全 文 ：植　　　物　　　研　　　究
BULLETIN OF BOTANICAL RESEARCH
第 19 卷　第 3 期 1999 年　7 月
Vol.19　No.3 July , 　1999
前体物对红豆杉培养细胞中紫杉醇生物合成的影响
李家儒1　曹孟德2　刘曼西2　陈辉蓉1　吴振斌1　王君健2
(1.中国科学院水生生物研究所 武汉 430072)　(2.华中理工大学生物工程系 武汉 430074)
摘　要　本文报道了添加 7种紫杉醇前体物/调节物后 ,红豆杉(T .chinensis(Pil-
ger)Rehd)TC158细胞系的反应 。在红豆杉细胞悬浮培养 25天时 ,分别加入不同
浓度乙酸钠 、苯甲酸钠 、L-苯丙氨酸 、甘氨酸 、丝氨酸 、α-蒎烯 、松节油 。试验结果
表明 ,各前体物对红豆杉细胞生长无明显影响 ,均不同程度地促进了紫杉醇的合成。
关键词　前体物;紫杉醇;细胞培养;红豆杉
EFFECTS OF PRECURSORS ON TAXOL BIOSYNTHESIS
IN CELL CULTURES OF TAXUS CHINENSIS
Li Jia-ru1　Cao Meng-de2　Liu M an-xi2　Chen Hui-rong1
Wu Zhen-bin1　Wang Jun-jian2
(1.Insititute of Hydrobiology , The Chinese Academy of Sciences , Wuhan 430072)
(2.Depar tment of Bioengineering , Huazhong University of Science and Technology , Wuhan 430074)
Abstract　The response of T .chinensis TC158 cell line to the addition of precurso rs/
regulators of taxol is described in this paper.On the 25th day af ter inoculat ion , cell
cultures w ere supplemented w ith dif ferent concentrations of one of the follow ing com-
pounds:sodium acetate , sodium benzoate , L-pheny lalanine , glycine , L-serine , α-
pinene and turpentine oil.The results demonst rated that taxol biosynthesis was im-
proved in different degrees , while the cell g row th w as not significantly affected by var-
ious precursors / regulators.
Key words　Precurso r;Taxol;Cell culture;Taxus chinensis
1　Introduction
Taxol is a complex diterpenoid f rom the genus Taxus that w as approved for t reatment a-
gainst ov arian and breast cancers and show s promise against some other cancers.The relative
收稿日期:1999-1-10
scarcity of the few natural resources and the expansion of the demand fo r taxol have stimulated
w ork forw ards developing cheap and reliable alternative sources of the drug .Taxol production
by cell culture is one of the potential approaches available to relieve of the crisis in supply of tax-
ol.
The key to taxol production by cell culture is the rapid grow th of the cell cultures and
higher y ield of taxol.The precusor is an impo rtant factor affecting the amount of plant sec-
ondary metablites.Taxol yield could be improved by adding precurso rs to the cell cultures of
Taxus.Fet to-Neto et al.reported on improved taxol yields significantly in callus and cell sus-
pension cultures of T . cuspidata follow ing feeding of precursors for the N - ben-
zoylpheny lisoserine side chain of taxol at C-13〔1〕.Gan FY et al.also reported that taxol yield
w as improved by adding phenylalanine , leucine and baccata Ⅲ to the cell cultures of T .yun-
nensis〔3〕.However , the effects of precursors on taxol biosynthesis in cell cultures of T .chi-
nensis(Pilger)Rehd have seldom been reported thus far.
Our objective has been to g ain a basic understanding of how the addition of precursor af fect
the taxol biosynthesis in cell cultures of T .chinensis.The response of T .chinensis TC158
cell line to the addi tion of precursors/ regulators of taxol is described in this paper.
2　Materials and methods
2.1　Plant material
T .chinensis TC158 cell line , an o rdinary single cell line , was set up and maintained by
the first author.
2.2　Cell suspension culture
Cell suspension cultures of T .chinensis (TC158)were incubated at 25℃±1℃ under
darkness on a rotary shaker at 110 r/min on MS medium supplemented w ith 0.5mg /L NAA ,
0.5mg/L BA , 2g/ L CH and 25g/L sucrose w ith five -week transfers.100ml of the medium
was aliquoted in each 250ml Erlenmeyer flask.The inoculum was appox.10g for each f lask.
2.3　The precursor preparation and addition
The water solution of sodium acetate , sodium benzoate and amino acids , ethanolic solu-
tions ofα-pinene , turpent ine oil w as sterilized by f iltering through 0.22μm sterile f ilters.On
the 25th day af ter inoculation , cell cultures w ere supplemented w ith 0 , 0.025 , 0.050 , 0.1 ,
0.2mmol/ L of one of the following compounds:sodium acetate , sodium benzoate , L -pheny-
lalanine , glycine , L-serine , andα-pinene , the addition of 1% turpentine oil(v/v)was at
levels of 0 , 0.1 , 0.25 , 0.5 , 1ml per flask , 5 f lasks per t reatment.
2.4　Analy tical Techniques
Cell cultures w ere harvested fo r g row th and taxol determintion on the 40th day after inoc-
ulation.
Cell g row th　Cell cultures w ere suction filtered , and then dried in an oven at 40℃ until
constant w eight for the cell g row th determinat ion , which w as changed into the co rresponding
value on the basis 10g inoculum.
3573 期　　　　　　　李家儒等:前体物对红豆杉培养细胞中紫杉醇生物合成的影响
Taxol ext raction and analy sis 　Powdered dry cell cultures w eighed were ex tracted in
methanol fo r 96 h and suction filtered through a 0.22μm filter.The f ilt rate w as analyzed by re-
versed phase high perfo rmace liquid chromatag raphy on the Novapak C-18 column(3.9mm×
150mm)at 25℃ previously described〔6〕.A mobil phase consisting of methanol :water (75:
25)at a flow rate of 0.5μl/min w as used.Taxol w as detected by monitoring absorbance at
227nm.Duplicate injections(3μl each)were made from every sample (ext ract)and the mean
of 2 duplication w as taken as the taxol yield of the sample.Authentic taxol w as obtained from
National Cancer Insti tute(USA).
3　Results
The results demonst rated that taxol biosynthesis w as improved in dif ferent deg rees(Fig.1
～ 7), while the cell g row th w as not significantly affected by various precursors / regulators.
F ig.1　Effects of gly cine on taxo l biosynthesis Fig.2　Effects of L-serine on taxol biosynthesis
Fig.3　Effects of L-pheny lalanine on taxol biosyn-
thesis
F ig.4 Effects of sodium benzoate on taxol biosynthesis
Fig.5 Effects ofα- pinene on taxol biosynthesis Fig.6 Effects of turpentine oil on taxol biosynthesis
Fig .1 ～ 2 show s that taxol yield tended to be higher in treatments wi th higher c oncentra-
358 植　　物　　研　　究　　　　　　　　　　　　　　　19 卷
Fig.7 Effects of sodium acetate on tax ol bio synthesis
tions of gly cine and L-serine.9.74μg/g (dry
w eight basis), the highest increase in the taxol
yield of cell suspension cultures , was observed
in the presence of L -phenylalanine at 0.05
mmol /L (Fig .3);11.55μg/g , sodium ben-
zoate 0.10mmol/L (Fig .4);24.39μg/g , α-
pinene 0.05mmol/L(Fig .5);47.36μg/g , tur-
pentine oil 0.1×10-4(v/v )(Fig.6);and 25.
06μg/g , sodium acetate 0.05mmol/L (Fig.7)
.Taxol in the control w as not detected.
4　Discussion
However , since the secondary products are
biosynthesized in multi-step enzymatic reaction in multiple secondary pathw ay s in plant cell
cultures , it is not easy to control the production even by modern sophisticated molecular biologi-
cal techniques.The taxol biosynthetic pathw ay includes three majo r steps:the formation of the
taxane ring skeleton , the synthesis of the side chain at C-13 , and finally the esterification of
the taxane ring sy stem with the side chain
〔4〕.Addi tion of known and presumed precurso rs /
regulators could relieve the key enzymatic block e.g .` bo ttleneck , or hinder the separation
and effective sto rage of endogenous intermidate , so as to enhance the objective product biosyn-
thesis.The experiment results demonst rated that taxol biosynthesis w as enhanced by known o r
presumed precursors to taxol adding to the cell suspension cultures.
Benzoic acid is the precursor to the side chain at C-13(2), and perhaps related to the ben-
zoylation of the diterpene tax ane ring skeleton at C-2.So the taxol biosynthesis w as promoted
by sodium benzoate feeding to the cell cultures of T .chinensis.
Fleming et al.indicated that the side chain of taxol is formed via β - phenylalanine〔2〕.
Leete et al.suggested a pathw ay fo r the biosynthesis of the side chain at C-13 from pheny lala-
nine via cinnamic acid pathw ay
〔5〕.The increase in taxol y ield in callus and cell cultures of T .
cuspidata by phenylalanine supplemented conf irmed Leete s view piont , as did the currently
experimental results.Perhaps the side chain at C -13 fo rmed via the aforesaid and other path-
ways , this possibili ty needs further research .
Serine and glycine are plant primary metabolites and are interchangable.The higher taxol
y ield in cell suspension cultures in the presence of glycine and serine is probably related to their
catabolism.Perhaps glycine is related to the biosynthesis and decoration of the diterpene taxane
ring skeleton by fi rst changing as serine , then pyruvic acid , and fo rmating acetyl CoA〔7〕 , en-
tering mevalonic acid pathw ay .Glycine and serine could also enter the shikimic acid pathway ,
resulting in phenylalanine and /or benzoic acid synthesis〔1〕.
Perhaps the promo tion of sodium acetate on taxol synthesis in cell cultures is related to its
entering into the mevalonic acid pathw ay , and the decoration and formation of the taxane ring
3593 期　　　　　　　李家儒等:前体物对红豆杉培养细胞中紫杉醇生物合成的影响
skeleton.
The main composit ions of turpentine oil areα-pinene (appro x.70%)and β -pinene(
approx .30%).Wender et al.reported that taxol analogues was synthesized wi th pinene.Pro-
motion effect of pinene on taxol biosy thesis maybe concern wi th the formation of the diterpene
taxane ring skeleton.Pinene is an end product in mono terpene biosynthesis.If pinene were to
feedback inhibit f low of geranyl py rophosphate , then more substrate might f low to geranylger-
anyl pyrophosphate and diterpenoid synthesis , and therefore taxol yield increased by pinene and
turpentine oil adding in cell cultures of T .chinensis.
Because of the complexi ty of taxol biosythesis , i t is very diff icult to investigate the ef fects
of precursors on taxol biosynthesis.The currently experimental results are just initial , the de-
velopment of the further thorough research w ould provide theoretical evidences for taxol pro-
duction by cell culture , and have an important meaning.
Acknowledgements
The authors thank Dr.Marcus J Darw ent , a visiting scientist , for his revision of this pa-
per.
Reference
1.Fet t-Neto AG , Melanson S J , Nicholson SA , Pennington JJ , DiCosmo F.Im proved taxol yield by aromatic carboxylic acid
feeding to cell cultures of Taxus cuspidata.Biotechnol.Bioeng., 1994 , 44(8):967～ 971
2.Fleming PE , Mocek U , Floss HG.Biosynthesis of taxoids.Mode of format ion of the taxol side chain.J.Am.C hem.Soc.
1993 , 115:805～ 807
3.Gan FY , Peng LP , Zheng GZ.Metabolic regulation on taxol of cell cultu re f rom Taxus yunnanesis.Acata Botanica yunnan-
ica , 1996 , 18(4):451～ 453(in C hinese)
4.Jazi ri M , Zhiri A , Guo YW et al.Taxus sp.cell , t issue and organ cultures as alternat ive sources for taxoids product ion:a
literature survey.Plant Cell , Tissue and Organ Cultu re , 1996 , 46:59～ 75
5.Leete E , Bodem GB.The biosynthesis of 3-dimethylamino-3-phenylpropanoic acid in yew.Tet rahedron Let t., 1966 ,
33:3925～ 3927
6.Li JR , Liu MX , Cao MD , et al.Effects of Penici llum cit rinum elicitor on the biosynthesi s of taxol in suspension cell cul-
tu res of Taxus chinensis , Bulletin of Botanical Research , 1998 , 18(1):78～ 82(in Chinese)
7.Sheng T , Wang JY.Biochemist ry (Vol.Ⅱ).Beijing:Higher educat ion press , 1991 , 424(in Chinese)
360 植　　物　　研　　究　　　　　　　　　　　　　　　19 卷