Process Biochemistry 46 (2011) 2334

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Review

Production of the anticancer drug taxol in Taxus baccata suspension cultures:

A review
Sonia Malik a , Rosa M. Cusid b , Mohammad Hossein Mirjalili c , Elisabeth Moyano d ,
Javier Palazn b , Mercedes Bonll b,
a

Departmento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, 13083-970 Campinas, Brazil
Laboratorio de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Spain
c
Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, 1983963113, Tehran, Iran
d
Departament de Ciencies Experimentals i de la Salut, Pompeu Fabra University, Av. Doctor Aiguader 80, 08003 Barcelona, Spain
b

a r t i c l e

i n f o

Article history:
Received 15 April 2010
Received in revised form 3 September 2010
Accepted 7 September 2010
Keywords:
Anti-cancer drug
Bioprocess engineering
Cell culture
Taxus baccata
Taxol
Secondary compounds

a b s t r a c t
Plant cell factories constitute an alternative source of high added value phytochemicals such as the
anticancer drug taxol (generic name paclitaxel), biosynthesized in Taxus spp. The growing demand for
taxol and its derivatives, due to a specic action mechanism and the scarcity of the taxane ring in nature,
has made this group of compounds one of the most interesting targets for biotechnological production.
This review is focused on recent advances in the production of taxol and related taxanes in Taxus baccata,
the taxol-producing European yew, using cell suspension culture technology. The review contains a brief
description of the botany and phytochemistry of T. baccata, as well as the chemical structure of taxol
and the molecular requirements for its anticancer effects. After a short overview of taxol production at
an industrial level, the review focuses on taxol biosynthesis in plant cells and the attempts to produce
taxol in T. baccata cell cultures, giving particular emphasis to the optimization steps that have improved
production, and including the most recently developed new tools. Finally, the future prospects for the
biotechnological production of taxol are also discussed.
2010 Elsevier Ltd. All rights reserved.

Contents
1.
2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Taxol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Taxol and anti-cancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Biosynthesis of taxol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.
Taxol demand and industrial production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.4.
Alternative methods for taxol production and the need for in vitro cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Approaches for in vitro production of taxol in T. baccata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.
Callus induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Establishment of cell suspension cultures and production of taxanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Approaches to increase the production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Selection of high yielding cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Optimization of culture conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.1.
Nutrient media and employment of a two-stage culture system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.2.
Carbohydrate source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.3.
Phytohormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Use of elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Addition of precursors, adsorbants or additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.1.
Synergistic effect of elicitors, additives or inducing factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.
In situ product removal and two-phase culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +34 93 4020267; fax: +34 93 4029043.

E-mail address: mbonll@ub.edu (M. Bonll).
1359-5113/$ see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.09.004

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S. Malik et al. / Process Biochemistry 46 (2011) 2334

4.6.
Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Scale-up studies in bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Metabolic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30
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1. Introduction

2.1. Taxol and anti-cancer activity

Taxus baccata or the European yew is distributed throughout

the temperate zones of the northern hemisphere. It is a smallto medium-sized evergreen tree that historically has been used
for weapon-making and medicine, and is poisonous except for
the fruit [1]. The genus Taxus belongs to the Class Pinopsida,
the Order Taxales and the Family Taxaceae. As the species are
highly similar, they are often easier to separate geographically
than morphologically. Typically, eight species are recognized: T.
baccata (European or English yew), T. brevifolia (Pacic yew or
Western yew), T. canadensis (Canadian yew), T. chinensis (Chinese yew), T. cuspidata (Japanese yew), T. oridana (Florida
yew), T. globosa (Mexican yew) and T. wallichiana (Himalayan
yew). There are also two recognized hybrids: Taxus media = T.
baccata T. cuspidata and Taxus hunnewelliana = T. cuspidata T.
canadensis [2].
The genus Taxus has generated considerable interest due to
its content of diterpene alkaloids, particularly taxol (known also
as the generic drug paclitaxel and by the registered trade name
Taxol BMS [Bristol-Myers Squibb]). The anticancer properties of
taxol were discovered in T. brevifolia extracts in 1971 [3], while
in 1979 Horwitz, working with T. baccata, found that the cellular
target of taxol was tubulin [4]. In their search for spindle poisons the Potier group in France found that the main taxane in
T. baccata (European Taxus) needles was 10-deacetylbaccatin III
(0.1% yield in the extracts). After studying the semi-synthesis of
taxol from this metabolic intermediate, they achieved the production of the analogous compound taxotere (also known by the
generic name of docetaxel and the registered trade name Taxotere
[Sano-Aventis]), which has the same action mechanism as taxol
[5]. Additionally, lignans, avonoids, steroids and sugar derivatives
have been synthesized in different parts of various Taxus species
[6]. Recent studies on Taxus extracts from needles found about
50 lignans, including neolignans, and a few terpenolignans [7,8].
Specically in T. baccata, ve lignans have been found: lariciresinol,
taxiresinol, 3 -demethylisolariciresinol-9 -hydroxyisopropylether,
isolariciresinol and 3-demethylisosalariciresinol. In vitro studies
have shown that larciresinol and isolarciresinol have a powerful inhibitory effect on tumor necrosis factor- (TNF-) [9] and
taxiresinol is reported to be highly protective against gastric lesions
[6].

The antitumor activity of taxol is mainly due to the side chain,

A ring, C2 benzoyl group and oxetane ring. The activity is maintained by the C3 amide-acyl group in the C13 chain [11] and
is enhanced by the hydroxyl group at C2 [5]. The interaction of
these constituents with -tubulin of the microtubule promotion of
polymerization produces cytotoxicity and microtubule stabilization [12].
Taxol inhibits cell proliferation by binding to the microtubule
surface, specically to the subunit of the tubulin heterodimers,
thus promoting its polymerization, even in absence of GTP
[5,13,14].
While the number of cancers being treated by taxol is expanding,
to date it has been principally used to treat metastatic carcinoma
of the ovary [15], metastatic breast cancer and non-small cell lung
cancer as well as in second-line treatment of AIDS-related Kaposis
sarcoma. Taxol is currently being studied for the treatment of diseases not related with cancer that require microtubule stabilization
and the avoidance of cell proliferation and angiogenesis, for example, psoriasis [16]. Taxol is also being studied for the treatment
of taupathies (affections in tau proteins), such as Alzheimers or
Parkinsonism linked to chromosome 17, among others [17].
In the search for alternative methods for producing taxol, the
similarly structured cephalomannine has been found to bear an Ntigloyl group instead of the N-benzoyl group at C3 without any
reduction of cytotoxicity and microtubule disassembly [18]. The
rst natural analogue of paclitaxel, 2-debenzoyl-2-tigloyl paclitaxel, has a modied ester group at C2 while retaining tubulin
binding activity, although it is less cytotoxic [19].

2. Taxol
Among secondary metabolites with anticancer activity, taxol,
a complex diterpene obtained from Taxus spp., is arguably the
most important. Its chemical name is 5, 20-epoxy-1,2,4,7,13hexahydroxytax-11-en-9one-4, 10-diacetate-2-benzoate 13 ester
with (2R,3S)-N-benzoyl-3-phenylisoserine; its molecular formula
is C47 H51 NO14 and molecular weight is 853.9 Da [10]. At the core
of taxol are the A, B and C ring systems, which have several functional groups including two OH groups, one benzoyl group, two
acetyl groups and an oxetane ring. Bound to the C13 of the core is
the side chain or C13 (2 R,3 S)-N-benzoyl-3 -phenylisoserine, with
a hydroxyl and a benzoyl functional group.

2.2. Biosynthesis of taxol

As a natural diterpenoid, taxol is formed exclusively from geranylgeranyl diphosphate (GGPP), which is synthesized from three
IPP molecules and the isomer dimethyl diphsophate (DMAPP) by
the enzyme geranylgeranyl diphosphate synthase (Fig. 1). This
enzyme is of special interest as it leads to the formation of a
branched point progenitor of a variety of diterpenoids and tetraterpenoids. According to Eisenreich et al. [20], the IPP involved in the
biosynthesis of the taxane ring is formed by the plastidic route.
However, other studies [2124] have shown the involvement of
the cytosolic pathway. Srinivasan et al. [25] suggested that cytosolic
IPP could play a role in taxol production in the initial growth phase
of Taxus cells. Additionally, Wang et al. [26], after supplementing T.
chinensis cell suspensions with two inhibitors of metabolite translocation, suggested that the translocation of IPP through the plastidic
membrane only occurs during the late growth phase of the culture.
A recent study in T. baccata cell cultures showed that while
taxol biosynthesis was blocked by the addition of fosmidomycin
(an inhibitor of the plastidic pathway), it was also reduced by mevinolin (an inhibitor of the cytosolic pathway), indicating that both
pathways could be involved [23].
The rst committed step of taxol biosynthesis is the cyclization
of geranylgeranyl diphosphate (GGPP) to the taxa-(4,5),(11,12)diene, a reaction catalyzed by taxadiene synthase (TS), a monomeric
protein of 79 kDa. The enzyme was puried and characterized

S. Malik et al. / Process Biochemistry 46 (2011) 2334

25

Fig. 1. Taxol biosynthetic pathway.

Adapted from Guo et al. [120] and Expsito et al. [22].

by Hezari et al. [27], and the gene that codies for TS has
been cloned and functionally expressed in E. coli by Wildung
and Croteau [28]. Afterwards, oxygen and acyl groups are added
to the taxane core by oxygenation at multiple positions mediated by cytochrome P450 mono-oxygenases. The hydroxylation
at the C5 position of the taxane ring by the enzyme cytochrome
P450 taxadiene-5-hydroxylase (T5H) results in the formation
of taxa-4(20),11(12)-dien-5-ol, which is the second step in taxol
biosynthesis [29]. T5H is a protein of 56 kDa with an N-terminal
sequence of insertion in the membrane of the endoplasmic reticulum. This enzyme, apart from its hydroxylating activity, also
conditions the migration of the double bond from 4(5) to 4(20).
Although these two metabolic steps, cyclization and hydroxylation,
are slow, they do not seem to be rate-limiting in taxol biosynthesis
[30].
The next step in the pathway is catalyzed by a specic taxadiene-5-ol-O-acetyl transferase (TDAT) that acylates
taxa-4(20),11(12)-dien-5-ol at the C5 position to form taxa4(20),11(12)-dien-5-yl-acetate. This enzyme is a protein of 50 kDa
that bears no N-terminal organellar targeting information [31].
The product of this reaction is then hydroxylated by the taxoid
10-hydroxylase (T10H) at C10. T10H is a P450-dependent
monooxygenase cloned and functionally characterized in yeast
[32].
Another Cyt P450-dependent hydroxylase leading to the formation of taxa-4(20),11(12)-dien-5-13-diol has been found [33].
The fact that this enzyme uses the same substrate as TDAT, the
taxa-4(20),11(12)-dien-5-ol, suggests that taxol biosynthesis is
not a linear pathway and that there are branch points that can
lead to other related taxoids. It has been observed that this alternative step is especially frequent in cell cultures elicited with methyl
jasmonate [34].

The taxoid 14-hydroxylase (T14H) is responsible for the formation of taxa-4(20),11(12)-dien-5-acetoxy-10-14-diol [35].
This enzyme does not use substrates already hydroxylated at the
C13 position, only those hydroxylated at the C10 position, suggesting that T14H cannot be involved in the production of taxol, which
does not present any hydroxylation at the C14 position.
The last steps in the taxol biosynthetic pathway, after the formation of taxa-4(20),11(12)-dien-5,10-diol 5-acetate, include
several hydroxylations at the C1, C2, C4 and C7 positions, oxidation of C9 and epoxidation at the C4C5 double bond. It is known
that the hydroxylations are mediated by Cyt P450 enzymes but not
exactly in which order. Taking into account the oxidation frequency
of the taxoids found in cell cultures, a probable sequence validated by phylogenetic analyses of previously cloned taxoid P450
oxigenases could be: C5, C10, C2, C9, C13, C7 and nally C1 [36].
However, rather than intermediates in taxol biosynthesis, some of
these taxoids might be commodities of the in vitro cultures.
Although different mechanisms for the oxetane ring formation
have been proposed [3739], it is currently accepted that the process involves epoxidation of the 4(20) double bond followed by
migration of the -acetoxy group from the C5 to the C4 position
together with the expansion of the oxirane to the oxetane group.
It is possible that this step precedes hydroxylation at C1 in taxol
biosynthesis, and in this case the hypothetical polyhydroxylated
intermediate would be a taxadien-hexaol rather than a heptaol
hydroxylated at C1 [40]. The enzyme that epoxidates the C4C20
double bond has not yet been functionally characterized and the
expansion of the oxirane-to-oxetane ring is also an incompletely
known step.
After the formation of the hypothetical polyhydroxylated precursor by the activity of the enzyme 2-O-benzoyl transferase
(DBT), a protein of 50 kDa, the next compound obtained is

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S. Malik et al. / Process Biochemistry 46 (2011) 2334

10-deacetylbaccatin III. Another identied transacetylation reaction in the taxol biosynthetic pathway involves hydroxylation at
the C10 position of the 10-DAB (10-deacetylbaccatin III), which
is catalyzed by the enzyme 10-deacetyl-baccatin III-10-O-acetyl
transferase (DBAT). It leads to the formation of a diterpene intermediate, baccatin III, using 10-DAB and acetyl CoA as substrates.
An essential step in the taxol biosynthesis is the esterication of
the C13 hydroxyl group of baccatin III with the -phenylalanoylCoA side chain. The side chain is obtained from the amino acid
-phenyalanine by the action of phenylalanine aminomutase
(PAM) [41]. An unknown ester CoA ligase probably activates the
compound so it can bind to baccatin III. The enzyme that catalyzes the conjugation of the -phenylalanoyl-CoA side chain to
baccatin III is C-13-phenylpropanoyl-CoA transferase (BAPT), yielding the compound 3 -N-debenzoyl-2 -deoxytaxol. This compound,
by the action of an unknown Cyt P450-dependent hydroxylase
that hydroxylates the C2 position and the enzyme 3 -N-debenzoyl2 -deoxytaxol N-benzoyl transferase (DBTNBT) that conjugates
benzoyl-CoA to 3 -N-debenzoyl-2 -deoxytaxol, yields taxol as the
nal compound. This enzyme can be exploited to improve the production of taxol in genetically engineered systems [42].
2.3. Taxol demand and industrial production
Taxol is one of the most successful anticancer drugs developed in the past 50 years. In 1999, worldwide sales for taxol
produced by Bristol-Myers Squibb (BMS) reached $1.5 billion.
Although this company reported a 24% decrease of taxol sales,
from $422 millions in 2006 to $385 millions in 2007 [BMS 2008
Annual Report], this reduction is primarily due to patent expiry
and increased generic competition in Europe, as well as generic
entry in Japan during the third quarter of 2006. Nevertheless,
the total market for taxol remains well above $1 billion per
year [www.strategyr.com/Bulk Paclitaxel Market Report.asp] and
continues to expand, with new clinical uses anticipated [43].
To combat the patent expiries, supergeneric versions of taxol,
such as Cell Therapeutics Xyotax (polyglutamate paclitaxel) and
Abraxis Oncologys Abraxane (nanoparticle albumin-bound paclitaxel), have been developed, offering signicant advantages over
taxol in terms of adverse effects and drug delivery. Sales of
Abraxane rose to $275 millions in 2009 [www.BioPortafolio.com:
emerging oncology treatments: a focus on targeted therapeutics
supergeneric reformulations and supportive care] reecting the
growing market for taxol and its derivatives.
2.4. Alternative methods for taxol production and the need for in
vitro cell culture
Since the discovery of taxol, considerable energy has been
invested in trying to increase its extraction. A serious obstacle to overcome is the low concentration (0.0010.05%) of taxol
found even in the most productive species, T. brevifolia. Since
it is necessary to take 10,000 kg of Taxus bark or 3000 yew
trees to produce only one kilogram of the drug [44] and a cancer patient needs approximately 2.53 g of paclitaxel [45], the
treatment of each patient consumes about eight 60-year-old yew
trees. Other Taxus species such as T. chinensis produce similar
results: CEC China Pharmaceuticals Ltd. reported that 10,000 kg
of leaves and bark of T. chinensis are required to isolate 1 kg
of taxol [http://www.21cecpharm.com/px/fac.htm]. Additionally,
extraction of taxol from yew trees requires a complex system
and specic purication techniques using advanced and expensive
technology.
Taking into account the above facts, together with the seasonal
variation in taxane concentration in Taxus [46] and the high demand
for the drug, there is an urgent need to nd other alternative

sources of taxol production. Since 1997, the Canadian Forest Service Atlantic Forestry Centre have been engaged in a program for
developing ecologically sustainable harvesting protocols of yews
in natural stands, converting elite cultivars of the wild species into
a commercially reared crop [47]. Similarly, in 2004, the company
Yewcare began to plant T. chinensis in the nature reserve of Da Huan
Mountain in the province of Yunan (China). Currently, this Taxus
plantation covers more than 30 km2 and is the largest Taxus yew
tree producer in the world [http://www.yewcare.com/index.php].
Another way to produce taxol is by chemical synthesis, rst
achieved by Holton and Nicolau in 1994 [4850]. However,
the complexity of the biosynthetic pathway and its low yield
limit its applicability. Another alternative is production by semisynthesis, which requires intermediates such as baccatin III or
10-deacetylbaccatin III, found in renewable needles of Taxus. BMS,
a leading global supplier of taxol, has a farm with 30 billion yews to
supply the bark and needles necessary for the extraction of intermediates [51]. In 2007, Indena developed the semisynthesis of taxol
in Europe by a patented process based on 10-deacetylbaccatin III,
which is extracted from T. baccata trees cultivated in the company
plantations [www.Indena.com: latest news from Indena, July 15th,
2009].
The semisynthetic analogue of taxol, docetaxel (registered
as taxotere by Sano-Aventis) is also synthesized from 10deacetylbaccatin III. The market for docetaxel exceeded $3 billion in
2009 but the Sano-Aventis patent expires in 2010 in Europe, 2012
in Japan and 2013 in the USA. Probably this is why the company is
currently investigating two new taxol derivatives, carbazitaxel and
larotaxel, whose biological action is an improvement on docetaxel
[http://www.oncology.sano-aventis.com/tcl/cp/en/index.jsp].
An alternative and environmentally sustainable source of taxol
and analogue compounds is plant cell cultures. This methodology
offers several advantages, not being subjected to weather, season or
contamination, and the material can be grown independently of its
original, potentially remote, location [36,52]. To increase the productivity of taxanes in plant cell cultures, different strategies can
be applied, such as optimization of culture conditions, selection of
high-producing cell lines, and the addition of elicitors or precursors. Currently, Python Biotech is the largest producer of paclitaxel
via plant tissue culture, employing a large-scale fermentor with a
capacity of up to 75,000 L [53]. Another company, Corean Samyang
Genex, uses Taxus plant cell cultures to produce paclitaxel with the
brand name of Genexol (http://www.genex.co.kr/Eng/).
In 1993, an endophytic taxol-producing fungus was discovered
in Taxus, but the production of taxol through fungal fermentation gives low and variable yields. Cytoclonal Pharmaceutics, Inc.
patented this process in 1994 and in 2001 signed an agreement
with BMS for the development of new technology based on microbial fermentation for the production of novel taxane therapeutics
[51].

3. Approaches for in vitro production of taxol in T. baccata

3.1. Callus induction
Calli, an undifferentiated mass of cells growing on solid medium,
is the starting material for growing suspension cultures. The rst
report on callus induction and proliferation from gametophytes of
T. baccata was published in 1973 by Rohr [54,55]. Later on David
and co-workers initiated callus cultures from mature stems and
studied the mineral and phytohormone composition of the culture medium to improve callus proliferation [56,57]. They also used
habituated tobacco calli as a nurse culture. Different explants viz.
cotyledons, hypocotyls, roots from young seedlings, young as well
as mature stems, gametophytes and needles, have been used for

S. Malik et al. / Process Biochemistry 46 (2011) 2334

27

Table 1
Establishment and maintenance of callus cultures in Taxus baccata and comparison of growth media, PGRs and culture conditions.
Explant*

Basal medium

Phytohormones and their

concentrations

Carbohydrate
(g/L)

Additives

Culture conditions

Remarks

Reference

st, n

B5, 2 X B5 vitamins

24 C, dark

Ascorbic acid
(50 mg/L)

24 C, dark

st

B5, 2 X B5 vitamins

Taxol production
within the callus
ranged between
0.113.1 mg/kg
Callus initiated after
1520 days

[59]

B5

Sucrose
(20.0) + glucose
(2.5) + fructose
(0.0025)
Sucrose (20.0)

Casein
hydrolysate
(1.0 g/L)

2,4-D (0.110.0 mg/L),

NAA (0.110.0 mg/L), IBA
(0.110.0 mg/L) + Kn (0.2
or 2.0 mg/L)
NAA (2.0 mg/L) + 2,4-D
(0.2 mg/L)
2,4-D (10.0 M) + Kn
(4.0 M) + GA (1.0 M)
for callus induction
NAA (10.0 M) + BAP
(0.5 M) for callus
growth and maintenance
2,4-D (6.0 mg/L) + Kn
(0.5 mg/L) for callus
induction

st, c, h, r

se, st

B5

B5

23 C, dark

Sucrose (30.0)

[71]
[117]

Sucrose
(5.0) + fructose
(5.0)

2,4-D (3 mg/L) for callus

growth and maintenance
2,4-D (3 mg/L) + Kn
(0.5 mg/L)

PVP (1.5 g/L)

22 2 C, dark

Growth index ranged

between 1.16 and 4.28.
Paclitaxel content was
23.2 mg/kg of dw after
26 days of culture in
line VI/Ha

[58]

PVP (1.5 g/L)

22 2 C, dark

Paclitaxel yield was

comparable to that
found in bark of the
intact plant.
0.0109 0.0037% (DW)
in slow-growing callus
line VI/Ha and
0.00006 0.00003%
(DW) in fast-growing
callus line V/Kle

[60]

B5: Gamborg et al. [61], SH: Schenk and Hildebrandt [63], *c: cotyledons, h: hypocotyls, n: needles, r: roots of young seedlings, s: young stem, se: seedlings, st: young stem
of mature trees.

callus induction (Table 1). It has been observed that young tissue
is more responsive or prone to callus initiation than mature plant
parts or young tissue from adult trees [5860]. Variability in growth
response as well as taxol production in callus cultures derived
from different genotypes has been demonstrated by Brunakova
et al. [58]. Different basal media such as Gamborg (1968, B5)
[61], Murashige and Skoog (1962, MS) [62], Schenk and Hildebrandt (1972, SH) [63] and Woody Plant Medium (WPM, 1981)
[64] have been employed for initiation and maintenance of callus
cultures. The medium is supplemented with various phytohormones as well as organic supplements and additives, including
casein hydrolysate, mannitol, polyninylpyrrolidone, ascorbic acid
and amino acids, to stimulate callus growth and proliferation. Out
of all the phytohormones, 2,4-dichlorofenoxyacetic acid (2,4-D) in
combination with Kinetin (Kn) is the best for callus induction [65].
Wickremesinhe and Arteca [59] cultured stems and needles on B5
medium with a double concentration of vitamins. B5 medium with
2,4-D (6 mg/L) + Kn (0.5 mg/L) and polyvinylpyrrolidone (PVP; 1.5%)
was favorable for callus induction and supplemented with a halfdose of 2,4-D for further growth and maintenance [58,60]. Table 1
lists the details of various media and combinations/concentrations
of Plant Growth Regulators (PGRs) as well as additives used for callus induction and maintenance. Brunakova et al. [58] induced callus
cultures using stems from different genotypes of the same Taxus
species and examined the increase in fresh weight when using two
different basal media i.e. B5 and modied MS. They found that B5
medium favored callus growth irrespective of the genotype, producing a callus growth index of 2.38 0.61 compared to 0.34 0.11
on modied MS medium after one subculturing. Callus growth was
optimized using MS or B5 media fortied with 2,4-D and Kn at content ratios of 1:0.1, 2:0.1, and 5:0.1. Histological studies showed
that both epidermis and mesophyll tissues divided to produce calli
in the leaf explants while cell division in cortical parenchyma and

cambium resulted in callus formation in stem explants [65]. Taxol

production in calli depends on morphology and age. Calli were
found to produce more taxol when old and brown than when young
and pale [5860].
3.2. Establishment of cell suspension cultures and production of
taxanes
Cell suspensions are initiated by inoculating friable calli into liquid medium and consist of single or small cell aggregates. These fast
growing systems can be used for large scale culture of plant cells to
obtain valuable products [6668]. Early studies by various research
groups showed that cells of Taxus spp. can produce taxol and related
compounds under optimized in vitro conditions, as covered by
several exhaustive reviews in the last decade [53,6974]. Various
strategies are being employed in continuing efforts to increase productivity (described in subsequent paragraphs). Table 2 depicts
different culture systems and media employed to establish cell
suspension cultures in T. baccata. Ma et al. [75] isolated four new
bioactive taxoids from cell suspension cultures and elucidated
the structures by spectroscopic analysis. In callus cultures of T.
baccata grown on MS agar gelled medium supplemented with different growth hormones, eight taxol analogues were identied
[76].
4. Approaches to increase the production
To improve the productivity of taxol and related taxanes
in cell cultures for commercial exploitation, efforts have been
focused on assaying the biosynthetic activities of cultured cells.
Approaches include optimizing cultural conditions, screening of
high yielding cell lines, optimization of growth and production
media, induction of secondary metabolite pathways by elicitors and

28

S. Malik et al. / Process Biochemistry 46 (2011) 2334

Table 2
Media employed for cell growth and taxane production in cell suspension cultures of Taxus baccata.
Culture type
Single stage

Reference

1.5 mg/L

[112]

39.75 mg/L

[71]

Two stage
Cell growth medium

B5 + NAA (5.0 M) + BAP

(0.01 M) + 2% sucrose + as
(50 mg/L) + glutamine (2.0 mM)
-

Yield

B5 + NAA (2.0 mg/L) +2,4-D

(0.2 mg/L) + 3% sucrose + VS
(0.1 mg/L) + AgNO3
(0.3 mg/L) + CoCl2 (0.3 mg/L),
addtition of sucrose (1%) + ac
(50 mg/L) at day 10 and addition of
sucrose (1%) + Phl (0.1 mM) at day
20
SH + NAA (5.0 M) + BAP
(0.5 M) + sucrose (1.5%) + glucose
(0.5%)

Taxane production medium

MJ (10 mg/L), SA (100 mg/L) and FE

(2.5 mg/L) at day 2530 in cell
growth medium

SH + NAA (5.0 M) + BAP (0.5

M) + sucrose (1.5%) + glucose
(0.5%) + MJ (100 M)

B5 + NAA (1.86 mg/L) + 2%

sucrose + 0.01% myo-inositol
B5 + NAA (10 M) + BAP (1.0 M)
B5 + NAA (2.0 mg/L) + BAP
(0.1 mg/L) + 0.5% sucrose + 0.5%
fructose
B5 + 2 B5 vitamins + 2,4-D
(4.0 mg/L) + Kn (1.0 mg/L) + GA3
(0.1 mg/L) + 3% sucrose + 0.01%
myo-inositol
B5 + NAA (2.0 mg/L) + BAP
(0.1 mg/L) + 0.5% sucrose + 0.5%
fructose

B5 + Picloram (2.0 mg/L) + Kn

(0.1 mg/L) + 3% sucrose + MJ
(100 M)

B5 + Picloram (2.0 mg/L) + Kn

(0.1 mg/L) + 3% sucrose + MJ (100
M) + cell entrapment in sodium
alginate (1.5%, 2.5%)

[83]

12.04 mg/L

[81]

20.05 mg/L

[117]
[22,78]

12.04 mg/L

[118]

Paclitaxel 13.20 mg/L, baccatin III

4.62 mg/L

[82]

[60]

B5 + 2,4-D (3 mg/L) + Kn
(0.5 mg/L) + 1.5% PVP
B5 + NAA (2.0 mg/L) + BAP
(0.1 mg/L) + 0.5% sucrose + 0.5%
fructose
B5 + NAA (2.0 mg/L) + BAP
(0.1 mg/L) + 0.5% sucrose + 0.5%
fructose

B5 + Picloram (2.0 mg/L) + Kn

(0.1 mg/L) + 3% sucrose
B5 + Picloram (2.0 mg/L) + Kn
(0.1 mg/L) + 3% sucrose

Paclitaxel 1.58 mg/L (1.68 mg/g

DW), baccatin III 0.32 mg/L
(0.35 mg/g DW)
Taxol 7.0 mg/L

[80]

[23]

2,4-D: 2,4-dicholorophenoxy acetic acid, ac: ammonium citrate, as: ascorbic acid, AgNO3 : silver nitrate, B5: Gamborg et al. [61], BAP: 6-benzylaminopurine, CoCl2 : cobalt chloride, FE: fungal elicitor, GA3 : gibberelic acid, Kn: kinetin, NAA: 1-naphthaleneacetic acid, MJ: methyl jasmonate, Phl: phenylalanine, Picloram: 4-amino-3,5,6-trichloropicolinic
acid, SA: salicyclic acid, SH: Schenk and Hildebrandt [63], VS: vanadyl sulphate.

precursors, using a two-phase culture system and immobilization

techniques.

tion of up to 0.0109 0.0037% on an extracted dry weight basis

[60].

4.1. Selection of high yielding cell lines

4.2. Optimization of culture conditions

Cells in suspension cultures generally show considerable variability in their capacity to produce secondary metabolites [77],
due to genetic variation or the heterogeneity associated with the
cells. The preliminary step in establishing a long-term cell culture is thus the selection and cloning of fast-growing cell lines
capable of producing taxol. Cell lines of T. baccata growing under
the same conditions show differing capacities for producing paclitaxel in suspension cultures [58]. It has been observed that the
production of paclitaxel is more affected by differences in biosynthetic activity among the cultured lines than by any other factor
[78]. Paclitaxel and baccatin III production in cell lines obtained
by mixing low-, medial- and high-producing cell lines was higher
than the mean productivity of individual lines before mixing [78].
Brunakova et al. [58] observed great variability (in terms of growth
and paclitaxel content) among callus cultures originating from the
same type of explants of different mother plants or from different parts of the same mother plant. Out of the nine well-growing
callus lines established after 18 months of cultivation, only one
showed improved production (23.2 g/g DW). In another study
by the same group, a cell line VI/Ha was selected and cloned
after 20 months of callus initiation, achieving a paclitaxel produc-

Dark conditions are suitable for the growth of cells and taxol
production [59,79]. Cell cultures grown under a 16/8 h photoperiod
showed a reduction in growth [58]. Suspension cultures have been
reported to turn a lime green color upon prolonged exposure to
continuous light but production of taxol did not take place [79].
4.2.1. Nutrient media and employment of a two-stage culture
system
Like other secondary compounds, taxol is produced in cell cultures when the exponential growth phase has ended and the
cells are in their stationary phase. Therefore, a two-stage system where the cells are rst cultured for biomass production and
then transferred to a medium favorable for taxane production is
an effective strategy for enhancing production. This system has
the added advantage in that it allows precursors and elicitors to
be added when secondary metabolite production is at its highest. The strategy has been successfully employed to improve the
production of paclitaxel and baccatin III in suspension cultures
of T. baccata (Table 2) [69,71,80,81]. Cells were cultured in B5
medium [61] supplemented with sucrose (0.5%), fructose (0.5%), 1naphthaleneacetic acid (NAA; 2.0 mg/L) and 6-benzylaminopurine

S. Malik et al. / Process Biochemistry 46 (2011) 2334

(BAP) (0.1 mg/L) for growth and then transferred to a medium with
3% sucrose, picloram (2.0 mg/L) and Kn (0.1 mg/L) for the production of paclitaxel and baccatin III [69,80,82].
4.2.2. Carbohydrate source
The growth and production of secondary compounds from cultured cells depends greatly on the source of carbon employed, its
concentration and on the biosynthetic pathway or process involved,
as well as the requirements of different plant species. Addition of
fructose (1%) to moderately-productive T. baccata cell cultures at
day 10 signicantly improved the fresh weight of cells in the later
stages of the run [79]. The various carbohydrate treatments result
in marked differences in taxol production in cell cultures, which is
enhanced by fructose treatment and suppressed by glucose. Therefore, it has been interpreted that the limiting step in taxol synthesis
is stimulated by the presence of fructose and inhibited by glucose
[79].

29

Other abiotic elicitors viz., vanadyl sulphate, silver nitrate,

cobalt chloride, arachidonic acid, ammonium citrate, and salicylic
acid have also been used to improve taxane production in T. baccata cell cultures. It was found that the addition of vanadium
sulphate (VSO4 ) to the culture medium signicantly stimulated callus growth as well as taxol and baccatin III content at the end of
culture period [69]. Cell suspension cultures grown from a selected
callus line were shown to enhance the production of taxol and baccatin III by a factor of 2.5 (5.213.1 g/g DW) and 3.6 (4.416.0 g/g
DW), respectively, upon treatment with 0.05 mM vanadium sulphate [69].
A biotic elicitor from Rhyzopus stelonifera fungus (25 mg/L)
used in combination with the abiotic elicitors methyl jasmonate
(10 mg/L) and salicylic acid (100 mg/L) was shown to improve taxol
production 16-fold when added at day 2530 of culture to a growth
medium [71].
4.4. Addition of precursors, adsorbants or additives

4.2.3. Phytohormones
Different concentrations and combinations of auxins (NAA,
2,4-D, 2,4,5-trichlorophenoxyacetic acid or 2,4,5-T, picloram) and
cytokinins (Kn, BAP) have been tested to achieve optimum cell
growth and taxol production from suspension cultures of T. baccata. 2,4-D or NAA (alone or together) in combination with Kn was
used for cell growth by Khosroushahi et al. [71], while NAA (5.0 M)
in combination with BAP (0.5 M) was found to be best for suspension cultures raised from stem-derived calli [83]. The optimum
concentration of PGRs for cell growth and production of taxol in cell
suspension cultures is summarized in Table 2. Picloram improved
cell growth but suppressed taxol production [79].
4.3. Use of elicitors
An elicitor is a substance that, when introduced in small
concentrations to a living cell system, initiates or improves the
biosynthesis of specic compounds and elicitation is a process of
induced or enhanced plant biosynthesis of secondary metabolites
due to the addition of trace amounts of elicitors [84]. Elicitors
can be classied into abiotic (such as metal ions, inorganic compounds) and biotic (including polysaccharides derived from plant
cell walls and micro-organisms and glycoproteins) depending on
their origin [84,85]. Elicitors have been used as an important
means of enhancing the production of taxanes in cell cultures of
Taxus species [25,8688]. Table 3 depicts the taxane production
in cell suspension cultures of T. baccata in response to various
treatments.
The accumulation of paclitaxel and related taxanes in Taxus
plants is thought to be a biological response to specic external
stimuli [86] and jasmonates have been reported to play an important role in a signal transduction process that regulates defense
genes in plants [8991]. It has been proposed that jasmonates are
key signal transducers leading to the accumulation of secondary
metabolites [92,93]. Methyl jasmonate has been used to increase
paclitaxel production in cell cultures of T. canadensis [9497] and
T. cuspidata [98]. The biosynthesis and accumulation of paclitaxel
and related taxanes in T. baccata are strongly promoted by jasmonic acid or its methyl ester [78,83,86]. The addition of methyl
jasmonate to the culture medium has increased the production of
paclitaxel (0.229%, 48.3 mg/L) and baccatin III (0.245%, 53.6 mg/L)
in cell cultures at week 2 compared to the control, which yielded
only 0.4 mg/L of both secondary compounds [86]. Moon et al. [83]
reported that the time course of taxane production after methyl
jasmonate addition differed from normal kinetics without elicitation. Baccatin III and 10-deacetyl baccatin III were detected
rst, followed sequentially by paclitaxel, 10-deacetyl taxol and
cephalomine [83].

The presence of glutamine (2.0 mM) is essential for cell growth

in suspension cultures. Callus growth was enhanced when the
medium was supplemented with 1 mM phenylalanine [69]. To prevent phenolic exudations, ascorbic acid (50 mg/L) was added to
culture medium [79]. Supplementation of the medium during the
rst phase with AgNO3 , VSO4 , CoCl2 , sucrose, phenylalanine and
ammonium citrate resulted in 5.6-fold higher taxol production
(13.75 mg/L) compared with the control (2.5 mg/L) [71].
4.4.1. Synergistic effect of elicitors, additives or inducing factors
As described above, different compounds or elicitors enhance
the production of taxol and related taxanes when applied individually [99]. They also have a pronounced effect on yield
when applied synergistically, due to their interaction with
different enzymes of the production pathway [71]. Medium supplementation with compounds such as AgNO3 , VSO4 , CoCl2 ,
sucrose, phenylalanine and ammonium citrate has an additive effect on taxol production in T. baccata [71]. Intermittent
supplementation of suspension cell cultures in stage I with
biomass growth factors (0.1 mg/L VSO4 + 0.3 mg/L AgNO3 + 0.3 mg/L
CoCl2 + 1% sucrose + 50 mg/L ammonium citrate + 0.1 mM phenylalanine) along with a mixture of elicitors viz. methyl jasmonate
(10 mg/L), salicyclic acid (100 mg/L) and fungal elicitor (2.5 mg/L) in
stage II resulted in a 16-fold higher yield of taxol (16.75 mg/L) with
minimal effect on cell viability compared to the control (2.45 mg/L)
[71] (Table 3).
4.5. In situ product removal and two-phase culture
Low yields of secondary metabolites released to the medium
may be the result of many factors, including feedback inhibition
of membrane transport, biosynthesis, gene activity, degradation
of the product by enzymatic or non-enzymatic processes in the
medium or cells and volatility of substances produced [100]. By
supplying an articial accumulation site in the form of a second phase (using an organic solvent or solid compound), it may
be possible to obtain higher yields by removing the metabolite
from the aqueous medium, and thereby shifting the intracellular/extracellular equilibrium [101,102]. According to Hooker and
Lee [103], in situ removal of secondary products from the medium
using a two-phase culture system facilitates their release from
intracellular organelles. In Taxus, it has been found that the accumulation of taxol in cells leads to feedback inhibition and product
degradation [104], hence its removal from the suspension cultures
is essential for improvement in productivity [105]. Release of taxol
and baccatin III from cells into the medium was enhanced 120%
and 97%, respectively (compared to the control) by the presence of

30

S. Malik et al. / Process Biochemistry 46 (2011) 2334

Table 3
Taxane production in cell suspension cultures (using shake asks and bioreactors) of Taxus baccata in response to various treatments.
Treatment

Culture type and capacity

Compounds

Taxanes

Yield/productivity

Reference

Elicitation

Shake ask (250 ml)

Taxol

13.75 mg/L

[71]

Elicitation + inducing
factors

Shake ask (250 ml)

Taxol

39.75 mg/L
1.02 mg/L/d

[71]

Elicitation

Shake ask (175 ml)

Vanadyl sulphate
(0.1 mg/L) + silver nitrate
(0.3 mg/L) + cobalt chloride
(0.3 mg/L) + phenylalanine
(0.1 mM)
Vanadyl sulphate
(0.1 mg/L) + silver nitrate
(0.3 mg/L) + cobalt chloride
(0.3 mg/L) + phenylalanine
(0.1 mM) + methyl
jasmonate
(10 mg/L) + salicyclic acid
(100 mg/L) + fungal elicitor
(2.5 mg/L)
Methyl jasmonate
(100 M)
Methyl jasmonate
(100 M)
Methyl jasmonate
(100 M)

Paclitaxel

20.05 mg/L

[78]

Elicitation

Shake ask (175 ml)

Elicitation

Shake ask (175 ml)

Elicitation

Shake ask (175 ml)

Elicitation + immobilization

Shake ask (175 ml)

Elicitation + immobilization

Stirred bioreactor (5 L)

Elicitation + immobilization

Airlift bioreactor (4 L)

Elicitation + immobilization

Wave bioreactor (2 L)

Additive
Additive

Shake ask (250 ml)

Pneumatically mixed
bioreactors (1 L)
Pneumatically mixed
bioreactors (1 L)
Shake ask (100 ml)

Additive
Cell suspension culture

[119]

Taxol

4.25 mg dm
2.4 mg dm3
7.09 mg/L on day
22
3.49 mg/L on day
22
8.8 mg/L

[23]

Paclitaxel

13.20 mg/L

[82]

Baccatin III

4.62 mg/L

Paclitaxel
Baccatin III
Paclitaxel

43.43 mg/L
(2.71 mg/L/d)
5.06 mg/L
12.03 mg/L

Paclitaxel
Baccatin III

20.79 mg/L
7.78 mg/L

[82]

Taxol
Taxol

0.3 mg/L
1.5 mg/L

[112]
[112]

Glutamine

Taxol

0.1 mg/L

[112]

Methyl jasmonate
(100 M)

Paclitaxel
Baccatin III

[86]

Cephalomannine
Total taxane
Taxol

48.3 mg/L
(0.229%)
53.6 mg/L
(0.245%) in 2
weeks
3.6 mg/L (0.017%)
36860 nmol/L
13.1 g/g DW

Baccatin III
Taxol

16.0 g/g DW
40 mg/L

Methyl jasmonate
(100 M)
Methyl jasmonate
(100 M) + sodium alginate
(1.5%)
Methyl jasmonate
(100 M) + sodium alginate
(2.5%)
Methyl jasmonate
(100 M) + sodium alginate
(2%)
Methyl jasmonate
(100 M) + sodium alginate
(2%)
Methyl jasmonate
(100 M) + sodium alginate
(2%)
Glutamine
Glutamine

Cell suspension culture

Shake ask (100 ml)

Vanadium sulphate
0.05 mM

Cell suspension culture

Shake ask (175 ml)

Taxol feeding 200 mg/L

vanadium sulphate [69]. Table 4 lists the total taxane production in

cells of T. baccata and its excretion into the medium.
A two-phase culture system has been successfully employed
with T. brevifolia [106] and T. cuspidata [107] but has not been
reported for T. baccata.
4.6. Immobilization
Immobilization is one of the most important strategies for
increasing cell production of secondary compounds. Immobilized
cells have advantages over freely suspended cells as immobilization provides high cell concentration per unit volume, better
cellcell contact and protection from uid shear stress, and prevents cell washout in continuous operations [108110]. Plant cells
are immobilized using different gels viz., alginate, carrageenan,

Paclitaxel
Baccatin III
Paclitaxel
Baccatin III

[80]

[82]

[82]

[69]

[22]

polyacrylamide, agarose, polyurethane foam, and hollow ber. Bentebibel et al. [82] used calcium alginate for immobilization of the
paclitaxel- and baccatin III-producing cells of T. baccata and found
that immobilization enhanced the production of paclitaxel and
baccatin III by factors of 3 and 2, respectively, compared to free
cells. The taxane yield depends on the concentration of alginate
e.g. the accumulation of paclitaxel was 13.20 mg/L, 10.85 mg/L, and
11.90 mg/L at the end of the culture period when using 1.5%, 2.0%
and 2.5% alginate, respectively [82]. However, maximum accumulation of baccatin III (4.62 mg/L) was achieved using 2.5% alginate
[82]. These observations reect differences in the levels of enzymes
induced by alginate concentrations due to variable calcium binding
capacity [111]. Immobilization of cells entrapped with 2% calcium
alginate beads substantially enhances taxane production under
optimized conditions in both shake ask and bioreactor cultures

S. Malik et al. / Process Biochemistry 46 (2011) 2334

31

Table 4
Total taxane production (cell associated + extracellular) and excretion into the media in cell suspension cultures of T. baccata.
Taxane

Treatment

Cell-associated

Extracellular

Total

Excretion (%)

Reference

Paclitaxel

PM

18.23 mg/L

1.68 mg/L

19.91 mg/L

8.4

[78]

Taxol

PM
Taxol feeding (200 mg/L)

0.82 mg/L
17.95 mg/L

0.38 mg/L
21.3 mg/L

1.20 mg/L
39.25 mg/L

31.6
54.2

[22]

Paclitaxel
Baccatin III

PM
PM

2.85 mg/L
0.67 mg/L

1.33 mg/L
1.46 mg/L

4.18 mg/L
2.13 mg/L

32
69

[82]

Paclitaxel
Baccatin III

Im (1.5% alginate)
Im (2.5% alginate)

12.94 mg/L
4.26 mg/L

0.26 mg/L
0.36 mg/L

13.20 mg/L
4.26 mg/L

2
8

Paclitaxel
Baccatin III

PM
PM

1.55 mg/dm3
0.73 mg/dm3

37
44

Paclitaxel
Baccatin III

100 M MJ
100 M MJ

4.25 mg/dm3
2.4 mg/dm3

37
44

Paclitaxel
Baccatin III

200 M MJ
200 M MJ

1.49 mg/dm3
0.75 mg/dm3

32
70

Paclitaxel
Baccatin III

Im + 100 M MJ
Im + 100 M MJ

13.20 mg/dm3
4.62 mg/dm3

2
8

Taxol

MJ
SA
FE

21.14 mg/L
18.65 mg/L
25.16 mg/L

0.003 mg/L
0.02 mg/L
0.015 mg/L

21.143 mg/L
18.67 mg/L
25.175 mg/L

[119]

[71]

FE: fungal elicitor, Im: immobilization, MJ: methyl jasmonate, PM: production medium, SA: salicylic acid.

(4-fold, 1.1-fold and 1.0-fold in Stirred, Airlift and Wave reactors,

respectively) [112].
5. Scale-up studies in bioreactors
For large-scale plant cell culture several bioreactor designs have
been suggested [113]. Bentebibel et al. [82] used Stirred, Airlift and
Wave bioreactors for the production of paclitaxel and baccatin III
from free as well as immobilized cells entrapped with calcium alginate. The maximum production of paclitaxel (43.43 mg/L at day 16,
5-fold higher than free cells) and baccatin III (5.06 mg/L at day 8,
1.6-fold higher than free cells) was found in the Stirred bioreactor, followed by Wave and Airlift bioreactors [82]. In the Airlift
bioreactor, the paclitaxel content in immobilized cells was almost
2-fold higher (12.03 mg/L at day 24) than in freely suspended cells
(6.94 mg/L at day 24). On the other hand, the highest content of
paclitaxel (only 20.79 mg/L at day 8) and baccatin III (7.78 mg/L
at day 16) were obtained from immobilized cells cultured in the
Wave bioreactor [82]. The paclitaxel productivity obtained in this
study using a Stirred bioreactor is one of the highest reported so far
by an academic laboratory for a Taxus species bioreactor culture.
Srinivasan et al. [103] used pneumatically mixed and stirred tank
bioreactors for paclitaxel production in cell cultures and compared
the yield with shake asks. A maximum production (1.5 mg/L) of
taxol was obtained in the pneumatically mixed bioreactor, which
was 5-fold greater than in the stirred tank bioreactor and shake
ask cultures. However, the growth kinetics and paclitaxel production in these reactors were similar to the shake asks, suggesting
that reactors with different congurations could be successfully
used for large-scale production of paclitaxel. Other shake ask data
may be applicable for scale-up studies. Navia-Osorio et al. cultivated cell suspension cultures of T. baccata var. fastigata in a 20 L
airlift bioreactor for 28 days in batch mode and compared the
growth rate and accumulation of taxol and baccatin III production.
Cultures of T. baccata were more effective than T. wallichiana in
excreting baccatin III into the medium. However, the total taxol
content in T. baccata was only 12.04 mg/L, which was lower than
in T. wallichiana (21.04 mg/L) [81]. Currently, bioreactors of up
to 75,000 L are being employed for the commercial production
of paclitaxel from cell cultures by Phyton Biotech, ESCAgenetic,
Samyang Genex, Nattermann (Germany) [72,114].

6. Metabolic engineering
It is possible to increase the production of paclitaxel and other
desirable taxanes either by the overexpression of genes controlling limiting steps or by suppressing the undesired taxanes by
employing antisense technology. With a full understanding of the
taxol biosynthetic pathway and the availability of the responsible
genes, it may be possible to bioengineer Taxus cell cultures for high
and commercially sustainable production rates of useful taxanes
[39,72].
In order to engineer the biosynthetic pathway, the time
course of expression of the genes 10-deacetylbaccatin III10-O-acetyltransferase (dbat) and 3 -N-debenzoyl-2 -deoxytaxol
N-benzoyltransferase (dbtnbt), which are involved in paclitaxel
biosynthesis and intracellular taxane accumulation, was studied
in callus and cell cultures [115,116]. It was shown that although
the increase in transcriptional activity of dbat and dbtnbt positively correlates with callus growth, the intracellular accumulation
of paclitaxel varied during subculture with the maximum occurring between the late linear and stationary phase. The advances in
taxol and related taxane production in T. baccata cell cultures are
highlighted in Table 5.
7. Future perspectives
Taxus baccata (European yew) has been one of the most frequent sources of taxol for studies of the biosynthetic pathway and
improved production of this anticancer drug. Taxol production in
T. baccata suspension cultures has been improved by optimizing
culture conditions, assaying several basic media, plant growth regulators, sugar supplements, etc, and cultures have been scaled up
to a bioreactor level for large-scale production. However, these
empirical methods have not been able to meet the increasing world
demand for taxanes, which according to Global Industry Analysts
will reach 1040 kg per year by 2012 [www.strategir.com: Bulk
Paclitaxel, a global strategic busines report]. A rational approach
might provide new insight into how the taxol biosynthetic pathway
is regulated, with genetic and metabolic engineering techniques,
differential genetic expression, transcription factors and key genes
leading to higher taxol yields. One aspect to take into account is the
mechanism of taxol excretion from cells, which could be enhanced

32

S. Malik et al. / Process Biochemistry 46 (2011) 2334

Table 5
Chronological studies of cell suspension cultures of T. baccata.
Year
1993
1994
1995
1996
1996
1998
1999
2002
2002
2004
2005
2005
2006

2006
2007
2008
2009
2009

Reference
Detection and evaluation of taxol in callus cultures
Four new bioactive taxoids were isoloted from a cell culture and their structures were elucidated by spectroscopic analyses
Scale up studies with cell suspension cultures were carried out using 1 L working volume pneumatically mixed and stirred tank
bioreactors
The response of cell suspension cultures to basic manipulation of culture conditions is described
Effects of methyl jasmonate and its analogs were studied on the accumulation of paclitaxel and related taxanes in cell suspension cultures
Studies on the kinetics of cell growth, production of paclitaxel and related taxanes after methyl jasmonate treatment were carried out
A callus line and its derived cell suspension cultures were treated with the abiotic elicitor VSO4 and its effect on the synthesis of taxol and
baccatin III was studied
Suspension cultures were grown in a 20 L airlift bioreactor (running for 28 days in batch mode) and their growth as well as capacity to
accumulate taxol and baccatin III was measured
Taxol transport in T. baccata L. cell suspension cultures was studied using [14 C]-taxol as a tracer. Taxol uptake was inhibited by
Na-orthovanadate and verapamil and Ca2+ was required for the active absorption of the molecule
Selection and cloning of a rapidly growing callus line with improved taxol production. The effect of the genotype on callus initiation,
growth and taxol production was studied
Effect of immobilization by entrapment with alginate on paclitaxel and baccatin III production in cell suspension cultures was studied and
scaling-up was carried out using different bioreactors
Stable-growing callus lines with different growth characteristics were selected after 12 years of culture. The ability to produce paclitaxel
and its analogues from these lines and their derived suspension cultures was demonstrated
Studies were carried out with cell lines showing different paclitaxel producing capacities. It was described how the production of
paclitaxel and baccatin III is affected when cell lines with different capacities are mixed and cultured in a production medium with or
without methyl jasmonate
To improve the production of taxol, cell suspension cultures were treated with a combination of inducing factors and their effects were
studied
It was discovered that isopentenyl diphosphate is a source for taxol and baccatin III biosynthesis in cell cultures of T. baccata
The time course of expression of two genes, dbat and dbtnbt, involved in paclitaxel biosynthesis and intracellular taxane accumulation
were studied in callus cultures
Effect of taxol feeding was studied on taxol and related taxane production in cell suspension cultures
Studies on gene expression proling in T. baccata seedlings and cell cultures were carried out

by employing a two-phase culture system, so far not assayed in

T. baccata cell suspensions. Future perspectives could be focused
on the simultaneous use of empirical and rational approaches
and assaying the two-phase culture system in order to develop a
biotechnological system for high taxol production.
Acknowledgements
Work in the Plant Physiology Laboratory (University
of Barcelona) was nancially supported by the Spanish
MEC (BIO2008-01210) and the Generalitat de Catalunya
(2009SGR1217).
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