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Process Biochemistry 46 (2011) 23–34
Contents lists available at ScienceDirect
Process Biochemistry
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / p r o c b i o
Review
Production of the anticancer drug taxol in Taxus baccata suspension cultures:A review
Sonia Malik a, Rosa M. Cusidó b, Mohammad Hossein Mirjalili c, Elisabeth Moyano d, Javier Palazón b, Mercedes Bonfill b,∗
a Departmento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, CP 6109, 13083-970 Campinas, Brazilb Laboratorio de Fisiologia Vegetal, Facultad de Farmacia, Universidad de Barcelona, Avda. Joan XXIII s/n, 08028 Barcelona, Spainc Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, 1983963113, Tehran, Irand Departament de Ciencies Experimentals i de la Salut, Pompeu Fabra University, Av. Doctor Aiguader 80, 08003 Barcelona, Spain
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 andits derivatives, due to a specific 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 reviewis focused on recent advances in the production of taxol and related taxanes in Taxus baccata,
the taxol-producing European yew, using cell suspension culturetechnology. The reviewcontains 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. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2. Taxol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.1. Taxol and anti-cancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.2. Biosynthesis of taxol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.3. Taxol demand and industrial production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.4. Alternative methods for taxol production and the need for in vitro cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3. Approaches for in vitro production of taxol in T. baccata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.1. Callus induction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.2. Establishment of cell suspension cultur es and production of tax anes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4. Approaches to increase the production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
4.1. Selection of high yielding cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.2. Optimization of culture conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4. 2. 1. Nutrient media and employment of a two- stage culture system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4.2.2. Carbohydrate source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.2.3. Phytohormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.3. Use of elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.4. Addition of precursors, adsorbants or additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4. 4. 1. Synergistic effec t of elicitor s, additives o r inducing fa ctors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4.5. In situ product removal and two-phase culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
∗ Corresponding author. Tel.: +34 93 4020267; fax: +34 93 4029043.
E-mail address: [email protected] (M. Bonfill).
1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.09.004
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4.6. Immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5. Scale-up studies in bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6. Metabolic engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
7. Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
1. Introduction
Taxus baccata or the European yew is distributed throughout
the temperate zones of the northern hemisphere. It is a small-
to 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 (Pacific yew or
Western yew), T. canadensis (Canadian yew), T. chinensis (Chi-
nese yew), T. cuspidata (Japanese yew), T. floridana (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 poi-
sons” the Potier group in France found that the main taxane inT. 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 pro-
duction of the analogous compound taxotere (also known by thegeneric nameof docetaxel andthe registeredtrade nameTaxotere®
[Sanofi-Aventis]), which has the same action mechanism as taxol
[5]. Additionally, lignans, flavonoids, 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].
Specifically in T. baccata, five 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 power-
ful inhibitory effect on tumor necrosis factor- (TNF-) [9] andtaxiresinol is reported to be highly protective against gastric lesions
[6].
2. Taxol
Among secondary metabolites with anticancer activity, taxol,
a complex diterpene obtained from Taxus spp., is arguably the
most important. Itschemicalname is 5, 20-epoxy-1,2,4,7,13-hexahydroxytax-11-en-9one-4, 10-diacetate-2-benzoate 13 ester
with (2R,3S )-N -benzoyl-3-phenylisoserine; its molecular formula
is C47H51NO14 and molecular weight is 853.9Da [10]. At the core
of taxol are the A, B and C ring systems, which have several func-
tional 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 (2R,3S )-N -benzoyl-3-phenylisoserine, with
a hydroxyl and a benzoyl functional group.
2.1. Taxol and anti-cancer activity
The antitumor activity of taxol is mainly due to the side chain,
A ring, C2 benzoyl group and oxetane ring. The activity is main-
tained 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 promotionof polymerization produces cytotoxicity and microtubule stabiliza-
tion [12].
Taxol inhibits cell proliferation by binding to the microtubule
surface, specifically to the subunit of the tubulin heterodimers,thus promoting its polymerization, even in absence of GTP
[5,13,14].
While thenumberof cancers being treated by taxolis expanding,
to date it has been principally used to treat metastatic carcinomaof the ovary [15], metastatic breast cancer and non-small cell lung
cancer as well as in second-line treatment of AIDS-related Kaposi’s
sarcoma. Taxol is currently being studied for the treatment of dis-
easesnot related withcancerthat require microtubule stabilization
and the avoidance of cell proliferation and angiogenesis, for exam-
ple, psoriasis [16]. Taxol is also being studied for the treatment
of taupathies (affections in tau proteins), such as Alzheimer’s 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 N -
tigloyl group instead of the N -benzoyl group at C3 without any
reduction of cytotoxicity and microtubule disassembly [18]. The
first natural analogue of paclitaxel, 2-debenzoyl-2-tigloyl pacli-
taxel, has a modified ester group at C2 while retaining tubulinbinding activity, although it is less cytotoxic [19].
2.2. Biosynthesis of taxol
As a natural diterpenoid, taxol is formed exclusively from ger-
anylgeranyl 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
branchedpoint progenitor of a variety of diterpenoids and tetrater-
penoids. 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 [21–24] have shown the involvement of
thecytosolic pathway.Srinivasanet al.[25] suggestedthat cytosolicIPP could play a role in taxol production in the initial growth phase
of Taxus cells. Additionally, Wang et al. [26], after supplementing T .
chinensis cellsuspensionswith twoinhibitors of metabolitetranslo-
cation, suggested that the translocation of IPP through the plastidic
membrane only occurs duringthe 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
(aninhibitor of theplastidic pathway), it was also reduced by mevi-
nolin (an inhibitor of the cytosolic pathway), indicating that both
pathways could be involved [23].
The first committed step of taxol biosynthesis is the cyclization
of geranylgeranyl diphosphate (GGPP) to the taxa-(4,5),(11,12)-
diene, a reactioncatalyzedby taxadienesynthase (TS),a monomeric
protein of 79 kDa. The enzyme was purified and characterized
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S. Malik et al. / Process Biochemistry 46 (2011) 23–34 25
Fig. 1. Taxol biosynthetic pathway.
Adapted from Guo et al. [120] and Expósito et al. [22].
by Hezari et al. [27], and the gene that codifies 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 medi-ated 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 formationof taxa-4(20),11(12)-dien-5-ol, which is the second step in taxolbiosynthesis [29]. T5H is a protein of 56 kDa with an N-terminalsequence of insertion in the membrane of the endoplasmic retic-
ulum. This enzyme, apart from its hydroxylating activity, also
conditions the migration of the double bond from 4(5) to 4(20).
Althoughthese 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 spe-
cific taxadiene-5-ol-O-acetyl transferase (TDAT) that acylates
taxa-4(20),11(12)-dien-5-ol at the C5 position to form taxa-4(20),11(12)-dien-5-yl-acetate.Thisenzymeis a protein of50 kDathat 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-dependentmonooxygenase cloned and functionally characterized in yeast
[32].
Another Cyt P450-dependent hydroxylase leading to the forma-
tion 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 isnot a linear pathway and that there are branch points that can
lead to other related taxoids. It has been observed that this alterna-
tive step is especially frequent in cell cultures elicited with methyl
jasmonate [34].
The taxoid 14-hydroxylase (T14H) is responsible for the for-mation 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, suggest-ingthat T14H cannotbe involved inthe production of taxol,whichdoes not present any hydroxylation at the C14 position.
The last steps in the taxol biosynthetic pathway, after the for-
mation of taxa-4(20),11(12)-dien-5,10-diol 5-acetate, includeseveral hydroxylations at the C1, C2, C4 and C7 positions, oxida-
tion 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 intoaccount the oxidationfrequency
of the taxoids found in cell cultures, a probable sequence vali-
dated by phylogenetic analyses of previously cloned taxoid P450
oxigenases could be: C5, C10, C2, C9, C13, C7 and finally 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 formationhave been proposed [37–39], it is currently accepted that the pro-
cess involves epoxidation of the 4(20) double bond followed by
migration of the -acetoxy group from the C5 to the C4 positiontogether 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 C4–C20
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 pre-
cursor by the activity of the enzyme 2-O-benzoyl transferase(DBT), a protein of 50kDa, the next compound obtained is
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26 S. Malik et al. / Process Biochemistry 46 (2011) 23–34
10-deacetylbaccatin III. Another identified transacetylation reac-
tion 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 inter-
mediate, baccatin III, using 10-DAB and acetyl CoA as substrates.
An essential step in the taxol biosynthesis is theesterification of
the C13 hydroxyl group of baccatin III with the -phenylalanoyl-CoA 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 cat-
alyzes the conjugation of the -phenylalanoyl-CoA side chain tobaccatinIII is C-13-phenylpropanoyl-CoA transferase (BAPT), yield-
ing the compound 3-N -debenzoyl-2-deoxytaxol. This compound,
by the action of an unknown Cyt P450-dependent hydroxylase
thathydroxylatesthe C2 positionand the enzyme 3-N -debenzoyl-
2-deoxytaxol N -benzoyl transferase (DBTNBT) that conjugates
benzoyl-CoA to 3-N -debenzoyl-2-deoxytaxol, yields taxol as the
final compound. This enzyme can be exploited to improve the pro-
duction of taxol in genetically engineered systems [42].
2.3. Taxol demand and industrial production
Taxol is one of the most successful anticancer drugs devel-
oped 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 Oncology’s Abraxane (nanoparticle albumin-bound pacli-taxel), have been developed, offering significant 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] reflecting 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 obsta-
cle to overcome is the low concentration (0.001–0.05%) of taxol
found even in the most productive species, T. brevifolia. Sinceit is necessary to take 10,000kg of Taxus bark or 3000 yew
trees to produce only one kilogram of the drug [44] and a can-
cer patient needs approximately 2.5–3 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 specific purification techniques using advanced and expensive
technology.
Taking into account the above facts, together with the seasonal
variationin taxane concentration in Taxus [46] andthehighdemand
for the drug, there is an urgent need to find other alternative
sources of taxol production. Since 1997, the Canadian Forest Ser-
vice – 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 thenature 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, first
achieved by Holton and Nicolau in 1994 [48–50]. However,
the complexity of the biosynthetic pathway and its low yield
limit its applicability. Another alternative is production by semi-
synthesis, 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 inter-
mediates [51]. In 2007, Indena developedthe 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 Sanofi-Aventis) is also synthesized from 10-deacetylbaccatin III.The market for docetaxelexceeded$3 billion in
2009 butthe Sanofi-Aventis patentexpires 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.sanofi-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, notbeing subjected to weather,season or
contamination, andthe material can be grownindependently of its
original, potentially remote, location [36,52]. To increase the pro-
ductivity 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 precur-sors. Currently, Python Biotech is the largest producer of paclitaxel
via plant tissue culture, employing a large-scale fermentor with a
capacity of up to 75,000L [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 fermenta-
tion 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 micro-
bial 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 massof cellsgrowing on solidmedium,
is the starting material for growing suspension cultures. The first
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 cul-
ture 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
http://www.strategyr.com/Bulk_Paclitaxel_Market_Report.asphttp://www.bioportafolio.com/http://www.21cecpharm.com/px/fac.htmhttp://www.yewcare.com/index.phphttp://www.indena.com/http://www.oncology.sanofi-aventis.com/tcl/cp/en/index.jsphttp://www.genex.co.kr/Eng/http://www.genex.co.kr/Eng/http://www.oncology.sanofi-aventis.com/tcl/cp/en/index.jsphttp://www.indena.com/http://www.yewcare.com/index.phphttp://www.21cecpharm.com/px/fac.htmhttp://www.bioportafolio.com/http://www.strategyr.com/Bulk_Paclitaxel_Market_Report.asp
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S. Malik et al. / Process Biochemistry 46 (2011) 23–34 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 2,4-D (0.1–10.0mg/L),
NAA (0.1–10.0mg/L), IBA
(0.1–10.0mg/L) + Kn (0.2
or 2.0mg/L)
Sucrose
(20.0) + glucose
(2.5)+ fructose
(0.0025)
Casein
hydrolysate
(1.0g/L)
24 ◦C, dark Taxol production
within the callus
ranged between
0.1–13.1 mg/kg
[59]
s B5 NAA (2.0 mg/L) + 2,4-D
(0.2mg/L)
Sucrose (20.0) Ascorbic acid
(50mg/L)
24 ◦C, dar k Callus initiat ed aft er
15–20 days
[71]
st B5, 2 X B5 vitamins 2,4-D (10.0M)+Kn
(4.0M)+ GA (1.0M)
for callus induction
Sucrose (30.0) 23 ◦C, dark – [117]
NAA (10.0M)+BAP(0.5M) for callus
growth and maintenance
Sucrose
(5.0)+ fructose
(5.0)
st, c, h, r B5 2,4-D (6.0 mg/L) +Kn
(0.5mg/L) for callus
induction
PVP (1.5 g /L) 22±2 ◦C, dark Growth index ranged
between 1.16 and 4.28.
Paclitaxel content was
23.2mg/kg of dw after
26 days of culture in
line VI/Ha
[58]
2,4-D (3 mg/L) for callus
growth and maintenance
se, st B5 2,4-D (3 mg/L) + Kn
(0.5mg/L)
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 youngtissue from adulttrees [58–60]. Variability in growth
response as well as taxol production in callus cultures derived
from different genotypes has been demonstrated by Brunakovaet al. [58]. Different basal media such as Gamborg (1968, B5)
[61], Murashige and Skoog (1962, MS) [62], Schenk and Hilde-
brandt (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 phytohor-
mones 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 (0.5mg/L)and polyvinylpyrrolidone (PVP; 1.5%)
was favorable for callus induction and supplemented with a half-dose 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 cal-
lus induction and maintenance.Brunakova et al. [58] induced callus
cultures using stems from different genotypes of the same Taxus
species andexamined the increase in fresh weight when using two
different basal media i.e. B5 and modified MS. They found that B5
medium favored callus growth irrespective of the genotype, pro-
ducinga callus growth indexof 2.38±0.61 compared to 0.34±0.11
on modified MS medium after one subculturing. Callus growthwas
optimizedusingMS or B5 media fortified with 2,4-D and Kn at con-
tent 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
foundto produce more taxol whenold andbrown thanwhenyoung
and pale [58–60].
3.2. Establishment of cell suspension cultures and production of
taxanes
Cell suspensions are initiatedby inoculating friable calli intoliq-
uidmediumand consist of singleor small cell aggregates. These fast
growing systems canbe used for large scale culture of plant cells to
obtain valuable products [66–68]. Earlystudies by various research
groups showed thatcells of Taxus spp.can produce taxoland related
compounds under optimized in vitro conditions, as covered by
several exhaustive reviews in the last decade [53,69–74]. Various
strategies are being employed in continuing efforts to increase pro-
ductivity (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 newbioactive 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 dif-
ferent growth hormones, eight taxol analogues were identified
[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 secondarymetabolite pathwaysby elicitorsand
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28 S. Malik et al. / Process Biochemistry 46 (2011) 23–34
Table 2
Media employed for cell growth and taxane production in cell suspension cultures of Taxus baccata.
Culture type Yield Reference
Single stage Two stage
Cell growth medium Taxane production medium
B5+ NAA (5.0M)+BAP
(0.01M)+2% sucrose+as
(50mg/L) + glutamine (2.0mM)
1.5mg/L [112]
- 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 (0.1mM) at day
20
MJ (10 mg/L), SA (100 mg/L) and FE(2.5mg/L) at day 25–30 in cell
growth medium
39.75mg/L [71]
– SH + NAA (5.0M)+BAP
(0.5M)+ sucrose (1.5%)+ glucose
(0.5%)
SH+ NAA (5.0M)+ BAP (0.5M) + sucrose (1.5%)+ glucose
(0.5%)+ MJ (100M)
[83]
B5+NAA (1.86mg/L)+2%
sucrose+ 0.01% myo-inositol
12.04mg/L [81]
B5+ NAA (10M)+ BAP (1.0M) [117]
B5+NAA (2.0mg/L)+BAP
(0.1mg/L)+ 0.5% sucrose+ 0.5%
fructose
B5+ Picloram (2.0 mg/L)+ Kn
(0.1 mg/L)+ 3% sucrose+ MJ
(100M)
20.05mg/L [22,78]
B 5+ 2×
B5 vitamins+ 2,4-D(4.0 mg/L)+ Kn (1.0mg/L)+ GA3(0.1 mg/L)+ 3% sucrose +0.01%
myo-inositol
12.04mg/L [118]
B5+NAA (2.0mg/L)+BAP
(0.1mg/L)+ 0.5% sucrose+ 0.5%
fructose
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%)
Paclitaxel 13.20mg/L, baccatin III
4.62mg/L
[82]
B5+ 2,4-D (3mg/L)+ Kn
(0.5mg/L)+ 1.5% PVP
[60]
B5+NAA (2.0mg/L)+BAP
(0.1mg/L)+ 0.5% sucrose+ 0.5%
fructose
B5+ Picloram (2.0 mg/L)+ Kn
(0.1mg/L)+ 3% sucrose
Paclitaxel 1.58mg/L (1.68mg/g
DW), baccatin III 0.32 mg/L
(0.35mg/g DW)
[80]
B5+NAA (2.0mg/L)+BAP
(0.1mg/L)+ 0.5% sucrose+ 0.5%
fructose
B5+ Picloram (2.0 mg/L)+ Kn
(0.1mg/L)+ 3% sucrose
Taxol 7.0mg/L [23]
2,4-D:2,4-dicholorophenoxy acetic acid,ac: ammonium citrate,as: ascorbic acid,AgNO3: silvernitrate, B5:Gamborget al. [61], BAP: 6-benzylaminopurine,CoCl2: cobalt chlo-
ride,FE: fungalelicitor,GA3: gibberelicacid, Kn:kinetin,NAA: 1-naphthaleneaceticacid,MJ: methyl jasmonate, Phl:phenylalanine, Picloram:4-amino-3,5,6-trichloropicolinicacid, SA: salicyclic acid, SH: Schenk and Hildebrandt [63], VS: vanadyl sulphate.
precursors, using a two-phase culture system and immobilization
techniques.
4.1. Selection of high yielding cell lines
Cells in suspension cultures generally show considerable vari-
ability 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 cul-
ture 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 pacli-taxel in suspension cultures [58]. It has been observed that the
production of paclitaxel is more affected by differences in biosyn-
thetic 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 differ-
ent 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 (23.2g/g DW). In another studyby the same group, a cell line VI/Ha was selected and cloned
after 20 months of callus initiation, achieving a paclitaxel produc-
tion of up to 0.0109±0.0037% on an extracted dry weight basis
[60].
4.2. Optimization of culture conditions
Dark conditions are suitable for the growth of cells and taxol
production [59,79]. Cell cultures grownunder 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 culturesystem
Like other secondary compounds, taxol is produced in cell cul-
tures when the exponential growth phase has ended and the
cells are in their stationary phase. Therefore, a two-stage sys-
tem where the cells are first 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 high-
est. 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%), 1-
naphthaleneacetic acid (NAA; 2.0 mg/L) and 6-benzylaminopurine
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S. Malik et al. / Process Biochemistry 46 (2011) 23–34 29
(BAP) (0.1 mg/L) for growthand then transferred to a medium with
3% sucrose, picloram (2.0mg/L) and Kn (0.1 mg/L) for the produc-
tion of paclitaxel and baccatin III [69,80,82].
4.2.2. Carbohydrate source
The growth and production of secondary compounds from cul-
tured cells depends greatly on the source of carbon employed, its
concentrationand on thebiosynthetic pathwayor 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 significantly 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. There-
fore, it hasbeen interpreted that thelimitingstep in taxol synthesis
is stimulated by the presence of fructose and inhibited by glucose
[79].
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. bac-
cata. 2,4-D or NAA (alone or together) in combination with Kn wasused forcellgrowthby Khosroushahi et al.[71], while NAA(5.0M)in combination with BAP (0.5M) was found to be best for sus-pension cultures raised from stem-derived calli [83]. The optimum
concentration of PGRs forcell growthandproductionof taxol incell
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 specific 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]. Elicitorscan be classified into abiotic (such as metal ions, inorganic com-
pounds) 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 [25,86–88]. 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 specific external
stimuli [86] and jasmonates have been reported to play an impor-
tant role in a signal transduction process that regulates defense
genes in plants [89–91]. It has been proposed that jasmonates are
key signal transducers leading to the accumulation of secondarymetabolites [92,93]. Methyl jasmonate has been used to increase
paclitaxel production in cell cultures of T. canadensis [94–97] and
T. cuspidata [98]. The biosynthesis and accumulation of paclitaxel
and related taxanes in T. baccata are strongly promoted by jas-
monic 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%, 53.6mg/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 elic-
itation. Baccatin III and 10-deacetyl baccatin III were detected
first, followed sequentially by paclitaxel, 10-deacetyl taxol and
cephalomine [83].
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. bac-
cata cell cultures. It was found that the addition of vanadium
sulphate (VSO4) to the culture medium significantlystimulated cal-
lus 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 andbac-
catin IIIby a factorof 2.5(5.2–13.1g/gDW) and3.6 (4.4–16.0g/gDW), respectively, upon treatment with 0.05 mM vanadium sul-
phate [69].
A biotic elicitor from Rhyzopus stelonifera fungus (25mg/L)
used in combination with the abiotic elicitors methyl jasmonate
(10mg/L) and salicylicacid (100mg/L) was shown to improve taxol
production 16-fold when added at day25–30 of culture to a growth
medium [71].
4.4. Addition of precursors, adsorbants or additives
The presence of glutamine (2.0mM) is essential for cell growth
in suspension cultures. Callus growth was enhanced when the
medium was supplemented with 1 mM phenylalanine [69]. To pre-
vent phenolic exudations, ascorbic acid (50mg/L) was added to
culture medium [79]. Supplementation of the medium during thefirst phase with AgNO3, VSO4, CoCl2, sucrose, phenylalanine and
ammonium citrate resulted in 5.6-fold higher taxol production
(13.75mg/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 indi-
vidually [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 sup-
plementation with compounds such as AgNO3, VSO4, CoCl2,
sucrose, phenylalanine and ammonium citrate has an addi-
tive effect on taxol production in T. baccata [71]. Intermittent
supplementation of suspension cell cultures in stage I withbiomass growth factors (0.1mg/L VSO4 +0.3mg/LAgNO3 +0.3mg/L
CoCl2 + 1% sucrose+ 50mg/L ammonium citrate + 0.1mM pheny-
lalanine) along with a mixture of elicitors viz. methyl jasmonate
(10mg/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 (16.75mg/L) with
minimal effecton cell viability compared to the control (2.45mg/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 themedium or cells and volatility of substances produced [100]. By
supplying an artificial accumulation site in the form of a sec-
ond 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 intracellu-
lar/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 accu-
mulation 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
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Table 3
Taxane production in cell suspension cultures (using shake flasks and bioreactors) of Taxus baccata in response to various treatments.
Treatment Culture type and capacity Compounds Taxanes Yield/productivity Reference
Elicitation Shake flask (250 ml) Vanadyl sulphate
(0.1mg/L)+ silver nitrate
(0.3mg/L)+ cobalt chloride
(0.3mg/L)+ phenylalanine
(0.1mM)
Taxol 13.75 mg/L [71]
Elicitation + inducing
factors
Shake flask (250 m l) Vanadyl sulphate
(0.1mg/L)+ silver nitrate(0.3mg/L)+ cobalt chloride
(0.3mg/L)+ phenylalanine
(0.1 mM)+ methyl
jasmonate
(10mg/L) + salicyclic acid
(100mg/L) + fungal elicitor
(2.5mg/L)
Taxol 39.75 mg/L
1.02mg/L/d
[71]
Elicitation Shake flask (175 ml) Methyl jasmonate
(100M)
Paclitaxel 20.05 mg/L [78]
Elicitation Shake flask (175 ml) Methyl jasmonate
(100M)
Paclitaxel
Baccatin III
4.25mgdm−3
2.4mgdm−3[119]
Elicitation Shake flask (175 ml) Methyl jasmonate
(100M)
Paclitaxel
Baccatin III
7.09mg/L on day
22
3.49mg/L on day
22
[80]
Elicitation Shake flask (175 ml) Methyl jasmonate
(100M)
Taxol 8.8 mg/L [23]
Elicit at ion + im mobilizat io n S hake flas k ( 175ml) Met hy l jasm on at e
(100M) + sodium alginate
(1.5%)
Paclitaxel 13.20 mg/L [82]
Methyl jasmonate
(100M) + sodium alginate
(2.5%)
Baccatin III 4.62 mg/L
Elicitation+ immobilization Stirred bioreactor (5L) Methyl jasmonate
(100M) + sodium alginate
(2%)
Paclitaxel
Baccatin III
43.43mg/L
(2.71mg/L/d)
5.06mg/L
[82]
Elicitation+ immobilization Airlift bioreactor (4L) Methyl jasmonate
(100M) + sodium alginate
(2%)
Paclitaxel 12.03 mg/L [82]
Elicitation+ immobilization Wave bioreactor (2L) Methyl jasmonate
(100M) + sodium alginate
(2%)
Paclitaxel
Baccatin III
20.79mg/L
7.78mg/L
[82]
Additive Shake flask (250 ml) Glutamine Taxol 0.3 mg/L [112]
Additive Pneumatically mixed
bioreactors (1 L)
Glutamine Taxol 1.5 mg/L [112]
Additive Pneumatically mixed
bioreactors (1 L)
Glutamine Taxol 0.1 mg/L [112]
Cell suspension culture Shake flask (100 m l) Methyl j asmonate
(100M)Paclitaxel
Baccatin III
48.3mg/L
(0.229%)
53.6mg/L
(0.245%) in 2
weeks
[86]
Cephalomannine 3.6 mg/L (0.017%)
Total taxane 36860 nmol/L
Cell suspension culture Shake flask (100 m l) Vanadium su lphate
0.05mM
Taxol 13.1g/g DW [69]
Baccatin III 16.0g/g DW
Cell suspension culture Shake flask (175 ml) Taxol feeding 200 mg/L Taxol 40 mg/L [22]
vanadium sulphate [69]. Table 4 lists thetotal 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 immobi-
lization provides high cell concentration per unit volume, better
cell–cell contact and protection from fluid shear stress, and pre-
vents cell washout in continuous operations [108–110]. Plant cells
are immobilized using different gels viz., alginate, carrageenan,
polyacrylamide, agarose,polyurethane foam,and hollow fiber. Ben-
tebibel 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. theaccumulation of paclitaxel was 13.20mg/L, 10.85mg/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 accumu-
lation of baccatin III (4.62 mg/L) was achieved using 2.5% alginate
[82]. These observations reflect 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 flask and bioreactor cultures
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S. Malik et al. / Process Biochemistry 46 (2011) 23–34 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 0.82 mg/L 0.38 mg/L 1.20 mg/L 31.6 [22]
Taxol feeding (200 mg/L) 17.95 mg/L 21.3 mg/L 39.25 mg/L 54.2
Paclitaxel PM 2.85 mg/L 1.33 mg/L 4.18 mg/L 32 [82]
Baccatin III PM 0.67 mg/L 1.46 mg/L 2.13 mg/L 69
Paclitaxel Im (1.5% alginate) 12.94 mg/L 0.26 mg/L 13.20 mg/L 2
Baccatin III Im (2.5% alginate) 4.26 mg/L 0.36 mg/L 4.26 mg/L 8
Paclitaxel PM 1.55 mg/dm3 37 [119]
Baccatin III PM 0.73 mg/dm3 44
Paclitaxel 100M MJ 4.25 mg/dm3 37
Baccatin III 100M MJ 2.4 mg/dm3 44
Paclitaxel 200M MJ 1.49 mg/dm3 32
Baccatin III 200M MJ 0.75 mg/dm3 70
Paclitaxel Im + 100M MJ 13.20 mg/dm3 2
Baccatin III Im + 100M MJ 4.62 mg/dm3 8
Taxol MJ 21.14 mg/L 0.003 mg/L 21.143 mg/L [71]
SA 18.65 mg/L 0.02 mg/L 18.67 mg/L
FE 25.16 mg/L 0.015 mg/L 25.175 mg/L
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 algi-
nate. 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 bioreac-
tor, followed by Wave and Airlift bioreactors [82]. In the Airliftbioreactor, the paclitaxel content in immobilized cells was almost
2-fold higher (12.03mg/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 oneof thehighest 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 flasks. 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
flask cultures. However, the growth kinetics and paclitaxel produc-tion in these reactors were similar to the shake flasks, suggesting
that reactors with different configurations could be successfully
used forlarge-scale production of paclitaxel. Othershake flask data
may be applicable for scale-up studies. Navia-Osorio et al. culti-
vated cell suspension cultures of T. baccata var. fastigata in a 20L
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 12.04mg/L, which was lower than
in T. wallichiana (21.04 mg/L) [81]. Currently, bioreactors of up
to 75,000L 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 control-
ling 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 maybe 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 III-
10-O-acetyltransferase (dbat ) and 3
-N -debenzoyl-2
-deoxytaxolN -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 posi-
tively correlates with callus growth, the intracellular accumulation
of paclitaxel varied during subculture with the maximum occur-
ring 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 fre-
quent sources of taxol for studies of the biosynthetic pathway andimproved 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 reg-
ulators, sugar supplements, etc, and cultures have been scaled up
to a bioreactor level for large-scale production. However, these
empiricalmethodshave not been able to meet the increasing world
demand for taxanes, which according to Global Industry Analysts
will reach 1040kg per year by 2012 [www.strategir.com: Bulk
Paclitaxel, a global strategic busines report]. A rational approach
might provide new insightinto howthe taxolbiosyntheticpathway
is regulated, with genetic and metabolic engineering techniques,
differential genetic expression, transcription factors and key genes
leading to highertaxol yields. One aspectto take into account is the
mechanism of taxol excretion from cells, which could be enhanced
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32 S. Malik et al. / Process Biochemistry 46 (2011) 23–34
Table 5
Chronological studies of cell suspension cultures of T. baccata.
Year Reference
1993 Detection and evaluation of taxol in callus cultures [59]
1994 Four new bioactive taxoids were isoloted from a cell culture and their structures were elucidated by spectroscopic analyses [75]
1995 Scale up studies with cell suspension cultures were carried out using 1 L working volume pneumatically mixed and stirred tank
bioreactors
[112]
1996 The response of cell suspension cultures to basic manipulation of culture conditions is described [79]
1996 Effects of methyl jasmonate and its analogs were studied on the accumulation of paclitaxel and related taxanes in cell suspension cultures [86]
1998 Studies on the kinetics of cell growth, production of paclitaxel and related taxanes after methyl jasmonate treatment were carried out [83]1999 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
[69]
2002 Suspension cultures were grown in a 20L airlift bioreactor (running for 28 days in batch mode) and their growth as well as capacity to
accumulate taxol and baccatin III was measured
[81]
2002 Taxol transport in T. baccata L. cell suspension cultures was studied using [14C]-taxol as a tracer. Taxol uptake was inhibited by
Na-orthovanadate and verapamil and Ca2+ was required for the active absorption of the molecule
[117]
2004 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
[58]
2005 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
[82]
2005 Stable-growing callus lines with different growth characteristics were selected after 1–2 years of culture. The ability to produce paclitaxel
and its analogues from these lines and their derived suspension cultures was demonstrated
[60]
2006 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
[78]
2006 To improve the production of taxol, cell suspension cultures were treated with a combination of inducing factors and their effects were
studied
[71]
2007 It was discovered that isopentenyl diphosphate is a source for taxol and baccatin III biosynthesis in cell cultures of T. baccata [23]
2008 The time course of expression of two genes, dbat and dbtnbt , involved in paclitaxel biosynthesis and intracellular taxane accumulation
were studied in callus cultures
[115]
2009 Effect of taxol feeding was studied on taxol and related taxane production in cell suspension cultures [22]
2009 Studies on gene expression profiling in T. baccata seedlings and cell cultures were carried out [116]
by employing a two-phase culture system, so far not assayed inT. 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 financially supported by the Spanish
MEC (BIO2008-01210) and the Generalitat de Catalunya
(2009SGR1217).
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