Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of taxol

Article abstract of DOI:10.1016/S0968-0896(01)00072-4

Taxadiene, the key inte mediate of paclitaxel (Taxol) biosynthesis, has been prepared enzymatically from isopentenyl diphosphate in cell-free extracts of Escherichi coli by ove expressing genes encoding isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase and taxadiene synthase. In addition, by the expression of three genes encoding four enzymes on the terpene biosynthetic pathway in a single strain of E. coli, taxadiene can be conveniently synthesized in vivo, at the unoptimized yield of 1.3 mg per liter of cell culture. The success of both in vitro and in vivo synthesis of taxadiene bodes well for the future production of taxoids by non-paclitaxel producing organisms through pathway engineering. Copyright

Full text of DOI:10.1016/S0968-0896(01)00072-4

Bioorganic & Medicinal Chemistry 9 (2001) 2237–2242
Engineering Escherichia coli for the Synthesis of Taxadiene,
a Key Intermediate in the Biosynthesis of Taxol$
Qiulong Huang,a,y Charles A. Roessner,a Rodney Croteaub and A. Ian Scotta,*
aCenter for Biological NMR, Department of Chemistry, PO Box 30012, Texas A& M University, College Station, TX 77842-3012, USA
bDepartment of Biochemistry and Biophysics, Washington State University, Pullman, WA99164, USA
Received 30 November 2000; accepted 19 January 2001
Abstract—Taxadiene, the key intermediate of paclitaxel (Taxol) biosynthesis, has been prepared enzymatically from isopentenyl
diphosphate in cell-free extracts of Escherichia coli by overexpressing genes encoding isopentenyl diphosphate isomerase, ger-
anylgeranyl diphosphate synthase and taxadiene synthase. In addition, by the expression of three genes encoding four enzymes on the
terpene biosynthetic pathway in a single strain of E. coli, taxadiene can be conveniently synthesized in vivo, at the unoptimized yield
of 1.3 mg per liter of cell culture. The success of both in vitro and in vivo synthesis of taxadiene bodes well for the future production
of taxoids by non-paclitaxel producing organisms through pathway engineering. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
with one molecule of DMADP to form GGDP, and (c)
taxadiene synthase (TS) for the cyclization of GGDP to
taxadiene.3 Genes encoding all three enzymes have pre-
viously been cloned and overexpressed in Escherichia
coli (see Table 1) but only minute amounts of taxadiene,
observed only as radioactive counts or small spots on
thin layer chromatography, have been obtained from
cell-free systems.4,5 Better yields have been achieved
with the cell-free conversion of IDP to the diterpene
Taxadiene [taxa-4(5),11(12)-diene, Fig. 1] is the key
intermediate in the biosynthesis of paclitaxel (Taxol),
one of the most potent chemotherapeutic agents known,
showing excellent activity against a range of cancers
including ovarian and breast cancer.1 The demand for
paclitaxel greatly exceeds the supply from both natural
sources and semisynthesis, so intensive efforts have been
directed toward finding alternate means of production
and to synthesize analogues with similar or increased
activity. Thus, taxadiene is important for studying the
biosynthesis of paclitaxel and other taxoids. However,
taxadiene is not readily available, requiring either
750 kg of dry bark from the Pacific yew (Taxus brevifo-
lia) for the isolation of only 1 mg of the compound or a
25-step chemical synthesis.2 Therefore, these procedures
are not efficient ways to produce substantial quantities
of taxadiene.
hydrocarbon, casbene, in
a multi-enzyme system
including casbene synthase, as verified by production of
sufficient amounts of casbene for analysis by 13C NMR
spectroscopy.6 Apparently, the main difference between
the two systems is the solubility of the overproduced
cyclases (taxadiene synthase is less soluble than casbene
synthase even when produced as a thioredoxin fusion
protein5), so we felt that the yield of taxadiene in a cell-
free system could be improved with a more soluble form
of taxadiene synthase. Furthermore, an in vivo system
for the de novo synthesis of taxadiene would be attrac-
tive, eliminating the need to pre-synthesize any of the
substrates. E. coli, however, produces GGDP at low
levels for use in isoprenoid biosynthesis. In E. coli, IDP
is derived from deoxyxylulose-5-phosphate (DXP, Fig.
1) rather than via the more common mevalonate path-
way,7 and it has been shown that the addition of deox-
The biosynthesis of taxadiene (Fig. 1) from isopentenyl
diphosphate (IDP), the universal precursor of all ter-
penes, requires three enzymes: (a) IDP isomerase for the
isomerization of IDP to dimethylallyl diphosphate
(DMADP), (b) Geranylgeranyl diphosphate (GGDP)
synthase for the condensation of three molecules of IDP
yxylulose to
a culture of E. coli increases the
intracellular isoprenoid concentration by a factor of 3–
4.8 Furthermore, overexpression in E. coli of the gene
encoding DXP synthase from Bacillus subtilis or Syne-
chocystis sp. 6803 has been shown to increase the levels
of carotenoids and isoprenoids.9 We report here the
*Corresponding author. Tel.: +1-979-845-3243; fax: +1-979-845-
5992; e-mail: scott@mail.chem.tamu.edu
yCurrent address: Abbott Lab, 100 Abbott Park Rd., Dept. 47G, Bldg.
Ap10-LL, Abbott Park, IL 60064-6098, USA.
$Dedicated to Peter Dervan, a pioneer in Chemical Biology.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00072-4

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Q. Huang et al. / Bioorg. Med. Chem. 9 (2001) 2237–2242
Figure 1. The biosynthetic pathway from pyruvate and glyceraldehyde-3-phosphate to taxadiene. Not all of the intermediates are known and some
have been omitted to save space. The enzymes catalyzing the numbered steps are: (1) deoxyxyulose phosphate synthase, (2) isopentenyl diphosphate
isomerase, (3) geranylgeranyl diphosphate synthase, and (4) taxadiene synthase.
obtainment of a more soluble form of taxadiene syn-
thase that has resulted in improved yields of taxadiene,
both in vitro and in vivo, and demonstrate that, in vivo,
overproduction of E. coli DXP synthase, in conjunction
with the other three enzymes, substantially increases the
yields of taxadiene, providing a convenient source of
milligram quantities of the target compound.
and form a ‘pseudo-mature’ enzyme resulted in mostly
soluble protein (Fig. 2A, lanes 1–4). While this work
was in progress, similar results were reported by Wil-
liams et al.10 The enzyme overproduced in
BL21DE3(pTS79H) was active, as demonstrated in
small scale incubations containing 13C-IDP and cell-free
extracts containing IDP isomerase, GGPD synthase,
and the truncated taxadiene synthase (Fig. 3). While it
appears that all of the IDP was converted to GGDP,
only about 70% of the GGDP was converted to tax-
adiene, possibly due to hydrolysis of the diphosphate
from GGDP or product inhibition of the enzyme.
However, attempts to improve the conversion of GGDP
to taxadiene by adding fluoride ion as a phosphatase
inhibitor, increasing the amount of taxadiene synthase,
or adding organic solvent and/or b-cyclodextrin to trap
the product were all unsuccessful.
Results
In vitro production of taxadiene
Our first objective for improved yields of taxadiene was
to obtain a more soluble, active form of the enzyme. In
the yew tree, taxadiene synthetase is synthesized with a
signal peptide, for transport into vacuoles, that is pre-
sumably removed during transport. To produce an
enzyme without a signal peptide, pcr primers were
designed to provide a product having a 50 NcoI restriction
site, a gene for taxadiene synthase that eliminates the
first 78 amino acids, a stop codon, and a 30 BamHI
restriction site. The pcr product was inserted into
pET23H2 to afford pTS79H. We found that removal of
the first 78 amino acids to eliminate the signal peptide
A large scale reaction was performed using crude IPP
(derived from 360 mg of isopentenyl tosylate) in a total
volume of 2L. A total of 10 mg of purified taxoid pro-
duct were obtained from the incubation. Surprisingly,
besides the expected taxa-4(5), 11(12)-diene as the major
product, a minor product was also formed (13% of the
total as calculated by GC–MS), which was shown to be

Q. Huang et al. / Bioorg. Med. Chem. 9 (2001) 2237–2242
Table 1. Strains and plasmids used in this study
2239
Plasmids
Relevant characteristics
Source or reference
pACYC184
pZS*24MCS1
pET15b
pET23a(+)
pET23H1
pET23H2
plysS
pTB42.1
pTS79H
pIDPI
pGGDPS
pIGS
P15 ori, CmR
Chang et al.14
Lutz et al.15
Novagen
pSC101 ori, KanR
T7 promoter, pBR322 ori, AmpR
T7 promoter, pBR322 ori, AmpR
modified pET23
modified pET23
T7 lysozyme gene in pACYC184
Taxdiene synthasea
truncated Taxadiene synthase
IDP isomerase gene in pET23
GGDP synthase gene in pET23H1
IDPI-GGDPS fusion in pET23H1
IDPI-GGDPS fusion in pACYC
IDPI-GGDPS fusion in plysS
DXP synthaseb gene in pET23H1
DXP synthase gene in pZS*24MCS1
Novagen
This study
This study
Novagen
Wildung et al.4
This study
This study
This study
This study
This study
This study
This study
This study
pACYC-IGS
plysS-IGS
pDXPS
pZS-DXPS
E. coli strains
Description (enzymes)
Source
TB1
BL21DE3
cloning host
T7 polymerase
Baldwin et al.16
Novagen
BL21DE3(plysS)
JM101(pFMH6)
JM101(pSM145)
BL21DE3(pDXPS)
BL21DE3(plysS)(pIDPI)
BL21DE3(pGGDPS)
BL21DE3(pTS79H)
BL21DE3(pIGS)
T7 polymerase, T7 lysozyme
IDP isomerasec
Novagen
Hahn et al.17
Math et al.18
This study
This study
This study
This study
This study
This study
This study
This study
GGDP synthased
DXP synthase
IDP isomerase
GGDP synthase
Taxadiene synthase
IDPI-GGDPS fusion
IDPI-GGDPS fusion
BL21DE3(pACYC-IGS)
BL21DE3(pTS79H)(pACYC-IGS) IDPI-GGDPS fusion, TS
BL21DE3(pTS79H)(plysS-IGS)(pZS-DXPS) DXPS, IDPI, GGDPS, TS
aGene isolated from Taxus brevifolia.
bdxs gene isolated from E. coli.
cidi1 gene isolated from Schizosaccharomyces pombe.
dcrtE gene isolated from Erwinia herbicola.
the isomeric taxa-4(20),11(12)-diene (Fig. 1) by com-
parison of its NMR spectrum with literature values.11
promoter and the gene for the fusion protein was blunt-
ended with T4 DNA polymerase and ligated into the
EcoRV site of either pACYC184 or plysS to give
pACYC-IGS and plysS-IGS, respectively, each with the
p15A origin of replication and, in the latter case, a
source of T7 lysozyme for tighter regulation expression
from the T7 promoter. Transformation of pACYC-IGS
into BL21DE3(pTS79H) resulted in a strain that pro-
duced both the fusion protein and taxadiene synthase in
soluble form (Fig. 2C, lanes 1 and 2). Overproduction
of IDP isomerase and GGDP synthase in E. coli
apparently increases the level of GGDP synthesis since,
for the first time, taxadiene has been produced in vivo
and isolated with yields approaching 0.5 mg from 1 L of
cells from the above engineered strain.
In vivo production of taxadiene
Initial attempts to detect in vivo taxadiene synthesis in
strains such as BL21DE3(pTS79H) overproducing only
taxadiene synthase were unsuccessful probably because
of limited amounts of the substrate, GGDP, in E. coli.
The first successful production of taxadiene in vivo was
accomplished by the simultaneous overproduction of
IDP isomerase, GGDP synthase and taxadiene syn-
thase, all from the T7 promoter. To this end, the genes
for IDP isomerase and GGDP synthase were moved
into the same plasmid compatible with pTS79H as fol-
lows: First, a 0.9 kb NdeI-HindIII fragment from
pSM145 bearing the crtE gene was inserted into
pET23H1 to give pGGDPS. Then a pcr product con-
sisting of the idi1 gene with a 50 XbaI site and a 30 NcoI
site was ligated into pGGDPS resulting in pIGS which
bears a crtE-idi1 gene fusion and produces a fusion
protein (Mr=61 Kd) containing both IDP isomerase
and GGDP synthase separated by a 21 amino acid
linker including a 6X his-tag. The fusion protein was
soluble (Fig. 2B, lanes 1 and 2) and displayed the
expected dual activities as demonstrated by the in vitro
synthesis of 13C-GGDP from 13C-IDP (not shown). A
2.1 Kb BspEI fragment from pIGS containing the T7
Co-production of DXP synthase with IDP isomerase,
GGDP synthase, and taxadiene synthase increases the
yield of taxadiene
To study the effect of supplying DXP synthase with the
other three enzymes, the E. coli dxs gene was cloned and
then inserted into a plasmid with yet a third origin of
replication. The gene was amplified by pcr using E. coli
genomic DNA as template and ligated into the NdeI-
BamHI sites of pET23H1 to afford pDXPS. Soluble
DXP synthase was overproduced (Fig. 2B, lane 3) at
high levels in strain BL21DE3(plysS)(pDXPS) and its

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Q. Huang et al. / Bioorg. Med. Chem. 9 (2001) 2237–2242
activity was demonstrated by the synthesis of DXP from
13C-labeled pyruvate and glyceraldehyde-3-phosphate
(Fig. 4). To provide an origin of replication compatible
with the other vectors already in use, a 2.3 kb BspEI
fragment from pDXPS containing the T7 promoter and
the DXP synthase gene was treated with T4 DNA
polymerase to provide blunt ends and ligated into
pZS*24-MSC1 that had been cleaved with BamHI and
blunt ended with T4 DNA polymerase. The resultant
plasmid, pZS-DXPS, has the pSC101 origin of replica-
tion and a lower copy number but, when transformed into
BL21DE3(plysS) produces soluble DXPS at levels equiva-
lent to those observed from pDXPS (Fig. 2B lane 4). pZS-
DXPS was transformed into both BL21DE3(pTS79H)
(pACYC-IGS) and BL21DE3(pTS79H)(plysS-IGS). The
latter strain grew better than the former, presumably due
to tighter regulation of expression from the T7
promoter by T7 lysozyme prior to induction, and pro-
duced all three proteins with four enzymatic activities
(Fig. 2D, lanes 1 and 2). Overproduction of DXP syn-
thase concommitant with IDP isomerase, GGDP syn-
thase, and taxediene synthase in this strain resulted in
the isolation of 1.3 mg of taxadiene from a 1 L culture, a
2.6-fold increase over that observed with over-
production of the latter three enzymes only.
Discussion
We have described the biosynthesis of milligram quan-
tities of taxadiene both in vitro and in vivo. The in vitro
system afforded the highest yields of the product (10 mg)
but is labor intensive requiring the synthesis of the sub-
strate and the preparation of cell-free extracts. The in
vitro system produces not only the true precursor of
paclitaxel, but also the taxa-4(20),11(12)-diene isomer.
This is the first report of the production of this isomer
in a cell-free taxadiene-synthesizing reaction and sug-
gests that there is either an equilibrium between the
two isomers or that the last deprotonation by the
enzyme is not completely regio-specific. A similar
phenomenon is observed with the enzyme pinene
synthase which produces both a-pinene and b-pinene
from geranyl diphosphate.12
The in vivo system is capable of producing milligram
quantities of taxadiene from common starting materials
such as glucose and glycerol and is, thus, more labor
and cost efficient. Overproduction of DXP synthase
simultaneously with IDP synthase, GGDP synthase and
taxadiene synthase afforded the best yields in vivo pre-
sumably due to shunting of the substrates, pyruvate
and glyceraldehyde-3-phosphate, down the isoprenoid
pathway. DXP, however, is also a precursor of thiamin
and pyridoxal phosphate. Experiments are under way
to study the effect of engineering the first truly com-
mitted enzyme of the pathway, DXP reductoisomerase,
which catalyzes the conversion of DXP to C-methyl-4-
erythritol-4-phosphate.13
Conclusion
E. coli can be genetically engineered for the production
of the rare terpenoid intermediate, taxadiene.
Experimental
Bacterial strains and plasmids
Figure 2. SDS-PAGE of cell-free extracts of E. coli strains over-
producing enzymes for the biosynthesis of taxadiene: (A) soluble
(lanes 1 and 3) and insoluble (lanes 2and 4) fractions of non-induced
(lanes 1 and 2) and IPTG-induced (lanes 3 and 4) BL21DE3(pTS79H)
overproducing truncated taxadiene synthase (arrow, Mr=90 Kd); (B)
lanes 1 and 2: soluble fractions of BL21DE3(pIGS), BL21DE3(plysS-
IGS), overproducing the IDP isomerase-GGPD synthase fusion pro-
The bacterial strains and plasmids used are listed in
Table 1.
Chemicals, enzymes, and buffers
tein (arrow, Mr=61 Kd); lanes
3 and 4: soluble fractions of
4-13C-IDP and unlabeled IDP were synthesized by
methods reported earlier.19 Sodium 3-13C-pyruvate was
from Aldrich, Milwaukee, WI, USA. Oligonucleotides
were synthesized by the Gene Technology Lab at Texas
BL21DE3(plysS)(pDXPS), and BL21DE3(plysS)( pZS-DXPS) over-
producing DXP synthase (arrow, Mr=66 Kd); (C) soluble (lane 1)
and insoluble (lane 2) fractions of BL21DE3(pTS79H)(pACYC-IGS);
(D) soluble (lane 1) and insoluble (lane 2) fractions of
BL21DE3(pTS79H)(plysS-IGS)(pZS-DXPS).

Q. Huang et al. / Bioorg. Med. Chem. 9 (2001) 2237–2242
2241
Figure 3. 13C NMR spectrum of the products synthesized from 4-13C-IDP (chemical shift=111.0 ppm) in a small scale cell-free reaction containing
IDP isomerase, GGDP synthase and truncated taxadiene synthase. The signals at 40.2 and 25.8 ppm are from GGDP and the signals at 121.9, 42.0,
31.1 and 30.4 ppm are from taxadiene. The major peaks are from acetonitrile (118.8 ppm) and glycerol (63.9, 73.3 ppm).
In vitro synthesis of taxadiene
A&M University. Molecular biology techniques were
performed according to standard protocols20 using
enzymes from New England Biolabs. PCR reactions
were performed with PfuTurbo from Stratagene. DNA
purification kits were purchased from Qiagen. 13C
NMR analyses were performed on a Bruker AM 500
spectrometer. Buffer A: 50 mM phosphate, pH 7.0,
10 mM KF, 1 mM DTT, 5 mM MgCl2; Buffer B: 30 mM
HEPES, pH 8.4, 10% glycerol, 5 mM ascorbate, 5 mM
Na2S2O5, 10 mM KF, 2mM dithiothreitol, 1 mM b-
cyclodextrin.
Small-scale reactions contained, in a total of 5 mL of
buffer B, 1 mg of 4-13C-IDP and the following cell-free
extracts: 200 mL from strain JM101(pFMH6), 600 mL
from strain JM101(pSM145), and 2000 mL from strain
BL21DE3(plysS)(pTS79H) containing IDP isomerase,
GGDP synthase, and taxadiene synthase, respectively.
The solution was incubated overnight at 30 ꢀC during
which time a milky white precipitate formed. The pre-
cipitate was pelleted by centrifugation at 13,000 g for
5 min, extracted with 400 mL acetonitrile-d3 and ana-
lyzed by 13C NMR spectroscopy.
Preparation of cell-free extracts
E. coli strains were incubated at 37 ꢀC with good aera-
tion in Luria-Bertani broth containing the appropriate
antibiotic(s) to an A600 of 0.6–1.0, induced by the addi-
tion of 50 mg/L IPTG, and incubated overnight at room
temperature. Antibiotics were added at the following
concentrations when required: ampicillin, 50 mg/mL,
kanamycin, 50 mg/mL and chloramphenicol, 34 mg/mL.
The cells were pelleted by centrifugation, resuspended in
buffer A or B (25 mL/L of cells) and lysed by sonication.
The lysate was centrifuged at 10,000 g for 10 min to
remove cell debris, and the cell-free extract used without
further purification as the source of enzymes.
Larger syntheses were scaled up to 2L of buffer B con-
taining 15 mL of IDP-isomerase extract, 60 mL of
GGDP-synthase extract and 200 mL of taxadiene syn-
thase extract derived from the above strains. To provide
unlabelled substrate, 360 mg of isopentenyl tosylate
were converted to IPP by the method of Davisson et
al.19 and added to the cell free extract without further
purification. After an overnight incubation at 30 ꢀC, the
taxadiene was extracted into hexane, concentrated and
purified by chromatography on a silica column with
hexane as eluant. The fractions containing taxadiene
were pooled and dried, and the amount of taxadiene
obtained was determined by weighing.
DXP synthase activity assay
In vivo synthesis of taxadiene
A mixture containing 1 mg sodium 3-13C-pyruvate, 5 mg
dl-glyceraldehyde-3-phosphate, 5 mg thiamine diphos-
phate and 100 mL of a cell-free extract containing DXP
synthase in a total of 1 mL of buffer A was incubated
at 37 ꢀC for various time periods. A 400 mL portion of
the solution was adjusted to 20% D2O for 13C NMR
analysis.
Strain BL21DE3(pTS79H)(pACYC-IGS) or strain
BL21DE3(pTS79H)(plysS-IGS)(pZS-DXPS) was grown
at 37 ꢀC in LB medium supplemented with 2% glucose
and antibiotics (ampicillin and chloramphenicol for the
former-ampicillin, chloramphenicol and kanamycin for
the latter) to an A600 of 0.6–0.9. The culture was cooled

2242
Q. Huang et al. / Bioorg. Med. Chem. 9 (2001) 2237–2242
product derived from pET23H1 was ligated into pET23
to give the sequence rbs-NdeI-6X his-NcoI-NheI.
Acknowledgements
pFMH6 and pSM145 were generous gifts from Dr. Dale
Poulter (University of Utah). We thank the Robert A.
Welch Foundation (Houston, TX) and the Texas
Advanced Technology and Research Program for
financial support.
References and Notes
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Figure 4. 13C NMR spectra of the products obtained in incubations
containing 13C-labeled pyruvate, glyceraldehyde-3-phosphate, and
cell-free extracts of BL21DE3(plysS)(pDPXS), as a source of DXP-
synthase, after 0 h (top), 3 h (middle), and 16 h (bottom) at 37 ꢀC. The
peaks at 25.8 and 26.3 ppm correspond to pyruvate and DXP, respec-
tively. With longer incubations, the DXP is dephosphorylated to
deoxyxlulose seen at 25.2 ppm.
to room temperature, induced by adding 50 mg/L of
IPTG, and incubated at room temperature for an addi-
tional 24 h. The cells were pelleted by centrifugation,
resuspended in 100 mL water, lysed by sonication, and
the taxadiene extracted into hexane and purified as
described above. Yield 1.3 mg/L of culture.
17. Hahn, F. M.; Poulter, C. D. J. Biol. Chem. 1995, 270,
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20. Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular
Cloning-ALaboratory Manual , 2nd ed. Cold Spring Harbor
Laboratory: Cold Spring Harbor, NY, 1989.
Two derivatives of pET23 were constucted to provide
plasmids an with N-terminal 6X his-tag and either NdeI
or NcoI sites for inserting pcr products. For construc-
tion of pET23H1, the 40 bp XbaI-NdeI fragment of
pET23 was replaced with the 96 bp XbaI-NdeI fragment
from pET15 to providing the sequence rbs-NcoI-6X his-
NdeI-NheI. For construction of pET23H2, a pcr