434
Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
Free cells
Free
protoplasts
Protoplasts
immobilized
in agarose gel
3.0
2.0
1.0
0
07
07
07
Cultivation time (day)
Taxol
TM
concentration (mg
l
1
)
Extracellular
Intracellular
Fig. 4
Production of Taxol
by
T. cuspidata
cells and free and immobilized protoplasts.
production by free cells and protoplasts
in a static culture. Protoplasts produced a
sixfold increase in Taxol
in the medium
compared to that of the free cells. In or-
der to improve the stability of protoplasts,
3.0% agarose gel was used as the artiF-
cial cell wall material so that they could
be cultivated by shaking culture without
disruption. Agarose gel provided an artiF-
cial mechanical support to the protoplasts
thus enabling them to withstand hydrody-
namic stress. The protoplasts immobilized
in agarose gel produced Taxol
and re-
leased the compound into the medium as
did protoplasts in static culture, yielding
about six times more Taxol
than free
cells.
The system described here may be use-
ful for the production of Taxol
and
other taxanes using different
Taxus
cell
lines. Agarose was employed as an artiF-
cial cell wall and 2,6-dichlorobenzonitrile,
for example, was used as inhibitor of
cell wall synthesis. This system has mer-
its in that various useful functions can
be freely incorporated into protoplasts
by using artiFcial cell walls with engi-
neered functions. By using an appropriate
artiFcial cell wall and an inhibitor of
cell wall synthesis, it may be possible
to further promote Taxol
production.
±or
example, Indole
alkaloids
produc-
tion by
C. roseus
protoplasts was syn-
ergistically enhanced by using alginate
gel as both elicitor and immobilization
carrier, and CaCl
2
was used for both
inhibitor of cell wall synthesis and sig-
nal transducer. Protoplasts with artiF-
cial cell walls have important potential
applications.
The numerous approaches described
above for enhancing taxane production
in cell suspension cultures may be ex-
amined for Taxol
production by pro-
toplast cultures. Protoplast cultures pro-
vide a valuable tool for detailed study
of biosynthetic pathways leading to efF-
cient production of Taxol
.±ur
thermore
,
protoplast cultures represent a sustain-
able and relatively clean source of en-
zymes
and
genes
involved
in
Taxol
biosynthesis.
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