Enhancement of Taxane Production in Hairy Root

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Journal of Plant Physiology 166 (2009) 19501954

SHORT COMMUNICATION

Enhancement of taxane production in hairy root

culture of Taxus x media var. Hicksii

Katarzyna Syklowska-Baranek, Agnieszka Pietrosiuk, Anna Kokoszka,Miroslawa Furmanowa

Department of Biology and Pharmaceutical Botany, Faculty of Pharmacy, Medical University of Warsaw, ul. Banacha 1,

02-697 Warsaw, Poland

Received 23 March 2009; received in revised form 4 May 2009; accepted 5 May 2009

KEYWORDS

10-deacetylbaccatinIII;Hairy roots;Paclitaxel;Taxus

SummaryThis study assessed the effect of two precursors (L-phenylalanine and p-aminobenzoic acid) used alone or in combination with methyl jasmonate, on the growthand accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy rootcultures of Taxus x media var. Hicksii. The greatest increase in dry biomass wasobserved after 4 weeks of culturing hairy roots in medium supplemented with 1 mM ofL-phenylalanine (6.2 g L1). Addition of 1 mM of L-phenylalanine to the medium alsoresulted in the greatest 10-deacetylbaccatin III accumulation (422.7 mg L1), whichwas not detected in the untreated control culture. Supplementation with 100 mM ofL-phenylalanine together with 100 mM of methyl jasmonate resulted in theenhancement of paclitaxel production from 40.3 mg L1 (control untreated culture)to 568.2 mg L1, the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yieldof paclitaxel (221.8 mg L1) was observed when the medium was supplemented with100 mM of the precursor in combination with methyl jasmonate.Baccatin III was not detected under the conditions used in this experiment and theinvestigated taxanes were not excreted into the medium.&2009 Elsevier GmbH. All rights reserved.

Introduction

Paclitaxel is the active substance of Taxols

(Bristol-Myers Squibb), a very valuable plant-derived drug that displays activity against differentcancers, including carcinomas of the ovary, breast,head and neck, bladder and cervix, melanomas,lung cancers and AIDS-related Kaposis sarcoma. In

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Corresponding author. Tel.: +48 22 5720933;fax: +4822 5720984.

E-mail address:[email protected](K. Syklowska-Baranek).
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addition, many clinical trials are underway to testthe efficacy of paclitaxel, used as monotherapy andin combination with other chemotherapeuticagents, against various other neoplasms (Nims etal., 2006). The base taxane structure is utilized asthe starting skeleton for molecules with improvedbiological properties (Kingston and Newman, 2007).

Plant cell cultures provide a very promisingapproach to the production of paclitaxel andrelated taxanes, and do not require forest har-vestation. Although production has been scaled-up,a serious drawback of cell suspension cultures istheir instability and the unpredictability of second-ary metabolite formation (Frense, 2007). Trans-formed or hairy roots, offer opportunities forproduction of important phytochemicals as theyare characterized by genetic and biochemicalstability and capacity for organogenesis-associatedsynthesis of metabolites (Georgiev et al., 2007).

The precursor feeding or elicitation strategy hasbeen also found to effectively stimulate taxaneaccumulation in both callus and suspension culturesofTaxusspecies (Zhong, 2002). However, yew hairyroot cultures have not been sufficiently explored asa potential source of taxanes.

In this paper we report the enhancement ofpaclitaxel and 10-deacetylbaccatin III (10-DAB III)production in transgenic root cultures of Taxus xmedia var. Hicksii Rehd. achieved through precur-sors and elicitor feeding.

Materials and methods

Transformed root cultures

Hairy root culture of Taxus x media var. Hicksiiwas derived from a single root developed at thewounded site of the leaf as described previously(Furmanowa and Syklowska-Baranek, 2000). Thehairy roots were cultivated in 250mL Erlenmeyerflasks containing 50mL of hormone-free DCR-Mmedium. This was the medium used by Gupta and

Durzan (1985)with the content of MgSO4increasedto 400mgL1 (Flores et al., 1993), and with theaddition of 500 mg L1 of L-glutamine. The culturewas maintained at 251C in the dark on a GioGyrotorys Shaker (New Brunswick Scientific Co.) at122 rpm.

Precursors and elicitor feeding

We investigated the effects of L-phenylalanine(PHE) and p-amino benzoic acid (PAB), either alone

or in combination with methyl jasmonate, on hairyroot growth and taxane production.

The precursors were added to the media prior toautoclaving, and methyl jasmonate was addedafter autoclaving. The 0.6770.12 g of fresh weightof 28-day-old hairy roots were placed into 250 mLErlenmeyer flasks with 50mL DCR-M medium

supplemented with precursors at concentrationsof 1 mM, 10 mM or 100 mM. The hairy roots from twoflasks were harvested after 48h, 1, 2, 3 and 4weeks, and their dry weight (D.W.) was determinedafter lyophilization.

In the second part of the experiment, the 4-week-old hairy roots were placed into mediasupplemented with precursors (1, 10 or 100mM)and 100 mM methyl jasmonate and cultured for 2weeks. Hairy roots were also cultured for 4 weeksin DCR-M medium without either precursors orelicitor to serve as controls.

HPLC analysis

The contents of paclitaxel, baccatin III and 10-deacetylbaccatin III were determined in powdereddry tissue of transformed roots and in the mediumsamples. Sample cleaning was performed using themethod described by Theodoridis et al. (1998a).The residue was re-dissolved in 100% methanol(300 mL). The resulting solution (25 mL) was inves-tigated by HPLC analysis using the DIONEX systemwith a UVD 340S diode array detector and an

automated sample injector (ASI-100). For com-pound separation, a NovaPak Phenyl column(150 3.9 mm) was eluted employing the gradientprogram described by Theodoridis et al. (1998b).The DAD spectrophotometer was set at a wave-length range from 215 to 275 nm. The taxanes wereidentified and quantified at 227 nm. The peaks wereassigned by spiking the samples with the standardsand comparing the retention times and UV spectra.The standard compounds paclitaxel and baccatin IIIwere purchased at Sigma and 10-deacetylbaccatinIII was donated by Prof. Jaziri from the Free

University of Brussels.All the experiments were conducted in triplicate,with the contents of two flasks assayed from eachtreatment.

Results

The aim of this study was to examine the effectsof the two precursors L-phenylalanine and p-aminobenzoic acid, alone and in combination with methyl

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Enhancement of taxane production in hairy root culture ofTaxus x media var. Hicksii 1951


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jasmonate, on the growth and taxane accumulationin transgenic root cultures (Figure 1).

Under the conditions assayed, the greatest drybiomass increase was observed after 4 weeks ofculturing hairy roots in the presence of theprecursor compounds (Table 1). Compared to thecontrol culture, the maximum dry biomass (6.2 g

L1

) was recorded when 1 mM of PHE was added tothe medium. In addition, the stimulatory effect of10 mM PAB was noted where the dry mass of hairyroots rose up to 5.3 g L1 at the end of the culture.

The media supplementation with the elicitorsignificantly (Po0.05, t-test) reduced the growthof cultured hairy roots compared to the controlculture and cultures supplemented with precursorsalone (Table 1).

PHE influenced taxane production in a dose-dependent manner. The addition of 1 mM PHE to themedia resulted in an 8-fold increase of paclitaxel

content after 4 weeks of culture. 10-DAB III was not

detected in the control (untreated) culture, whilemedium supplementation with 1 mM of PHE pro-duced the greatest 10-DAB III accumulation underthe conditions of this experiment (Tables 2 and 3).In the presence of 10 mM of PHE, paclitaxel and 10-DAB III were determined in roots only at 48h ofculture and there were no taxanes in the hairy root

cultures growing in the medium supplemented with100 mM of PHE alone. However, the combination of100 mM PHE with 100 mM methyl jasmonate yieldedthe greatest (almost 28-fold) increase in paclitaxelproduction (Table 2).

Paclitaxel and 10-DAB III accumulated in hairyroots during the entire culture period carried out inthe media supplemented with PAB. In most cases,the amounts of the investigated taxanes weresignificantly higher (Po0.05,t-test) than in controlcultures (Table 3). The highest paclitaxel contentwas detected when hairy roots grew in the media

supplemented with 10 or 100 mM PAB in combinationwith methyl jasmonate, 85.6 and 130.5 mg g1 D.W.,respectively. No taxanes were found in the tissue ofhairy roots when 1 mM PAB and methyl jasmonatewere added to the medium.

In this study, baccatin III was not detected eitherin the root tissues or in the post-culture media.None of the examined taxanes was excreted intothe media.

Discussion

The beneficial effect of precursor and elicitorfeeding on paclitaxel and related taxane accumula-tion in Taxus cell culture is well documented(Zhong, 2002; Pinol et al., 2007). The strategy ofcombining of several precursors and/or elicitors

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Figure 1. Transgenic roots ofTaxus x media var. Hicksiicultivated 4 weeks in liquid hormone-free DCR-M medium(Photo I.Rudnicki).

Table 1. Dry mass (g L1) increase ofTaxus x media var. Hicksii transformed roots cultivated in DCR-M mediumsupplemented with precursors or precursors and elicitor.

Time L-phenylalanine p-amino benzoic acid

1 mM 10 mM 100 mM 1 mM 10 mM 100 mM

48 h 1.270.1 1.370.1 1.570.5 1.470.3 1.570.3 1.870.31 week 1.670.3 1.770.2 1.570.3 2.470.4 2.370.4 2.170.12 weeks 2.170.3 2.270.8 2.470.2 3.170.7 3.270.2 2.470.53 weeks 3.270.4 1.970.1 1.870.4 3.670.8 3.470.4 3.2270.54 weeks 6.270.4 3.570.5 3.370.5 3.770.9 5.370.7 3.8470.5

L-phenylalanine+methyl jasmonate 100 mM p-amino benzoic acid+methyl jasmonate 100 mM

2 weeks 1.770.7 1.570.7 1.770.8 1.570.4 1.570.3 1.570.4

Data are means7SE.Control growth of 4 week-old hairy roots in DCR-M medium without precursors or elicitor: 3.4 g L170.7.

K. Syklowska-Baranek et al.1952


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Table 2. Taxane content in hairy root culture of Taxus x media var. Hicksii carried out in DCR-M medium supplemented w11.670.9 mg g1 D.W., 40.376 mg L1; 10-DAB III and baccatin III were not detected).

L-phenylalanine

Time 1 mM 10 mM 100 mM

Paclitaxel 10-deacetylbaccatin III Paclitaxel 10-deacetylbaccatin III Paclitaxel

mg g1 D.W. mg L1 mg g1D.W. mg L1 mg g1 D.W. mg L1 mg g1D.W. mg L1 mg g1 D.W.

48 h 15.375 20.875 116.977 158.179 1 week 2 weeks 3 weeks 18.872 55.377 22.373 65.376 4 weeks 52.377 335.679 65.878 422.7718

L-phenylalanine+methyl jasmonate 100 mM2 weeks 41.476 48.074 76.875 89.176 319.7714

Table 3. Taxane content in hairy root culture ofTaxus x media var. Hicksii carried out in DCR-M medium supplemented with p11.670.9 mg g1 D.W., 40.376 mg L1; 10-DAB III and baccatin III were not detected).

p-amino benzoic acid

Time 1 mM 10 mM 100 mM

Paclitaxel 10-deacetylbaccatin III Paclitaxel 10-deacetylbaccatin III Paclitaxel

mg g1 D.W. mg L1 mg g1 D.W. mg L1 mg g1 D.W. mg L1 mg g1 D.W. mg L1 mg g1D.W.

48 h 33.376 48.176 39.473 47.374 23.474 37.875 24.872 34.774 83.775 1 week 8.272 19.873 20.273 50.576 12.773 28.876 30.174 68.175 27.774

2 weeks 15.274 37.176 33.574 37.173 4.271 13.074 18.174 56.475 6.571 3 weeks 20.576 79.778 16.373 52.475 16.874 56.877 30.176 101.679 12.572 4 weeks 16.574 60.175 30.674 136.1711 18.2 78.478 11.873 42.274 12.172

p-amino benzoic acid+methyl jasmonate 100 mM2 weeks 85.679 145.7712 11.775 22.372 130.579


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seems to be promising. However, to date, taxaneproduction in cultures ofTaxushairy roots has beendescribed in only two studies. Huang et al. (1997)examined 5 clones ofT. brevifoliatransgenic roots.The highest paclitaxel content was determinedafter 3 weeks of culture (480 mg g1 D.W.) and thecompound was also found in the medium (13 mg

L1

). In our previous study, the greatest paclitaxelyield (420 mg g1 D.W.) was achieved after a 3-weekelicitation with methyl jasmonate (Furmanowaand Syklowska-Baranek, 2000), but the elicitordid not cause any increase in the 10-DAB IIIcontent.

Nims et al. (2006) observed increased transcriptabundance of genes involved in late steps of thetaxane biosynthetic pathway (10-DAB III, baccatinIII and paclitaxel formation) after methyl jasmo-nate addition. The enhanced enzyme activitycorrelated with the induction of taxane accumula-

tion. However, low activity of enzymes engaged in10-DAB III biosynthesis was also noted in unelicitedcultures. In our experiments, neither 10 DAB III norbaccatin III were found in the unelicited controlculture, but paclitaxel was detected after 4 weeksof culture.

Our results may suggest that the regulation ofgene expression engaged in the taxane biosyntheticpathway may differ in Taxus cell suspensioncultures and Taxus hairy root cultures. The studyalso demonstrates the suitability of the precursorand elicitor feeding strategy for augmentation ofpaclitaxel and 10-deacetylbaccatin III production in

Taxus x media var. Hicksii hairy root cultures.

Acknowledgements

The Medical University of Warsaw Research Fundis thanked for financial assistance. We are gratefulto Prof. Mondher Jaziri from the Laboratory ofBiotechnology and Plant Morphology, Free Univer-sity of Brussels for the standard substance of 10-

deacetylbaccatin III.

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Enhancement of Taxane Production in Hairy Root