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

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Process Biochemistry 46 (2011) 23–34

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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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24 S. Malik et al. / Process Biochemistry 46 (2011) 23–34

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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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


5/12


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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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]


(0.1mg/L)+ 0.5% sucrose+ 0.5%

fructose


(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]


(0.1mg/L)+ 0.5% sucrose+ 0.5%

fructose


(0.1mg/L)+ 3% sucrose

Paclitaxel 1.58mg/L (1.68mg/g

DW), baccatin III 0.32 mg/L

(0.35mg/g DW)

[80]


(0.1mg/L)+ 0.5% sucrose+ 0.5%

fructose


(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


7/12


(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]


(100M)

Paclitaxel

Baccatin III

4.25mgdm−3

2.4mgdm−3[119]


(100M)

Paclitaxel

Baccatin III

7.09mg/L on day

22

3.49mg/L on day

22

[80]


(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%)


Methyl jasmonate


(2.5%)

Baccatin III 4.62 mg/L

Elicitation+ immobilization Stirred bioreactor (5L) Methyl jasmonate


(2%)

Paclitaxel

Baccatin III

43.43mg/L

(2.71mg/L/d)

5.06mg/L

[82]

Elicitation+ immobilization Airlift bioreactor (4L) Methyl jasmonate


(2%)


Elicitation+ immobilization Wave bioreactor (2L) Methyl jasmonate


(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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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

http://www.strategir.com/http://www.strategir.com/


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