432
Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
for Taxol
biosynthesis. However, the use
of three different PAL inhibitors had lit-
tle or inhibitory effect on Taxol
yield,
suggesting that more speciFc steps of the
phenylpropanoid pathway need to be eval-
uated in order to achieve practical results.
Inhibitors
have
also
been
used
to
study the contribution of the mevalonate
and
1-deoxy-D-xylulose
(DXP)
pathway
to Taxol
and baccatin III accumula-
tion in
T. baccata
cell suspensions. Both
mevinolin (inhibitor of the mevalonate
branch, acting on HMG-CoA reductase)
and fosfomidomycin (inhibitor of the DXP
branch, acting on DXP reducto- isomerase)
caused decreases in Taxol
accumulation,
whereas baccatin III was only reduced by
fosfomidomycin. The results indicate that
both pathways may be used for Taxol
assembly with a predominance of the non-
mevalonate one.
6.5
Gas Composition
The gas phase of flasks and bioreactors
is often a determinant of secondary prod-
uct accumulation. The effects of gas phase
composition on Taxol
was examined in
shake-flask suspensions of
T. cuspidata
.
Low head space oxygen (10% v/v) pro-
moted early production of Taxol
,whereas
high CO
2
(10% v/v) had an inhibitory
effect. The same authors examined the
role of ethylene, a gaseous phytohormone
involved in various stress responses, in
Taxol
production. Gas composition also
affected sugar, calcium, and phosphate
uptake, which may have influenced tax-
ane metabolism. The best combination for
Taxol
production was 10% (v/v) oxygen,
0.5% carbon dioxide, and 5 ppm ethy-
lene. However, ethylene may have a dual
effect on Taxol
yield. In fungal elicitor-
induced cell suspension of
T. chinensis
and
T. yunnanensis
,e
t
h
y
l
e
n
eb
i
o
s
y
n
t
h
e
-
sis inhibitors (e.g. Co and Ni salts) and
the ethylene action inhibitor AgNO
3
en-
hanced Taxol
yield (up to threefold by
combining Co and Ag); the opposite was
observed with the ethylene-releasing com-
pound ethrel. Such positive and negative
effects were observed when adjuvants were
added before or simultaneously with the
elicitor, becoming progressively negligible
when added 24 h after elicitation. Control
of ethylene action and production during
fungal elicitation appears to favor Taxol
accumulation.
6.6
Protoplast Cultures
In general, protoplasts have not been
used for useful metabolite production but
as an important material for cell fusion
and genetic transformation. One of the
major advantages of using protoplasts
is that the products are released freely
into the broth with the double conse-
quences of increasing overall productivity
and facilitating down stream processing.
±urthermore, since many high molecu-
lar weight substances (including elicitors)
that are otherwise not accessible to the
cells (due to the presence of cell wall) can
directly access the cell membrane in proto-
plasts, the composition and concentrations
of the metabolites produced by protoplasts
m
a
yv
a
r
yf
romtho
s
ep
rodu
c
edb
yc
e
l
l
s
.
However, protoplasts are very fragile and
cannot be used for long-term production
process, and active protoplasts easily re-
generate their cell walls during cultivation.
In order to solve these problems, var-
ious gels were used as immobilization
carriers (providing stability to the pro-
toplasts) combined with an inhibitor of
cell wall synthesis. Thus, active protoplasts
were maintained for a long time without
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