In vitro enzymatic synthesis of baccatin III with novel and cheap acetyl donors by the recombinant taxoid 10β-O-acetyl transferase

Article abstract of DOI:10.1080/10242422.2018.1549235

Despite the importance of baccatin III as a precursor to paclitaxel, a widely used chemotherapeutic agent, efficient enzymatic synthesis methods are lacking. Therefore, in this study, the recombinant taxoid 10β-O-acetyl transferase was prepared to produce baccatin III in vitro. The recombinant enzyme could use vinyl acetate, butyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate, and isoamyl acetate as novel and cheap alternative acetyl group donors to replace the expensive acetyl CoA for the enzymatic synthesis of baccatin III. A molecular docking study further confirmed that these acetyl donors could reasonably bind to the enzyme molecule. Using the aqueous two-phase bio-catalytic reaction system, hexane and ethyl acetate could increase the yield of product baccatin III by 2.8% and 1.1% respectively. This approach using novel and cheap acetyl donors is promising for the enzymatic synthesis of baccatin III for the future industrial production of paclitaxel.

Full text of DOI:10.1080/10242422.2018.1549235

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
In vitro enzymatic synthesis of baccatin III with
novel and cheap acetyl donors by the recombinant
taxoid 10β-O-acetyl transferase
Lin-Feng You, Jia-Jun Huang, Shu-Ling Lin, Tao Wei, Qian-Wang Zheng, Bing-
Hua Jiang, Jun-Fang Lin & Li-Qiong Guo
To cite this article: Lin-Feng You, Jia-Jun Huang, Shu-Ling Lin, Tao Wei, Qian-Wang Zheng, Bing-
Hua Jiang, Jun-Fang Lin & Li-Qiong Guo (2019): Inꢀvitro enzymatic synthesis of baccatin III with
novel and cheap acetyl donors by the recombinant taxoid 10β-O-acetyl transferase, Biocatalysis
and Biotransformation, DOI: 10.1080/10242422.2018.1549235
To link to this article: https://doi.org/10.1080/10242422.2018.1549235
Published online: 11 Jan 2019.
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2018.1549235
RESEARCH ARTICLE
In vitro enzymatic synthesis of baccatin III with novel and cheap acetyl
donors by the recombinant taxoid 10b-O-acetyl transferase
a,b
a
a
a,c
a,c
d
Lin-Feng You , Jia-Jun Huang , Shu-Ling Lin , Tao Wei , Qian-Wang Zheng , Bing-Hua Jiang ,
a,c
a,c
Jun-Fang Lin and Li-Qiong Guo
a
b
College of Food Science & Institute of Food Biotechnology, South China Agricultural University, Guangzhou, China; Chongqing Key
Laboratory of Catalysis and Functional Organic Molecule, Chongqing Technology and Business University, Chongqing, China;
c
d
Research Center for Micro-Ecological Agent Engineering and Technology of Guangdong Province, Guangzhou, China; Department
of Pathology Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA
ABSTRACT
ARTICLE HISTORY
Despite the importance of baccatin III as a precursor to paclitaxel, a widely used chemothera-
peutic agent, efficient enzymatic synthesis methods are lacking. Therefore, in this study, the
recombinant taxoid 10b-O-acetyl transferase was prepared to produce baccatin III in vitro. The
recombinant enzyme could use vinyl acetate, butyl acetate, sec-butyl acetate, isobutyl acetate,
amyl acetate, and isoamyl acetate as novel and cheap alternative acetyl group donors to replace
the expensive acetyl CoA for the enzymatic synthesis of baccatin III. A molecular docking study
further confirmed that these acetyl donors could reasonably bind to the enzyme molecule.
Using the aqueous two-phase bio-catalytic reaction system, hexane and ethyl acetate could
increase the yield of product baccatin III by 2.8% and 1.1% respectively. This approach using
novel and cheap acetyl donors is promising for the enzymatic synthesis of baccatin III for the
future industrial production of paclitaxel.
Received 13 August 2017
Accepted 6 November 2018
KEYWORDS
Enzymatic synthesis; taxoid
1
0b-O-acetyl transferase;
baccatin III; acetyl group
donors; paclitaxel
Introduction
synthesis of paclitaxel requires large volumes of sol-
vents and is therefore expensive and has a major eco-
logical impact. Biotransformation is an alternative
approach to eliminate complex steps and reduce pro-
duction costs (Wang et al. 2011).
Paclitaxel (commercial and trade name is Taxol), a
highly-oxygenated diterpenoid natural product first
isolated from Taxus brevifolia, is one of the most suc-
cessful chemotherapeutic agents, showing excellent
activity against a range of cancers including ovarian
and breast cancers (Barton et al. 2016; Li et al. 2016).
The demand for paclitaxel has largely exceeded its
supply from natural sources, and intensive efforts have
focused on the development of methods for paclitaxel
synthesis that are less destructive than direct extrac-
tion from yew (Flores-Bustamante et al. 2010; Guerra-
Bubb et al. 2012). To this end, semi-synthesis is a
highly efficient and practical solution (Patel 2017).
The C-10 deacetylase from Nocardioides luteus SC
1
3913 has been successful for the enzymatic conver-
sion of 10-DAB to baccatin III (Patel et al. 2000). Since
then, few additional trans-acetylases with specific
activity towards 10-DAB have been identified, except
the taxoid 10b-O-acetyl transferase (i.e. 10-deacetyl
baccatin III-10-O-acetyl transferase, DBAT) (Kevin and
Rodney 2000; Loncaric et al. 2006). Previous studies
have shown that DBAT catalyzes the formation of bac-
catin III by transferring an acetyl group from acetyl-
CoA to 10-deacetyl-baccatin III (Guerra-Bubb et al.
2012). DBAT is highly regioselective at C-10 and is a
rate-limiting enzyme in paclitaxel biosynthesis (Ondari
and Walker 2008; Nasiri et al. 2016).
1
0-Deacetylbaccatin III (10-DAB), which is readily
and constantly available in sufficient quantities
0–4800 lg/g) (Ghassempour et al. 2009), has been
(
employed in the pharmaceutical industry for the last
three decades as a precursor for the semi-synthesis of
Taxol. Regioselective protection of the 7-hydroxyl
group is needed prior to acetylation at the C-10
hydroxyl group during the semi-synthesis of paclitaxel
These previous findings suggest that DBAT is an
excellent candidate for the enzymatic synthesis of
paclitaxel. Based on the metabolite pools of paclitaxel
intermediates, several studies have focused on
(Hu et al. 2008; Zhou et al. 2010). The chemical semi-
CONTACT Jun-Fang Lin
linjf@scau.edu.cn; Li Qiong Guo
guolq@scau.edu.cn
College of Food Science & Institute of Food Biotechnology, South
China Agricultural University, Guangzhou, Guangdong 510642, China
ß 2019 Informa UK Limited, trading as Taylor & Francis Group

2
L.-F. YOU ET AL.
methods to enhance baccatin III production in vivo,
including the over-expression of the dbat gene in
Taxus cells (Zhang et al. 2011) and Flammulina velu-
tipes (Han et al. 2014). Despite promising results, these
approaches are inconvenient for large-scale baccatin III
production, and more efficient genetic transformation
methods are required. DBAT was first purified from
the root cells of T. baccata (Zocher et al. 1996), T. chi-
nensis (Menhard and Zenk 1999), and T. cuspidate
introduced into Escherichia coli BL21(DE3). Cultures of
the transformed E. coli BL21 (DE3) were induced with
1 mM IPTG at 28 C for 3 h. Cells were harvested by cen-
ꢀ
trifugation and then lysed. Protein of interest was puri-
2
þ
fied using a Ni -NTA Qiagen column according to the
manufacturer’s instructions. The molecular weight of
the purified protein was determined by SDS-PAGE,
using an 8% polyacrylamide gel and stained with
Coomassie Brilliant Blue R-250.
(Pennington et al. 1998). However, the purified acetyl-
transferases are insufficient for the large preparation
of baccatin III in vitro, since the purification process is
rather complicated and only yields small amounts of
protein with low activity. In this study, we evaluated
the enzymatic synthesis of baccatin III using recombin-
ant DBAT of T. x media. Furthermore, since acetyl-Co
is prohibitively expensive as a co-substrate, we
explored several novel and cheap acetyl donors for
the conversion of 10-DAB to baccatin III. This approach
provides a promising alternative for baccatin III bio-
synthesis from 10-DAB.
Enzyme assay
The protein concentration was determined using the
Bradford Protein Assay Kit (Tiangen, Beijing, China)
with bovine serum albumin as the standard. The reac-
tion mixture in 1 mL of water contained 10 lL of puri-
fied enzyme in 50 mM potassium phosphate buffer
(pH 7), 5 mM MgCl , 400 lM 10-DAB, and 400 lM ace-
2
ꢀ
tyl CoA. The mixture was incubated at 30 C for 1 h.
The reaction was terminated by the addition of 1 mL
of methanol. The reaction mixture was then extracted
ꢀ
with ether, evaporated in vacuo at 4 C and the residue
was re-suspended in 1 mL of methanol before the
high-performance liquid chromatography (HPLC) assay.
All assays were performed in triplicate.
Materials and methods
Materials
Commercial reagents were used without further purifi-
cation. 10-DAB, baccatin III, and acetyl-coenzyme A
were all obtained from Sigma-Aldrich (Shanghai,
China). Vinyl acetate, isopropenyl acetate, butyl acet-
ate, sec-butyl acetate, isobutyl acetate, amyl acetate,
and isoamyl acetate were purchased from Guangzhou
Product measurement
Both 10-DAB and baccatin III were quantified by HPLC-
UV as described previously, with modifications (Han
et al. 2014; Nasiri et al. 2016). Briefly, samples were
loaded onto
a
Luna C18 Analytical Column
(Phenomenex, Torrance, CA), eluted at 1.0 mL per min,
Qiyun Biotech Ltd. (Guangzhou, China).
A
Gel
and detected at 227nm. Criteria for identification
included the retention time and authentic standard.
The identity of the enzymatic synthesis product was fur-
ther verified by UPLC-MS (ultra-performance liquid chro-
matography-tandem mass spectrometry), according to
the identical mass spectrum for the authentic standard.
Extraction Kit and Plasmid Mini Kit I were purchased
from Omega Bio-Tek (Guangzhou, China). Ex Taq DNA
polymerase, T4 DNA ligase, premixed protein marker,
and restriction enzymes were obtained from Takara
(Dalian, China). The pGEM-T Easy plasmid was pur-
chased from Invitrogen (Guangzhou, China).
Characterization of recombinant DBAT
Preparation of DBAT
Effect of temperature on enzyme activity
The full-length coding sequence of DBAT (GenBank
accession: JQ029678) was amplified using the primers
ꢀ
DBAT activity levels at different temperatures (10–60 C)
were determined by HPLC against 10-DAB and acetyl-
CoA. Each enzymatic setup (5 mM MgCl , 400 lM 10-
DAB, 400 lM acetyl CoA, and pH =7) was incubated at
0
1
3
3EcoRF (5 -TGGAATTCATGGCAGGCTCAACAGAATTTG-
0
2
) with an EcoRI site (underlined) at the putative initial
Met residue and 13NotR (5 -TGTGCGGCCGCTCAAGGTT
TAGTTACATATTT-3 ) with a NotI site (underlined) at the
0
the assay temperature for 60 min before HPLC.
0
0
3
end. The amplified PCR fragment was sub cloned
Effect of pH on enzymatic activity
into the pGEM-T Easy Vector. The plasmid was then
digested with EcoRI and NotI, and the excised fragment
was sub cloned into the pET32a(þ) expression vector.
The resulting plasmid, termed pET32a-dbat, was
DBAT activity levels at different pH values (pH 4–9)
were determined in different citrate-phosphate buffers
for 60 min at 30 C. Each enzymatic setup (5 mM
ꢀ

BIOCATALYSIS AND BIOTRANSFORMATION
3
MgCl , 400 lM 10-DAB, and 400 lM acetyl CoA) was
incubated at 30 C for 60 min. Baccatin III generated by
results were visualized using the PyMOL molecular
viewer (Schr o€ dinger Inc., New York, NY).
2
ꢀ
the process was then measured by HPLC.
Results and discussion
Effect of metal ions on enzyme activity
Preparation of baccatin III with
recombinant DBAT
To study the effect of metal ions on enzyme activity,
DBAT was mixed with 10-DAB (200 lM), acetyl CoA
DBAT catalyzed the formation of baccatin III in the
Taxol biosynthetic pathway (Figure 1(a)). The vector
pET32a-dbat containing a 6 ꢁ His coding sequence
was applied to express recombinant DBAT, according
to previous methods (Fang and Ewald 2004). An ana-
lysis of the recombinant enzyme is shown in
Figure 1(b), indicating the soluble expression of the
recombinant protein. A UPLC-MS analysis revealed
that the enzymatic acetylation product had the same
ion characters as those of authentic baccatin III
(Figure 1(c,d)), proving that the recombinant DBAT
was a functional candidate for baccatin III catalysis
in vitro.
(200 lM), and the respective chemicals (5 mM) in
potassium phosphate buffer (50 mM, pH 7), and then
ꢀ
the mixture was incubated at 30 C for 60 min before
the HPLC assay.
Acetylation of 10-deacetyl baccatin III to baccatin
III in a biphasic system
The aqueous two-phase system is beneficial for the
purification of biomolecules owing to its low inter-
facial tension and high water content, preserving
enzyme activity (Grilo et al. 2016). Two-phase extract-
ive catalysis was used to enhance baccatin III produc-
tion. Trichloro-methane, hexane, and ethyl acetate
were used as the extractive solvents. The catalysis pro-
cess was performed in a total volume of 10 mL (5 mL
aqueous phase plus 5 mL organic solvent). Each
Biochemical characterization of DBAT
Effects of temperature, pH, and metal ions on
DBAT activity
enzymatic setup (50 mM MgCl , 4 mM 10-DAB, and
2
ꢀ
4
mM acetyl CoA) was incubated at 30 C for 60 min,
As shown in Figure 2(a), DBAT exhibited the highest
ꢀ
and baccatin III generated in the aqueous and organic
phases was determined by HPLC.
activity at 30 C. The activity decreased rapidly as the
ꢀ
temperature increased above 40 C or declined below
ꢀ
2
0 C. In addition, the maximum enzyme activity was
Substrate specificity of DBAT
observed at pH 7.0 (Figure 2(b)). Furthermore, all
2þ
2þ
metal ions deactivated DBAT while Mg , Fe
and
signifi-
The substrate specificity for DBAT was determined
using seven novel acetyl group donors in an aqueous
system, including vinyl acetate, isopropenyl acetate,
butyl acetate, sec-butyl acetate, isobutyl acetate, amyl
acetate, and isoamyl acetate. These substrates
2
þ
Co
decreased
enzyme
activity
cantly (Figure 2(c)).
Kinetic properties
For kinetic evaluations, the concentrations of 10-DAB
0.5–200 lM) and acetyl CoA (0.5–200 lM) were inde-
2
þ
(
(
0.5–3%, v/v) were mixed with 10-DAB (400 lM), Mg
(
5 mM), and purified DBAT, respectively. Each enzym-
pendently varied, maintaining the alternative substrate
at a concentration of 200 lM. Each enzymatic step
including the purified protein (0.5 mg/mL) was incu-
ꢀ
atic step was incubated at 30 C for 60 min, and bacca-
tin III generated in both the aqueous and organic
phases was determined by HPLC.
ꢀ
bated at 30 C for 60 min, and the generated baccatin
III was determined by HPLC. The Km values for 10-DAB
and acetyl CoA were 74.9 lM and 73.4 lM, while kcat
Molecular docking
ꢂ1
values for 10-DAB and acetyl-CoA were 14.69 s and
To further validate the binding mode of these novel
donors to DBAT, a docking study based on the hom-
ology structure of DBAT was performed using AutoDock
Vina 1.1.2 (Trott and Olson 2010). The derivatives were
prepared and optimized using AutoDock Tools (Morris
et al. 2009; Lin et al. 2018). Further, a receptor grid
ꢂ1
1
6.12 s , respectively, as determined by a multiple
regression analysis.
Aqueous/organic biphasic system for the
acetylation of 10-DAB
(
60 ꢁ 60 ꢁ 60 Å) was generated around the DBAT active
Three solvents, i.e. hexane, trichloro-methane, and
ethyl acetate were evaluated to avoid inhibitory
effects of the substrate and/or product on the
site. The lowest energy conformation of the most highly
populated cluster was selected for analyses. Finally,

4
L.-F. YOU ET AL.
Figure 1. Enzymatic transformation of baccatin III. (a) Enzymatic synthesis of baccatin III with recombinant DBAT. (b) SDS-PAGE
analysis of recombinant DBAT. Lane M, marker proteins; lane 1, total soluble protein after IPTG induction; lane 2, purified enzyme.
(
c) UPLC-MS analysis of the biosynthetic product (retention time 2.13 min) generated by recombinant DBAT with 10-DAB and ace-
þ
þ
þ
þ
þ
tyl CoA as substrates. (d) The diagnostic mass spectral fragment ions were at m/z 625 (M þN þ Na ), 609 (M þNa ), 587 (M ),
þ
þ
5
27 (M -CH COOH), and 509 (M -CH COOH-H O).
3 3 2
biocatalytic reaction (Vanb ꢀe sien et al. 2016). Trichloro-
methane used as the extraction solvent decreased the
yield of baccatin III, while hexane and ethyl acetate
increased the yield by 2.8% and 1.1%, respectively,
when compared with controls (Table 1). These results
suggested that an aqueous two-phase system
consisting of hexane or ethyl acetate is suitable for
baccatin III bio-convention.
Substrate specificity of DBAT
Previous studies have shown that propionate-CoA
and butyrate-CoA could convert 10-DAB to baccatin

BIOCATALYSIS AND BIOTRANSFORMATION
5
Figure 2. Effects of temperature (a), pH (b), and metal ions (c) on DBAT activity.
Table 1. Screening of organic solvents in the biphasic system
for the acetylation of 10-DAB.
Table 2. Acylation of various acetyl analogues by DBAT.
Substrate
Conversion
Experiments
Reference
log p
(Laane
et al. 1987)
Partition coefficient
Acetyl-CoA
80.01%
0.07%
0.36%
1.52%
0.74%
0.91%
0.56%
ꢃ1.30%
ꢃ2.60%
–
In vitro
In vitro
In vitro
In vitro
In vitro
In vitro
In vitro
In vivo
In vivo
In vitro
In vitro
This study
Organic
solvent
Conversion
[%]
Vinyl acetate
Butyl acetate
sec-butyl acetate
Isobutyl acetate
Amyl acetate
Isoamyl acetate
Propionyl acid
Butyryl acid
This study
This study
This study
This study
This study
This study
(Loncaric et al. 2007)
(Loncaric et al. 2007)
(Loncaric et al. 2006)
(Loncaric et al. 2006)
10-DAB
Baccatin III
None
n-Hexane
Trichloro-methane
Ethyl acetate
–
–
–
80.0 ± 1.5
82.8 ± 1.3
41.8 ± 1.1
81.1 ± 1.8
3.50
0.86
0.68
0.89
1.57
1.84
0.94
1.68
2.12
Propionyl-CoA
Al.Butyryl-CoA
–
Figure 3. Acetyl-CoA analogues as potential acetyl donor sub-
strates of DBAT: Acetyl-CoA (1), vinyl acetate (2), isopropenyl
acetate (3), butyl acetate (4), sec-butyl acetate (5), isobutyl
acetate (6), amyl acetate (7), isoamyl acetate (8).
Figure 4. Enzymatic acylation of 10-DAB using various novel
esters (0.5%–3%). 10-DAB was used as an acetyl acceptor
at 400 lM.
III analogues with different acylation patterns at C10
in vivo (Loncaric et al. 2006). Thus, seven different
acetyl derivatives were used as novel acetyl donors
to produce baccatin III in vitro (Figure 3). As shown
in Table 2, recombinant DBAT exhibited different
specificities to different acetyl substrates, e.g. vinyl
acetate, butyl acetate, sec-butyl acetate, isobutyl
acetate, amyl acetate, and isoamyl acetate; no
detectable activity of DBAT towards isopropenyl
acetate was observed. Furthermore, the highest
reaction yield was obtained as 1.52% when using
Docking studies
According to a previous study, His162 acts as a gen-
eral base catalyst and deprotonates the substrate 10-
DAB, which results in the transfer of an acetyl group
from donors to 10-DAB (Li et al. 2017). The binding
mode of each compound positioned in the groove of
the DBAT active site showed reasonable attachment
to the carbonyl group, establishing possible hydropho-
bic interactions with the active site of the hydrophobic
residues of DBAT (Figure 5). To gain a better under-
standing of how acetyl donors respond to baccatin III
2
.0%
sec-butyl
acetate
as
an
acetylating
agent (Figure 4).

6
L.-F. YOU ET AL.
Figure 5. Docking of different acylation donors and DBAT, respectively. a: vinyl acetate; b: butyl acetate; c: isobutyl acetate;
d: sec-butyl acetate; e: amyl acetate; f: isoamyl acetate. Both residue His162 and ligand are rendered as stick models.
production, the binding affinity and the distance
between the substrate and enzyme, were simulated
by docking studies. We observed a linear relationship
active site of hydrophobic residues of DBAT. These
novel and cheap acetyl donors are potential alterna-
tives for the semi-synthesis of baccatin III.
between affinity and the production of baccatin III
2
(
[baccatin III]= ꢂ2E-05 ꢁ affinity ꢂ4.0253, R =0.8984)
when evaluating various acetyl donors. A lower affinity
was associated with increased baccatin III production.
Furthermore, a similar trend was observed when con-
sidering the distances between the eN of H162 in
DBAT and the C atom of the carbonyl group in acetyl
donors ([baccatin III]= ꢂ4E-05 ꢁ distance þ6.0339,
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Science and Technology
2
R =0.8654). These results indicated that virtual screen-
Program
014B050505018, 2014B020205003; and the National Natural
Science Foundation of China under Grant 31071837,
1372116, and 31572178.
of
Guangdong
Province
under
Grant
ing is an effective approach to identify acetyl donors
by molecular docking.
2
3
Conclusions
References
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with DBAT among six commercially available chemi-
cals tested in the present study. Docking studies
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