全 文 ：Received 15 Aug. 2003 Accepted 17 Feb. 2004
Supported by the National Natural Science Foundation of China (20176038).
* Author for correspondence. Tel (Fax): +86 (0)22 27403888; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (6): 730-737
Acting Point in Taxol Biosynthesis Pathway of Elicitor in Suspension
Cultures of Taxus chinensis var. mairei
MIAO Zhi-Qi, YUAN Ying-Jin*, REN De-Feng, WEI Zuo-Jun, WU Jin-Chuan
(Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology,
Tianjin University, Tianjin 300072, China)
Abstract: A new method was developed to determine the acting paths of elicitors based on the analysis
of intermediate response mode in suspension cultures. It was found that the acting path of an elicitor,
defined as the biosynthesis step whose rate was significantly enhanced upon the addition of the elicitor,
was located between two adjacent intermediates whose concentrations changed in opposite directions.
The validity of this method was verified in the biosynthesis of taxol from suspension cultures of Taxus
chinensis (Pilg.) Rehd. var. mairei (Lemee et Levl.) Cheng et L. K. Fu. The acting paths of methyl jasmonate,
silver nitrate and ammonium citrate were determined to be located at the step from baccatin Ⅲ to 10-
deacyl taxol. There are three possibilities for salicylic acid and arachidonic acid: (1) improving the biosyn-
thesis of 10-deactyl baccatin Ⅲ (10-DAB), (2) inhibiting the degradation of taxol and (3) inhibiting the
degradation of cephalomannine. But it is difficult to elucidate the exact acting paths of these two elicitors
based on the present method.
Key words: acting path; elicitor; intermediate response mode; suspension culture; taxol; Taxus chinensis
var. mairei
Metabolic control analysis, developed by Kacser and
Burns (1973) and Heinrich and Rapoport (1974), is com-
monly used to locate a bottleneck in a linear pathway. The
genetic machinery of cells is then altered through recombi-
nant DNA to improve the activity of the enzymes cata-
lyzing the bottleneck step. Besides the genetic method
(Croteau et al., 1995), elicitation is another effective way to
improve the activity of enzymes especially those induced
in the biosynthesis pathway of secondary metabolites.
Considering the biosynthesis of taxol (Christen et al.,
1991; Fett-Neto et al., 1994; Hezari et al., 1997), an anti-
cancer drug (Wani et al., 1971), it was reported (Miroslawa
et al., 1997) that the taxol concentration under the elicita-
tion of methyl jasmonate at 100 mmol/L was 38 times higher
than that in the control culture without elicitor addition.
Other elicitors such as silver nitrate, ammonium citrate and
arachidonic acid have also been shown to be effective in
improvement of the taxol production (Choi et al., 1996;
Srinivasan et al., 1996; Miao et al., 2000). Although it has
been recognized that elicitors enhance the production of
taxol by improving the activity of some enzymes, there is a
few better understanding of the enzyme activation by the
individual elicitors due to the complexity of the taxol bio-
synthesis pathway, which results in the difficulty of
determining the suitable elicitor and its amount to add into
the culture medium.
In a previous paper (Yuan et al., 2002), we proposed the
concept of the acting path of an elicitor and clarified that
two elicitors with different acting paths could give a syner-
getic effect in improving taxol production while those with
the same acting path could not. The biosynthesis pathway
of taxol was simplified into a linear one by neglecting the
branched pathway of cephalomannine and assuming that
the enzymatic reactions follow the classical Monod equa-
tion (Yuan et al., 2002). However, based on the above
assumptions, the concentration of 10-deactyl baccatin Ⅲ
(10-DAB) can not be well predicted when the enzymes cata-
lyzing the transformation from baccatin Ⅲ to taxol was ac-
tivated as a result of elicitation. In addition, it is inconve-
nient to measure the quasi-steady-state contents of inter-
mediates as the operation is complicated and time consum-
ing for the conventional batch or fed-batch culture systems.
Therefore, it is necessary to develop an easier method to
determine the acting paths of elicitors for production of
secondary metabolites in suspension cultures.
In this study, the acting path of an elicitor is analyzed
based on the intermediate response mode in pathways with
a linear or branched topology. The assumptions used in
MIAO Zhi-Qi et al.: Acting Point in Taxol Biosynthesis Pathway of Elicitor in Suspension Cultures of Taxus chinensis var. mairei 731
the previous paper (Yuan et al., 2002) were no longer needed
and the product inhibition was considered. The acting path
of the elicitor was determined by the change of intermedi-
ates in contents compared with their contents at the quasi-
steady-state. Therefore, the present method is more rea-
sonable and convenient in determining acting paths of
elicitors. The validity of the new method was verified by
application in suspension cultures of Taxus chinensis var.
mairei for taxol production.
1 Materials and Methods
1.1 Chemicals
Methyl jasmonic acid, arachidonic acid and taxol sample
were purchased from Sigma (St. Louis, MO). The 10-deacetyl
baccatin Ⅲ, baccatin Ⅲ and cephalomannine were from
Xi’an Tiancheng Drugs and Bioengineering Company. All
other chemicals used were of analytical grade and obtained
commercially.
1.2 Cell line and culture conditions
YH strain of Taxus chinensis (Pilg.) Rehd. var. mairei
(Lemee et Levl.) Cheng et L. K. Fu was provided by Insti-
tute of Botany, The Chinese Academy of Sciences, Beijing,
China. Cell line was transferred from solid B5 medium
supplemented with 25 g/L sucrose at pH 5.8 before auto-
claving to the liquid growth medium in 250 mL Erlenmeyer
flasks and cultured (25 ℃, in darkness, 100 r/min) for four
weeks to activate the cells. The growth medium was re-
placed by a fresh one every week to provide sufficient nu-
trients to cells. The cells were harvested and used in the
subsequent experiments.
1.3 Taxane extraction and measurement
The harvested sample was separated by centrifugation
and the cell pellet was collected. Two hundred milligram
cells were powdered for 15 min in 20 mL cyclohexane : metha-
nol (1:1, V/V) and ultrasonicated for 20 min. Then, the mix-
ture was extracted three times with 100 mL methylene chloride.
The methylene chloride phase was respectively col-
lected and evaporated at room temperature. The remaining
taxanes were resuspended in 1 mL methanol and filtered
through a 0.2-mm polymeric filter prior to HPLC analysis.
The above methanol solution was injected for taxol
analyses on a reverse-phase column (Kromasil C18, 5 mm,
250 mm×4.6 mm). The mobile phase consisted of metha-
nol : water (65:35, V/V) and the flow rate was 1 mL/min.
Taxane was quantitated at 227 nm (Yuan et al., 1998). The
authentic taxane, including 10-deacetyl baccatin Ⅲ,
baccatin Ⅲ, cephalomannine and taxol, was purchased from
Sigma and outer samples were used to confirm the product.
The retention time of the above four kinds of taxanes is
4.950 min, 6.142 min, 25.775 min and 28.917 min, respectively.
1.4 Design of the response mode experiment
Seed cells were cultured in 250 mL Erlenmeyer flasks at
25 ℃ in the dark with continuous shaking at 100 r/min for
12 d in the control experiment. Each flask contained 3 g
fresh cells and 40 mL growth medium filtered through a 0.2-
mm cellulose acetate membrane after autoclaving. Half of
the medium was replaced with a fresh one every day to
maintain the medium compositions almost unchanged in
the batch cultures and to simulate a continuous culture.
After a 6-day culture, the content of key intermediates
in taxol biosynthesis kept constant and reached a control
steady state (Fig.3). Then, part of the cells cultured for 10 d
was harvested and cultured in a medium containing appro-
priate quantity of elicitors, while the left cells were used as
control. Both control and elicitation cultures were carried
out for 12 d following the same procedures of seed cell
culture described as above.
Samples were collected every day to analyze the con-
tents of taxanes by HPLC. All data were the average of
triplicate samples and the errors were within ±10%.
2 Results
2.1 Analysis of intermediate response mode
When a metabolic pathway is at a steady state, the con-
tent of intermediates involved in the pathway was deter-
mined by the activity of enzyme catalyzing each biochemi-
cal reaction involved in the pathway. If the activity of one
enzyme is changed, the system will reach a new steady
state with different concentrations of the intermediates. By
analysis of the content change of intermediates as a result of
outer stimulus, the enzyme, whose activity is influenced by
the factor, can be identified. Therefore, the acting path of the
elicitor, which affects the enzyme activity, can be determined.
2.1.1 Analysis of intermediate response mode in a linear
pathway Considering a linear pathway consisting of n
reversible enzymatic reactions (Fig.1) containing n inter-
mediates Xi (i=1, 2, … n). The activity of the enzyme i,
defined as the maximum rate of the synthesis of Xi (i=1, 2,
… n), is denoted by Ri. The net rates for the n enzymatic
reactions can be expressed as:
ri=Ri fi,s (Xi-1) fi,p (Xi) i = 1, 2, ⋯ n (1)
Here fi,s (Xi-1) (i=1, 2, ⋯ n) and fi,p (Xi) (i=1, 2, ⋯ n)
represent the contributions to the reaction rate ri (i=1, 2, ⋯
n) from substrates and products, respectively.
The following constraints should be satisfied:
Ri > 0 i = 1, 2, ⋯ n (2)
1 > fi,p (Xi) > 0 i = 1, 2, ⋯ n (3)
1 > fi,s (Xi-1) > 0 i = 1, 2, ⋯ n (4)
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004732
> 0 i = 1, 2, ⋯ n (5)
> 0 i = 1, 2, ⋯ n (6)
When a steady state is established, the overall flux of
substances through the pathway r should be equal to cer-
tain fold of the net rate of each enzymatic reaction:
r = r1= r2= ri =⋯ = rn (7)
Here, gi is the number of carbon in intermediate Xi ; r is a
function of Ri (i =1, 2, ⋯ n) and meets the following differ-
ential equations:
≥0 i = 1, 2, ⋯ n (8)
≤0 i = 1, 2, ⋯ n (9)
According to Fig.2, equation 9 can be rewritten as
- = ( - )≤0 i = 1, 2, ⋯ n (10)
Therefore, the contents of taxane at a steady state, de-
noted by Xi* (i =1, 2, ⋯ n), should also be a function of Ri
(i=1, 2, ⋯ n):
Xi*=yi (R1, R2, ⋯ Rn) i = 1, 2, ⋯ n (11)
To examine the steady-state content change of interme-
diates with respect to the activity changes of enzymes, we
define the response coefficients ( ) of intermediates at
steady state by:
(12)
It is obvious, from equation 12, that a positive value of
(i=1, 2, ⋯ n; j=1, 2, ⋯ n) should lead to an increase of
the ith intermediate steady-state level if the activity of the
jth enzyme is enhanced by an elicitor and vice versa.
The positive or negative signs of the response coeffi-
cients of intermediates at a steady state can be interpreted
as follows: It is reasonable that the content of the primary
substrate X0 would not be influenced strongly by the
change of Ri (i=1, 2, ⋯ n), because only less part of X0
runs into the pathway of secondary metabolism than that
running into the primary metabolism. Another reason, sup-
porting the above hypothesis, is that the content of the
primary substrate is higher than its subsequent secondary
metabolites. The above assumption can be summarized into
the following equation:
= 0 i = 1, 2, ⋯ n (13)
(1) The sign of
The following differential equation can be obtained from
equation 1 when i is 1.
=f1, s(X0*) (f1, p(X1*)+R1 ) (14)
Thus, is obtained from the above equation and its
sign can be determined on the basis of inequations of 10, 2,
4 and 5.
=
= ≥0 (15)
(2) The sign of (i=1, 2, ⋯ n)
The following differential equation can be obtained from
equation 1 when i is 1.
= R1 f1, s (X0*) i = 2, 3, ⋯ n (16)
Fig.1. A linear pathway with n intermediates and n+1 enzy-
matic steps.
¶ fi,p (Xi)
¶Xi
¶ fi,s (Xi-1)
¶Xi-1
1
g n
¶ r
¶Ri
¶ 2r
¶Ri2
¶ r
¶Ri
r
Ri
1
g i
¶ ri
¶Ri
ri
Ri
Fig.2. Schematic diagram of the relationship between the over-
all flux of substances at steady state and the enzyme activity in a
linear pathway.
¶ X*
¶R
¶ Xi*
¶Rj
¶ X0*
¶Ri
¶ X1*
¶R1
¶ X1*
¶R1
¶ r1
¶R1
¶ X1*
¶R1
¶ f1, p (X1*)
¶X1*
¶ X1*
¶R1
¶X1*
¶Ri
1
f1, s (X0*)
¶ r1
¶R1
- f1, p (X1*)
¶ f1, p (X1*)
¶X1*
R1
¶ r1
¶R1
r1
R1
-
¶ f1, p (X1*)
¶X1*
R1 f1, s (X0*)
¶ r1
¶R1
¶ f1, p (X1*)
¶X1*
¶ X1*
¶Ri
1
g 1
1
g 2
1
gi
MIAO Zhi-Qi et al.: Acting Point in Taxol Biosynthesis Pathway of Elicitor in Suspension Cultures of Taxus chinensis var. mairei 733
So, (i=2, 3, ⋯ n) can be deduced from the above
equation and its sign can be judged on the basis of
inequations of 7, 8, 2, 4 and 5.
= ≤0 i = 2, 3, ⋯ n (17)
(3) The sign of (i=1, 2, ⋯ n)
= =
=t = gn t
≥0 (18)
(4) The sign of (i =2, 3, ⋯ n; j =1, 2,⋯ i)
The differential of ri (i =2, 3, ⋯ n-1) to Ri (j =1, 2,⋯ i)
can be expressed according to equations 1 and 7 as
=gi+1 =Ri+1 (
fi+1, p (Xi+1*)+fi+1, s (Xi*) ) (19)
So, the following equations should be met:
fi+1, p (Xi+1
*)+
fi+1, s (Xi*) )≥0 (20)
fi+1, p (Xi+1*)≥
- fi+1, s (Xi
*) (21)
Thus, the sign of (i =2, 3, ⋯ n-1; j = 1, 2,⋯ i) is
obtained
≥
i = 2, 3, ⋯ n-1; j =1, 2,⋯ i (22)
For convenience of subsequent usage, we define a pa-
rameter ai
ai= - ≥0 i =1, 2,⋯ n (23)
Therefore, the equation 22 can be written as follows:
≥ai+1 ≥0 (24)
≥ai+1 ≥ai+1ai+2 ≥⋯≥
P am ≥0 (25)
≥0 i=2, 3, ⋯ n-1; j=1, 2, ⋯ i (26)
(5) The sign of (i =2, 3,⋯ n-1; j = i+1, i+2, ⋯ n)
The differential of ri (i =2, 3,⋯ n-1) to Rj (j = i+1, i+2, ⋯
n) can be expressed according to equations 1 and 7 as
= gi = Ri ( fi,p (Xi*)+
fi, s (Xi-1
*) (27)
Therefore, the following inequation should be met:
fi,p (Xi*)+
fi, s (Xi-1
*) ≥0 (28)
≤- (29)
≤ (30)
≤ ≤ ≤ ⋯ ≤
≤0 (31)
≤ 0 i = 2, 3,⋯ n-1; j = 1, 2, ⋯ i-1 (32)
In summary, if an elicitor increases the activity of an
enzyme in a linear pathway, all steady-state levels of the
intermediates before the reaction catalyzed by the enzyme
should decrease and those after the reaction should
increase. All steady-state levels of the intermediates be-
fore the enzymatic reactions should increase and those
after the reactions should decrease when the activity of
¶ X1*
¶Ri
¶ X1*
¶Ri
¶ r1
¶Ri
R1 f1, s (X0*)
¶ f1, p (x1*)
¶x1*
¶ Xn*
¶Ri
¶ Xn*
¶Ri
¶ Xn*
¶rn
¶rn
¶Ri
¶ (Xn (t=0) + ∫ rndt)
¶rn
¶rn
¶Ri
t=0
t
¶rn
¶Ri
¶r
¶Ri
¶ Xi*
¶Rj
¶ ri+1
¶Rj
¶ r
¶Rj
¶ fi+1,s (Xi*)
¶Xi*
¶ Xi*
¶Rj
¶ Xi+1*
¶Rj
¶ fi+1, p (Xi+1*)
¶ Xi+1*
¶ fi+1, s (Xi*)
¶Xi*
¶ Xi*
¶Rj
¶Xi+1*
¶Rj
¶ fi+1, p (Xi+1*)
¶ Xi+1*
¶ fi+1, s (Xi*)
¶Xi*
¶ Xi*
¶Rj
¶ Xi+1*
¶Rj
¶ fi+1, p (Xi+1*)
¶ Xi+1*
¶ Xi*
¶Rj
¶ fi+1, p (Xi+1*)
¶ Xi+1*
- fi+1, s (Xi*)
¶ fi+1,s (Xi*)
¶ Xi*
fi+1, p (Xi+1*)
¶Xi+1*
¶Rj
¶ fi, p (Xi*)
¶ Xi*
fi, s (Xi-1*)
¶ fi, s (Xi-1*)
Xi-1*
fi, p (Xi*)
¶Xi*
¶Rj
¶Xi+1*
¶Rj
¶Xi*
¶Rj
¶Xi+1*
¶Rj
¶Xi+2*
¶Rj
¶Xn*
¶Rj m=i+1
n
¶ Xi*
¶Rj
¶Xi*
¶Rj
¶ ri
¶Rj
¶ r
¶Rj
¶fi, s (Xi-1*)
¶Xi-1*
¶ Xi-1*
¶Rj
¶fi, p (Xi*)
¶Xi*
¶ Xi*
¶Rj
¶fi, s (Xi-1*)
¶Xi-1*
¶ Xi-1*
¶Rj
¶fi, p (Xi*)
¶Xi*
¶ Xi*
¶Rj
¶ Xi*
¶Rj
¶fi, s (Xi-1*)
¶Xi-1* ¶ Xi-1
*
¶Rj
fi,p (Xi*)
fi, s (Xi-1
*)
¶fi, p (Xi*)
¶Xi*
¶ Xi*
¶Rj
1
a i
¶ Xi-1*
¶Rj
¶ Xi*
¶Rj
1
a i
¶ Xi-1*
¶Rj
1
a i
¶ Xi-2*
¶Rj
1
ai-1
¶ Xi*
¶Rj
1
P am
m=i
m=1
¶ Xi*
¶Rj
¶ Xi*
¶Rj
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004734
the enzyme was inhibited by an elicitor.
i =1, 2⋯n; j = 1, 2⋯ n (33)
Therefore, the acting path of an elicitor in the linear
pathway should locate between the two adjacent interme-
diates whose levels at the steady state changed in oppo-
site directions.
2.1.2 Analysis of intermediate response mode in a
branched pathway Figure 3 is the schematic diagram of a
branched pathway. Based on equation 34,
≥0 j=1, 2, ⋯5 (34)
the following equations can be easily obtained:
≥0 j =1, 2, ⋯5 (35)
≤0 j =2, 3, 4, 5 (36)
≤0 (37)
≤0 (38)
Because , thus
R2 f2,s (X1*) f2,p (X2*) = R4 f4,s (X2*) (39)
f2,s (X1
*) = (40)
The following equation can be obtained by differential
the both side of equation 40 to X1*
= (41)
= ≥0 (42)
Therefore
= ≤0 i = 2, 4; j = 3, 5 (43)
Because , the following equation is obtained
following the same method:
= ≥0 (44)
= ≤0 i = 3, 5; j =2, 4 (45)
For simplicity, the subbranch containing the acting path
of an elicitor is called the key subbranch. Therefore, all
steady-state levels of the intermediates in non-key sub-
branch should decrease when an elicitor activates an en-
zyme in the key subbranch. Meanwhile, if an elicitor inhib-
its an enzyme in the key subbranch, the levels of all inter-
mediates in the non-key subbranch should increase. The
key subbranch is the one in which the steady-state levels
of the intermediates change in a different direction under
elicitation. And the acting path of the elicitor is located
between two adjacent intermediates whose steady-state
contents change in a different direction.
2.1.3 Generalization of the intermediate response mode
The major limitation of the response mode analysis of inter-
mediates is the basis on a steady state of the pathways. So,
experiments to measure the response modes of intermedi-
ates at steady state must be carried out in a continuous
culture mode to keep the medium compositions unchanged.
The drawback of the steady-state assumption applied
in the response mode analysis of intermediates can be over-
come if the following assumption holds true:
Assuming that the derivatives of the intermediate lev-
els versus time are continuous during the transfor-
mation from the control steady state to the elicitation steady
state, and the derivatives equal to zero only at the above
two steady states. In other way, Xi (i =1, 2, ⋯ n) are
¶ Xi*
¶Rj
¶ Xi*
¶Rj
≥0 j≤i
≤0 j >i
{
¶ Xj*
¶Rj
¶ Xj*
¶R1
¶ X1*
¶Rj
¶ X2*
¶R4
¶ X3*
¶R5
Fig.3. Branched pathway with two sub-branches.
1
g2
1
g4r2 = r4
1
g4
1
g2
g4
g2
R2
R4
f4,s (X2*)
f2,p (X2*)
g4
g2
R2
R4
¶ f2,s (X1*)
¶X1*
f4,s (X2*)
f2,p (X2*)
¶X1*
¶ X2*
¶X1*
¶ X2*
¶ X1*
g4
g2
¶ f2, s (X1*)
¶ X1*
R2
R4
f4, s (X2*)
f2, p (X2*)
¶
¶X2*
¶ Xi*
¶Rj
¶ Xi*
¶X1
¶ X1*
¶Rj
1
g3
r3= r5
1
g5
¶ X3*
¶ X1*
g5
g3
¶ f3, s (X1*)
¶ X1*
R3
R5
f5, s (X3*)
f3, p (X3*)
¶
¶X3*
¶ Xi*
¶Rj
¶ Xi*
¶X1
¶ X1*
¶Rj
¶Xi
¶t
¶
MIAO Zhi-Qi et al.: Acting Point in Taxol Biosynthesis Pathway of Elicitor in Suspension Cultures of Taxus chinensis var. mairei 735
monotonous functions of time. Thus,
DXi*
≥0 (46)
In which DXi* is the difference between content of Xi at
the control steady state and the elicitation steady state.
It is seen from inequation 46 that one can use the changes
of intermediate levels in a transient state to determine the
response mode and locate the acting path of an elicitor in
addition to those in the steady state as described in the
previous section.
2.2 Response mode of taxanes under elicitation
2.2.1 Determination of the time when taxol biosynthesis
reaches a steady state Figure 4 shows the time course of
taxane contents in the control experiment. The concentra-
tions of taxanes markedly changed within the first 6 d and
altered less afterwards, indicating that a steady state was
established at the 6th day. So, the control cells cultured for
10 d were thought as arriving in the steady state, harvested
and used as the seed cells in the response mode experiment.
2.2.2 Response mode of taxanes under elicitation Figure
5 shows the change of taxane contents with time after stimu-
lation by an elicitor. The relative contents of taxanes were
defined as the ratio of taxane contents in elicitation to those
in control experiments. It is seen from Fig.5 that the as-
sumption described by equation 46 is applicable in the elici-
tation experiment. Thus, the changes in taxane contents,
which can be measured more conveniently and rapidly, can
be used to analyze and determine the acting path of an
elicitor in comparison with using the changes of taxane
contents at the steady-state.
According to the response mode of taxanes (Fig.5), the
¶ Xi
¶t
Fig.4. Time course of taxane concentrations in the control
culture. Culture conditions: B5 medium, 25 ℃, 100 r/min, 12 d,
half of the medium was replaced with a fresh one everyday. ◇,
10-deactyl baccatin Ⅲ (10-DAB); □, baccatin Ⅲ; △,
cephalomannine; 〇, taxol.
Fig.5. Time course of taxane concentration in elicitation culture.
Culture conditions: B5 medium, 25 ℃, 100 r/min, 12 d, half of the
medium was replaced with a fresh one everyday. The relative
content of taxanes was defined as the ratio of taxane contents
elicitation to those in control groups. A. Salicylic acid (0.1 mg/L).
B. Arachidonic acid (0.1 mg/L). C. Methyl jasmonate (1.0 mg/L ).
D. Ammonium citrate (1.0 mmol/L). E. Silver nitrate (10.0
mmol/L). ◇, 10-deactyl baccatin Ⅲ (10-DAB); □, baccatin Ⅲ;
△, cephalomannine; 〇, taxol.
Acta Botanica Sinica 植物学报 Vol.46 No.6 2004736
elicitors are divided into two groups. The first one includes
silver nitrate, methyl jasmonate and ammonium citrate,
whose introduction increased the content of taxol and
cephalomannine while decreased the contents of 10-DAB
and baccatin Ⅲ. The second one consists of salicylic acid
and arachidonic acid whose addition increased all contents
of 10-DAB, baccatin Ⅲ, cephalomannine and taxol.
2.3 Acting path of elicitors in taxol biosynthesis pathway
The widely accepted pathway (Fleming et al., 1993) for
taxol biosynthesis is a branched one (Fig.6) and can be
divided into seven sections by five intermediates. The re-
sponse modes of taxanes stimulated by elicitors with act-
ing paths in any of the sections are listed in Table 1.
On the basis of relationship listed in Table 1 and the
response mode of taxanes obtained in Taxus culture in-
duced by certain elicitor, it is seen that the acting paths of
methyl jasmonate, silver nitrate, and ammonium citrate lo-
cated between baccatin Ⅲ and N-deacyl taxol, and their
addition improved the transformation from baccatin Ⅲ to
N-deacyltaxol. However, there are three possible acting
paths for salicylic acid and arachidonic acid. The first one
is the improvement of the biosynthesis of 10-DAB, the sec-
ond is the inhibition of the taxol degradation and the third
is the inhibition of the degradation of cephalomannine. In
addition, it was found that silver nitrate, methyl jasmonate
and ammonium citrate with an acting path between baccatin
Ⅲ and N-deacyltaxol were most effective among the five
elicitors in terms of improving taxol production. This might
indicate that in this study, the step from baccatin Ⅲ to N-
deacyltaxol was the rate-limiting step of taxol biosynthesis
in B5 medium.
3 Discussion
Due to the complexity of biosynthesis processes, it is
difficult or even impossible to determine the acting path of
an elicitor by examining the activity changes of all enzymes.
Therefore, the analysis of the intermediate response mode
is an alternative method to locate the acting path of an
elicitor based on the response modes of intermediates in-
duced by the elicitor. It was found that in the acting path of
an elicitor, the levels of two adjacent intermediates changed
in opposite directions. The addition of methyl jasmonate,
silver nitrate or ammonium citrate enhanced the transfor-
mation from baccatin Ⅲ to 10-deacyltaxol. However, it is
still difficult to determine the exact acting paths of salicylic
acid and arachidonic acid among the three possibilities,
i.e., improving the biosynthesis of 10-DAB, inhibiting the
degradation of taxol or cephalomannine based on the
Table 1 Putative possible acting paths of elicitors and the corresponding changes in taxanes contents based on analysis of the
intermediate response mode
Acting path of elicitor Regulating direction
Change of taxane content induced by elicitor
10-DAB Baccatin Ⅲ Cephalomannine Taxol
Biosynthesis of 10-DAB Upward ↑ ↑ ↑ ↑
Downward ↓ ↓ ↓ ↓
From 10-DAB to baccatin Ⅲ Upward ↓ ↑ ↑ ↑
Downward ↑ ↓ ↓ ↓
From baccatin Ⅲ to N-deacyl taxol Upward ↓ ↓ ↑ ↑
Downward ↑ ↑ ↓ ↓
From N-deacyl taxol to taxol Upward ↓ ↓ ↓ ↑
Downward ↑ ↑ ↑ ↓
From N-deacyl taxol to cephalomannine Upward ↓ ↓ ↑ ↓
Downward ↑ ↑ ↓ ↑
Degradation of taxol Upward ↓ ↓ ↓ ↓
Downward ↑ ↑ ↑ ↑
Degradation of cephalomannine Upward ↓ ↓ ↓ ↓
Downward ↑ ↑ ↑ ↑
↑, increase; ↓, decrease; 10-DAB, 10-deactyl baccatin Ⅲ.
Fig.6. Schematic representation of taxol biosynthesis pathway. 10-DAB, 10-deactyl baccatin Ⅲ.
MIAO Zhi-Qi et al.: Acting Point in Taxol Biosynthesis Pathway of Elicitor in Suspension Cultures of Taxus chinensis var. mairei 737
present method.
The result can be used to explain why taxol production
under synergetic elicitation of salicylic acid and ammonium
citrate is 44% higher than the sum of taxol induced by the
above elicitor respectively (Yuan et al., 2002). Because the
above two kinds of elicitors have different acting points in
taxol biosynthesis pathway, their addition can improve the
biosynthesis of 10-DAB and the transformation from 10-
DAB to taxol, a clear synergetic effect (Yuan et al., 2002) on
taxol biosynthesis occurred. Therefore, the acting path re-
sult of elicitor can also be used to select elicitor to improve
the production of secondary metabolites, and the strategy
has been proved effectively (Yuan et al., 2002).
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