Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
429
same cell line suspension. The occurrence
of product excretion into the medium is
useful for industrial application of plant
cell culture technology, since it facilitates
downstream processing (especially when
using immobilized cells).
From the mid-1990s on, a large research
effort from various groups brought Taxol
and taxane productivity levels in cell cul-
tures to progressively higher values, in
some cases reaching industrially interest-
ing amounts. In the next section, different
proven strategies to promote taxane yield
in cell cultures are described.
6
Techniques to Improve Taxol
Yield in Cell
Culture
6.1
Precursor and Sugar Feeding
The problem of overall low Taxol
yields,
particularly at higher growth rates, can be
partly overcome by feeding Taxol
precur-
sors to the cell cultures. The appropriate
combination of type, amount, and feeding
time of precursors able to enter the cells
can signi±cantly increase the yield of sec-
ondary metabolites in plant cell cultures,
provided the biosynthesis of the metabolite
of interest is at least partly limited by poor
activity of enzymes involved in precursor
formation or appropriate supply of precur-
sor to the biosynthetic reaction. In two-
month-old callus cultures of
T. cuspidata
,
phenylalanine supplementation at 0.1 mM
in B5bPVP can double the Taxol
yield in
relation to regular B5bPVP without affect-
ing growth signi±cantly; similar results
can be obtained with cell suspensions af-
ter a 25-day growth period in medium
supplemented with the same amount of
this amino acid. Phenylalanine acts as a
precursorforthesidechainofTaxol
and
the benzoyl moiety at C-2.
Other
potential
precursors
for
the
Taxol
side chain were investigated in
feeding experiments to both callus and
cell suspension cultures of
T. cuspidata
.
Compounds that signi±cantly enhanced
Taxol
yields in cultures were phenylala-
nine, benzoic acid, hippuric acid, serine,
and glycine. Impacts on Taxol
yields were
concentration dependent. Taxol
yields
were increased by two to four times when
thepu
ta
t
ivep
recu
rso
rswe
rep
rov
ideda
t
concentrations of 0.2 mM for callus cul-
tures and 0.05 mM for cell suspension
cultures; all cultures were fed at the onset
of the stationary phase of growth. The best
results were obtained with phenylalanine
or benzoic acid feeding.
In a related approach, replacement of
2,4-D by Indole 3 acetic acid–glycine or
Indole 3 acetic acid–phenylalanine conju-
gates yielded a two and threefold increase
in Taxol
production by cell cultures of
T. wallichiana
(Himalayan yew), respec-
tively. Results suggest that precursor feed-
ing is a promising approach to increase
Taxol
yields in yew cell cultures without
detrimental effects on biomass generation.
Kinetic studies of taxane production in
cell cultures showed that carbohydrate
availability is closely related to biomass
increase in
Taxus
cell cultures. Feeding
of sucrose to
Taxus chinensis
cultures ini-
tially kept at 20 gL
1
and fed-batch system
increased growth and taxane production;
intermittent feeding of various concen-
trations of sucrose or maltose improved
Taxol
production in the same species.
A
nim
p
o
r
t
a
n
ta
s
p
e
c
tt
oc
o
n
s
i
d
e
ri
s
the
osmotic
effect
of
sugars
on
tax-
ane production. It has been shown that
high concentration of sucrose (60 gL
1
),
combinations of sucrose and mannitol,
as
well
as
nonsugar
osmotic
agents
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