transformation occurred in the weak basic conditions
during the incubation.
To evaluate whether the epimerization resulted from the effect
of pH values or enzymatic transformation, the substrate 7-epi-10-
DAB III (1) was incubated in the medium with 3 different pH values
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