# Engineering Isoprenoid Biosynthesis inArtemisia annua L. for the Production of Taxadiene: A Key Intermediate of Taxol

Authors: Meiya Li; Fusheng Jiang; Xiangli Yu; Zhiqi Miao
Journal: BioMed Research International
 (2015)

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## Abstract

Taxadiene is the first committed precursor to paclitaxel, marketed as Taxol, arguably the most important anticancer agent against ovarian and breast cancer. InTaxus, taxadiene is directly synthesized from geranylgeranyl diphosphate (GGPP) that is the common precursor for diterpenoids and is found in most plants and microbes. In this study, Artemisia annua L., a Chinese medicinal herb that grows fast and is rich in terpenoids, was used as a genetic engineering host to produce taxadiene. The TXS (taxadiene synthase) gene, cloned from Taxus and inserted into pCAMBIA1304, was transformed into Artemisia annua L. using the Agrobacterium tumefaciens-mediated method. Thirty independent transgenic plants were obtained, and GC-MS analysis was used to confirm that taxadiene was produced and accumulated up to 129.7 μg/g dry mass. However, the high expression of TXS did not affect plant growth or photosynthesis in transgenic Artemisia annua L. It is notable that artemisinin is produced and stored in leaves and most taxadiene accumulated in the stem of transgenic Artemisia annua L., suggesting a new way to produce two important compounds in one transgenic plant: leaves for artemisinin and stem for taxadiene. Overall, this study demonstrates that genetic engineering of the taxane biosynthetic pathway in Artemisia annua L. for the production of taxadiene is feasible.


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## Body

## 1. Introduction

Taxanes are a class of polycyclic diterpenes produced by many species of yew. Among these taxanes, paclitaxel, also known as Taxol, is the most important chemotherapy drug in the treatment of ovarian and breast cancers and diseased vasculature [1, 2]. The annual sale of Taxol and its analogue Taxotere reached up to 3.7 billion in 2006. However, only limited amounts can be obtained from the currently available sources. Total synthesis of Taxol was successfully finished in 1994; however, the complex structure of Taxol results in too low yield to be cost effective [3]. Semisynthesis methods start from more abundant and readily available precursors, such as 10-deacetylbaccatin III and baccatin III fromTaxus, but the limited availability of natural yew trees, slow growth rate of cultivated ones, and the low yield of taxanes result in a high price for Taxol [4]. Meanwhile, excessive exploitation of wild trees creates environmental damage and has been prohibited in many countries. It is considered that cell cultures and entophytic fungi fermentation are good sources of Taxol or its intermediates [5–7]; however, the processes of cell culture are not easy and yield is low.Metabolic engineering is a useful strategy for production of natural product in plant, such as producing tanshinone inSalvia miltiorrhiza hairy root cultures [8] and enhancing the production of tropane alkaloids in transgenicAnisodus acutangulushairy root cultures [9]. Taxadiene is the first taxane in the pathway of Taxol biosynthesis, which is synthesized from geranylgeranyl diphosphate (GGPP) directly. Taxadiene synthase, located in plastids, catalyzes the cyclization of GGPP to taxadiene [10, 11]. Because no routine transformation systems are currently available forTaxus species, researchers have explored the possibility of transferring the pathway to microbial or fast-growing plant species that are easier to genetically manipulate to produce taxoids by metabolic engineering. By expressing GGPP synthase and a truncated taxadiene synthase gene, taxadiene can be synthesized inEscherichia coli [12]. Further, eight Taxol biosynthetic genes were transformed and expressed inSaccharomyces cerevisiae to obtain advanced taxanes, such as taxadiene-5α-acetoxy-10β-ol [13]. Because the universal diterpenoid progenitor, GGPP, is rich in most plants, syntheses of taxanes from transgenic plants, such asArabidopsis[14] and tomato [15], have been investigated. However, constitutive expression of taxadiene synthase caused concomitant growth retardation and reduced levels of photosynthetic pigments in both plant systems. But in ginseng roots [16], the expression of taxadiene synthase gene did not affect the phenotype or growth.In this study,Artemisia annuaL., a Chinese annual herb, was engineered to produce taxadiene for several reasons.A. annuaL. has an abundant amount of terpene precursors, such as IPP and FPP, with total terpenoids in leaves recorded up to 3% of the dry mass [17]. As a traditional medical plant [18] which has been cultured in China for many years,A. annua L. grows fast, can reach more than 1.8 m in height, and has a high yield of biomass. Moreover, an efficient genetic transformation system has been established forA. annua L. To regulate the metabolism of terpenoid, divert some GGPP to taxadiene biosynthesis and construct a new system to produce taxadiene and even some more advanced taxanes (Figure 1); the taxadiene synthase gene was transformed intoA. annua.Figure 1
The biosynthetic pathway of secondary metabolites, including taxadiene and paclitaxel in transgenicA.annua.


## 2. Materials and Methods

### 2.1. Transformation ofArtemisia annua L.

Seeds ofA. annua. were collected from Youyang, Chongqing, China. The seeds were surface-sterilized in 75% ethanol for 1 min followed by treatment with 20% (v/v) sodium hypochlorite (NaOCl) for 20 min, washed 3 to 4 times with sterile distilled water, and sown onto MS0 medium [Murashige and Skoog (MS) basal medium supplemented with sucrose (30 g/L) and phytoagar (Sigma) (2.6 g/L)] [19] in Petri dishes (9 cm diameter). Plants were grown with a 16 h light/8 h dark photoperiod and 8,000 Lux (metal halide source) of light at 25°C. When reaching 5 cm in height, germinated seedlings were collected and the leaves were cut into 0.5 cm diameter pieces and used as the explants inAgrobacterium tumefaciens-mediated leaf disc transformation [20]. The plant binary expression vector pCAMBIA1304::p35s-TXS-nos, derived from pCAMBIA1304 in which the GUS reporter gene was replaced with theTXS gene, was used for transformation intoAgrobacterium tumefaciensstrain EHA105.After coculture in MS0 and hygromycin selection in MS1 (MS0 + 0.5 mg/L 6-BA + 0.05 mg/L NAA + 10 mg/L hygromycin + 500 mg/L carbenicillin), the hygromycin-resistant shoots were regenerated and transferred to rooting medium MS2 (1/2 MS0 + 5 mg/L hygromycin + 250 mg/L carbenicillin) [20]. After roots were formed (about 14–21 d), the rooted plantlets were transferred to soil and grown under controlled conditions in the growth chamber. After 2-3 months, transformed plants were transplanted to the field and covered by plastic film for further growth.


### 2.2. PCR Analysis ofTXS andhptII Genes in Transgenic Plants

The taxadiene synthase gene (TXS) from transgenicA. annua plants, which were about 10 cm in height, was identified by PCR analysis using genomic DNA isolated by the CTAB method [21]. Primers p35S (5′-GAT GAC GCA CAA TCC CAC T-3′) and TXSR (5′-CGT TTC GTG AGA GTT CTA CTT ACC-3′) were used to identify the introducedTXS gene, and primershptIIF (5′-GAC ATT GTT GGA GCC GAA A-3′) andhptIIR (5′-TGC TTG ACA TTG GGG AGT T-3′) amplified thehptII gene. The PCR reaction was carried out using the following mixture [forward primer (10 μM) 0.5 μL, reverse primer (10 μM) 0.5 μL, 10 × PCR buffer 2.5 μL, dNTPs (2.5 mM) 2.0 μL, MgCl2 (25 mM) 1.5 μL, DNA Template (100 ng/μL) 2.0 μL,Taq enzyme (5 U/μL) 0.2 μL, add ddH2O up to 25 μL] under the following conditions: 94°C for 5 min followed by 32 cycles of amplification (94°C for 45 s, 58°C for 45 s, and 72°C for 45 s) and 72°C for 10 min, resulting in 567 bp and 383 bp DNA fragments for theTXSandhptII genes, respectively.


### 2.3. DNA Isolation and Southern Blot Analysis

Genomic DNA from transgenic and control plants were isolated and purified according to the CTAB method. Approximately 40μg of DNA per sample was digested withHindIII, fractionated by 1.0% agarose gel electrophoresis, transferred onto a positively charged Hybond-N+ nylon membrane (Amersham Biosciences, UK), and hybridized with an alkaline phosphatase-labeled partialhptII sequence as the probe. The probe (383 bp) was generated by PCR usinghptII as the template, with primershptIIF1 andhptIIR1. Probe labeling (alkaline phosphatase), hybridization, and signal detection were performed using Amersham AlkPhos Direct Labeling Reagents (GE Healthcare, UK) and the CDP-Star Detection Module following the manufacturer’s instructions (Amersham Biosciences, UK). The hybridized signals were visualized by exposure to Fuji X-ray film at room temperature for 2-3 h.


### 2.4. RNA Isolation and Semiquantitative RT-PCR Analysis

RNA was extracted from upper leaves of control and transgenicA. annua plants using the RNAprep Plant Kit [TIANGEN Biotech (Beijing) Co., Ltd.]. DNA contamination was removed with DNaseI (TaKaRa) following the protocol provided by the manufacturer. The cDNA synthesis was completed from the RNA samples using reverse transcriptase reagent (TaKaRa) according to the manufacturer’s instructions.A 291 bp fragment of theTXSgene (corresponding toTXS cDNA from nucleotides 335 to 626) was prepared by PCR amplification from cDNA with the following primers: TSF1 (5′-CGT TTC GTG AGA GTT CTA CTT ACC-3′) and TSR1 (5′-CCG AGA GGG CGA TAA CAG A-3′). The gene-specific primers for ubiquitin conjugation (UBC) gene are UBC-F (5′-CAC ACT TGA GGT TGA GTC CAG-3′) and UBC-R (5′-CAT AAC ATT TGC GGC AGA TAG-3′). The PCR reaction was carried out using the following mixture [forward primer (10 μM) 0.5 μL, reverse primer (10 μM) 0.5 μL, 10 × PCR buffer 2.5 μL, dNTPs (2.5 mM) 2.0 μL, MgCl2 (25 mM) 1.5 μL, DNA Template (100 ng/μL) 2.0 μL,Taq enzyme (5 U/μL) 0.2 μL, add ddH2O up to 25 μL] under the following conditions: 94°C for 5 min followed by 35 cycles of amplification (94°C for 30 s, 58°C for 30 s, and 72°C for 30 s) and 72°C for 10 min.Following the separation of the PCR products on ethidium bromide stained 1% agarose gels, the bands at 291 bp were quantified. Each band was normalized against the intensity of the UBC control band obtained with the same cDNA sample.


### 2.5. Quantification of Taxadiene by Gas Chromatography-Mass Spectrometry (GC-MS)

To detect the new taxanes arising after the transformation ofTXS gene-taxadiene, leaves of 3 independent transgenicA. annua plants and a wild control plant were collected after growing in the field for about 2.5 months and 6.5 months. Leaves were weighed and milled to a fine power in liquid N2, followed by addition of 15 mL of hexane and treatment with ultrasonication for 30 min at 75 Hz. The extract was filtered, and the plant leaves were reextracted twice more according to the same method. These extracts were pooled and concentrated to 5 mL and then washed through a small (1 × 5 cm) silica gel column. The hexane elute was collected and concentrated to 1 mL.1μL of the hexane solution was analyzed by GC-MS (Perkin Elmer, Auto System XL GC/Turbo Mass MS) equipped with the capillary column DB-5MS (30 m × 0.25 mm × 0.25 μm) under the following condition: injection port temperature, 285°C; carrier gas, He (99.999%) 1.0 mL min−1; no split ratio; the column temperature increase at a speed of 10°C/min from 80°C to 300°C after a 2 min delay and then maintenance for 16 min at 300°C; electron ionization voltage, 70 eV. The taxadiene was analyzed according to NIST98 and WILEY7.0 mass spectrum database, by comparison to a nonadecane (Sigma) internal standard (1 μg/mL), to estimate total taxadiene content.


### 2.6. Quantification of Artemisinin Using High Performance Liquid Chromatography-Evaporative Light Scattering Detection (HPLC-ELSD)

Leaves ofA. annua collected as described above were dried at 45°C and ground. The leaf powder (0.1 g/sample) was extracted with ethanol (1 mL) by ultrasonication (twice, 15 min each, 75 Hz), and then centrifuged for 10 min at 12,000 rpm to remove the suspended particles. The final supernatant was filtered through a 0.22 μm filter.The prepared samples were analyzed by Waters Alliance 2695 HPLC system coupled with Waters 2420 ELSD detector. The HPLC condition was Waters C18 column using water : methanol (40 : 60, v/v) mixture as a mobile phase at a flow rate of 1 mL/min. The ELSD condition was optimized at a nebulizer-gas pressure of 50 psi and drift tube temperature of 45°C, and the gain was set at 7. The authentic artemisinin purchased from Sigma (St. Louis, USA) was used as the standard control. For each sample, the injection volume was 20 μL, and the results were analyzed with the Empower data system.


### 2.7. Observation of the Growth Influence ofTXS on Transgenic and Wild Plants

To investigate the influence of transformedTXS gene on the growth ofA. annua, transgenic and wild plants were grown in the greenhouse for two months and then transplanted to the field for another eight months. After about 2.5 months, the height, number of branch stems, and diameter of the stalk were measured. After collecting the seeds, whole plants were weighed and a thousand seeds from different plants were germinated to compare the germination capacity.


## 2.1. Transformation ofArtemisia annua L.

Seeds ofA. annua. were collected from Youyang, Chongqing, China. The seeds were surface-sterilized in 75% ethanol for 1 min followed by treatment with 20% (v/v) sodium hypochlorite (NaOCl) for 20 min, washed 3 to 4 times with sterile distilled water, and sown onto MS0 medium [Murashige and Skoog (MS) basal medium supplemented with sucrose (30 g/L) and phytoagar (Sigma) (2.6 g/L)] [19] in Petri dishes (9 cm diameter). Plants were grown with a 16 h light/8 h dark photoperiod and 8,000 Lux (metal halide source) of light at 25°C. When reaching 5 cm in height, germinated seedlings were collected and the leaves were cut into 0.5 cm diameter pieces and used as the explants inAgrobacterium tumefaciens-mediated leaf disc transformation [20]. The plant binary expression vector pCAMBIA1304::p35s-TXS-nos, derived from pCAMBIA1304 in which the GUS reporter gene was replaced with theTXS gene, was used for transformation intoAgrobacterium tumefaciensstrain EHA105.After coculture in MS0 and hygromycin selection in MS1 (MS0 + 0.5 mg/L 6-BA + 0.05 mg/L NAA + 10 mg/L hygromycin + 500 mg/L carbenicillin), the hygromycin-resistant shoots were regenerated and transferred to rooting medium MS2 (1/2 MS0 + 5 mg/L hygromycin + 250 mg/L carbenicillin) [20]. After roots were formed (about 14–21 d), the rooted plantlets were transferred to soil and grown under controlled conditions in the growth chamber. After 2-3 months, transformed plants were transplanted to the field and covered by plastic film for further growth.


## 2.2. PCR Analysis ofTXS andhptII Genes in Transgenic Plants

The taxadiene synthase gene (TXS) from transgenicA. annua plants, which were about 10 cm in height, was identified by PCR analysis using genomic DNA isolated by the CTAB method [21]. Primers p35S (5′-GAT GAC GCA CAA TCC CAC T-3′) and TXSR (5′-CGT TTC GTG AGA GTT CTA CTT ACC-3′) were used to identify the introducedTXS gene, and primershptIIF (5′-GAC ATT GTT GGA GCC GAA A-3′) andhptIIR (5′-TGC TTG ACA TTG GGG AGT T-3′) amplified thehptII gene. The PCR reaction was carried out using the following mixture [forward primer (10 μM) 0.5 μL, reverse primer (10 μM) 0.5 μL, 10 × PCR buffer 2.5 μL, dNTPs (2.5 mM) 2.0 μL, MgCl2 (25 mM) 1.5 μL, DNA Template (100 ng/μL) 2.0 μL,Taq enzyme (5 U/μL) 0.2 μL, add ddH2O up to 25 μL] under the following conditions: 94°C for 5 min followed by 32 cycles of amplification (94°C for 45 s, 58°C for 45 s, and 72°C for 45 s) and 72°C for 10 min, resulting in 567 bp and 383 bp DNA fragments for theTXSandhptII genes, respectively.


## 2.3. DNA Isolation and Southern Blot Analysis

Genomic DNA from transgenic and control plants were isolated and purified according to the CTAB method. Approximately 40μg of DNA per sample was digested withHindIII, fractionated by 1.0% agarose gel electrophoresis, transferred onto a positively charged Hybond-N+ nylon membrane (Amersham Biosciences, UK), and hybridized with an alkaline phosphatase-labeled partialhptII sequence as the probe. The probe (383 bp) was generated by PCR usinghptII as the template, with primershptIIF1 andhptIIR1. Probe labeling (alkaline phosphatase), hybridization, and signal detection were performed using Amersham AlkPhos Direct Labeling Reagents (GE Healthcare, UK) and the CDP-Star Detection Module following the manufacturer’s instructions (Amersham Biosciences, UK). The hybridized signals were visualized by exposure to Fuji X-ray film at room temperature for 2-3 h.


## 2.4. RNA Isolation and Semiquantitative RT-PCR Analysis

RNA was extracted from upper leaves of control and transgenicA. annua plants using the RNAprep Plant Kit [TIANGEN Biotech (Beijing) Co., Ltd.]. DNA contamination was removed with DNaseI (TaKaRa) following the protocol provided by the manufacturer. The cDNA synthesis was completed from the RNA samples using reverse transcriptase reagent (TaKaRa) according to the manufacturer’s instructions.A 291 bp fragment of theTXSgene (corresponding toTXS cDNA from nucleotides 335 to 626) was prepared by PCR amplification from cDNA with the following primers: TSF1 (5′-CGT TTC GTG AGA GTT CTA CTT ACC-3′) and TSR1 (5′-CCG AGA GGG CGA TAA CAG A-3′). The gene-specific primers for ubiquitin conjugation (UBC) gene are UBC-F (5′-CAC ACT TGA GGT TGA GTC CAG-3′) and UBC-R (5′-CAT AAC ATT TGC GGC AGA TAG-3′). The PCR reaction was carried out using the following mixture [forward primer (10 μM) 0.5 μL, reverse primer (10 μM) 0.5 μL, 10 × PCR buffer 2.5 μL, dNTPs (2.5 mM) 2.0 μL, MgCl2 (25 mM) 1.5 μL, DNA Template (100 ng/μL) 2.0 μL,Taq enzyme (5 U/μL) 0.2 μL, add ddH2O up to 25 μL] under the following conditions: 94°C for 5 min followed by 35 cycles of amplification (94°C for 30 s, 58°C for 30 s, and 72°C for 30 s) and 72°C for 10 min.Following the separation of the PCR products on ethidium bromide stained 1% agarose gels, the bands at 291 bp were quantified. Each band was normalized against the intensity of the UBC control band obtained with the same cDNA sample.


## 2.5. Quantification of Taxadiene by Gas Chromatography-Mass Spectrometry (GC-MS)

To detect the new taxanes arising after the transformation ofTXS gene-taxadiene, leaves of 3 independent transgenicA. annua plants and a wild control plant were collected after growing in the field for about 2.5 months and 6.5 months. Leaves were weighed and milled to a fine power in liquid N2, followed by addition of 15 mL of hexane and treatment with ultrasonication for 30 min at 75 Hz. The extract was filtered, and the plant leaves were reextracted twice more according to the same method. These extracts were pooled and concentrated to 5 mL and then washed through a small (1 × 5 cm) silica gel column. The hexane elute was collected and concentrated to 1 mL.1μL of the hexane solution was analyzed by GC-MS (Perkin Elmer, Auto System XL GC/Turbo Mass MS) equipped with the capillary column DB-5MS (30 m × 0.25 mm × 0.25 μm) under the following condition: injection port temperature, 285°C; carrier gas, He (99.999%) 1.0 mL min−1; no split ratio; the column temperature increase at a speed of 10°C/min from 80°C to 300°C after a 2 min delay and then maintenance for 16 min at 300°C; electron ionization voltage, 70 eV. The taxadiene was analyzed according to NIST98 and WILEY7.0 mass spectrum database, by comparison to a nonadecane (Sigma) internal standard (1 μg/mL), to estimate total taxadiene content.


## 2.6. Quantification of Artemisinin Using High Performance Liquid Chromatography-Evaporative Light Scattering Detection (HPLC-ELSD)

Leaves ofA. annua collected as described above were dried at 45°C and ground. The leaf powder (0.1 g/sample) was extracted with ethanol (1 mL) by ultrasonication (twice, 15 min each, 75 Hz), and then centrifuged for 10 min at 12,000 rpm to remove the suspended particles. The final supernatant was filtered through a 0.22 μm filter.The prepared samples were analyzed by Waters Alliance 2695 HPLC system coupled with Waters 2420 ELSD detector. The HPLC condition was Waters C18 column using water : methanol (40 : 60, v/v) mixture as a mobile phase at a flow rate of 1 mL/min. The ELSD condition was optimized at a nebulizer-gas pressure of 50 psi and drift tube temperature of 45°C, and the gain was set at 7. The authentic artemisinin purchased from Sigma (St. Louis, USA) was used as the standard control. For each sample, the injection volume was 20 μL, and the results were analyzed with the Empower data system.


## 2.7. Observation of the Growth Influence ofTXS on Transgenic and Wild Plants

To investigate the influence of transformedTXS gene on the growth ofA. annua, transgenic and wild plants were grown in the greenhouse for two months and then transplanted to the field for another eight months. After about 2.5 months, the height, number of branch stems, and diameter of the stalk were measured. After collecting the seeds, whole plants were weighed and a thousand seeds from different plants were germinated to compare the germination capacity.


## 3. Results and Discussion

EHA105 harboring the plasmid pCAMBIA1304::p35s-TXS-nos was used to introduce theTXS gene into the young leaves ofA.annuaviaA. tumefaciens-mediated transformation. After hygromycin selection and regeneration, 67 independent hygromycin-resistant plantlets were obtained (Figure 2), and among them 40 independent transgenic plants were confirmed to have theTXSandhptII genes by PCR. The consistent presence of theTXSandhptII genes indicated that both were transformed into plant cells as an intact T-DNA sequence. Therefore, theTXS copy number can be determined by anhptII gene probe in Southern hybridization. The probe hybridized to the supercoiled pCAMBIA1304::p35s-TXS-nos but did not hybridize to wildA.annua. Young plant leaves from the transgenic T1 generation were harvested and checked forTXS gene insertion by Southern blot analysis. The results confirmed the T-DNA integration in all independent lines of transgenicA.annua. Single bands were detected in four lines (3, 50, 55, and 56) and double bands in five lines (9, 11, 19, 21, and 38) (Figure 3(a)). The transgenic lines with less than twoTXS gene copies were expected to express the transgene at a higher level, and the following experiments were focused on these lines.Pictures ofA. annua at different stages of transformation and regeneration. (a) Screening; (b) growing in the bottle; (c) shoot regeneration; (d) growing in the greenhouse.
(a)
(b)
(c)
(d)Southern blot and semiquantitative RT-PCR analysis of transgenicA.annua.(a) Southern blot analysis ofA.annua transformed with pCAMBIA1304::p35s-TXS-nos. Lane assignment: 1 λ/HindIII DNA molecular marker; 2–10 genomic DNA isolated from independent transgenicA.annua lines 3, 9, 11, 55, 56, 19, 21, 38, and 50; 11 pCAMBIA1304-TXS; 12 genomic DNA of untransformed wildtypeA.annua (b) and (c) semiquantitative RT-PCR analysis for the expression ofTXS in independent transgenicA.annua lines 50, 55, 11, 38, 3, 19, and 56. ck indicates the untransformed wildtype controlA.annua; UBC: ubiquitin conjugation gene;TXS: taxadiene synthase gene.
(a)
(b)
(c)TheTXS gene expression in young leaves of transgenic lines was further analyzed by semiquantitative RT-PCR. A housekeeping gene,UBC, was used as a reference to normalize the transgene expression level.TXS expression was detected in approximately 30 transgenic lines; however, there were significant differences inTXS expression between lines (Figures 3(b)/3(c)). The highestTXS expression was detected in transgenic line 55, which was approximately 3 times higher than that in lines 11 and 56 and contained only one transgene copy.Taxadiene was purified from hexane extracts of lines 55, 56, and 11 using a silica gel column and then analyzed by GC-MS. The ion flow data atm/z 122, reported as the most abundant broken ion of taxadiene [10], was used for identifying and quantifying taxadiene in hexane extracts. The data showed that the hexane extracts from all three transgenic lines gave a GC-MS peak at 17.29 min that was absent from the wild control (Figure 4(a)). The major mass spectrum ions were atm/z 122, 121, 123, 107, and 272, which is consistent with the published data on taxadiene [22] (Figure 4(b)). A similar peak was also detected in the yew hexane extracts following the same preparation. Together these results indicated that the transgenicA.annuais synthesizing taxadiene. Using nonadecane (Sigma, Cat. 39756-36-0) as an internal standard, the contents of taxadiene were estimated from 4.4 to 129.7 μg/g dry weight in the transgenicA.annua lines. In transformed tomato withTXS gene, the contents of taxadiene was 20 μg/g dry weight [15] and in transgenic ginseng roots was 9.1 μg/g dry weight [16]; the highest taxadiene yield in transgenicA.annua was gotten from line 55, since the expression of theTXS gene in line 55 is much higher than others, which may indicate that the higher the gene expressed, the more the taxadiene produced.Figure 4
(a) GC-MS analysis for taxadiene in the crude hexane extracts from yew and transgenic and wildtypeA.annua. The GC profiles of the ion current atm/z 122 from hexane extracts of a yew tree, theA.annua transgenic line 55, and the wildtype control ofA.annua. (b) The mass spectrum of the peak with retention time of 17.29 min, matching the published MS data of taxadiene. (c) Artemisinin analysis in transgenic and untransformedA.annua plants by HPLC-ELSD. Plants that were grown in the field for 2.5 and 6.5 months, respectively, were harvested to determine the content of artemisinin in wild control and transgenicA.annua. ck: the wildtype; 3, 5, 11, 19, 30, 55, and 56: independent transgenic lines ofA.annua. (mean ± SD, n
=
3). (d) The amount of taxadiene in 2.5- and 6.5-month-oldA.annua transgenic line 55 (mean ± SD, n
=
3). (e) Taxadiene content in the stem and the leaves of 6.5-month-oldA.annua transgenic line 55 (mean ± SD, n
=
3).Artemisinin is the main terpenoid in wildA.annua. However, most IPP, the universal terpenoid building block, in transgenicA.annua is diverted into the taxadiene pathway as the result of the introduction of taxadiene synthase. Therefore the biosynthesis of artemisinin may be competitively inhibited in transgenicA.annua. To investigate the interaction between the biosynthesis of taxadiene and artemisinin in transgenicA.annua, the contents of artemisinin in transgenic and wild lines ofA.annua were measured by HPLC-ELSD. The results revealed that transgenic lines 55 and 56 produced more taxadiene and less artemisinin after growing in the field for 2.5 months: the artemisinin content was 0.057% in line 55 and 0.066% in line 56, while the content of artemisinin reaches up to 0.58% in the wild control (Figure 4(c)). Since FPP is the common precursor to taxadiene and artemisinin and its content is limited in the 2.5-month-old transgenicA.annua, the correlation between the decrease of artemisinin and the increase of taxadiene suggests that the introduction of taxadiene synthase changed the FPP metabolic flux. It is likely that more FPP is conscribed to produce GGPP, some of which was further bound to taxadiene synthase and converted into taxadiene, which decreased the biosynthesis of artemisinin. However, more FPP is produced inA.annua after growing in the field for 6.5 months andwhen it is blooming. At this stage, the transgenicA.annua produced more taxadiene than, but a similar amount of artemisinin to, the wildtype when it is blooming (Figures 4(c)/4(d)).It is reported that artemisinin is produced by the trichome cell in the leaves, floral buds, and flowers ofA.annua but not in the stem [23, 24]. Usually the leaves ofA.annua are harvested for extracting artemisinin and the stems are discarded as waste. Our research shows that the content of taxadiene in the stems is much higher than that in the leaves of transgenicA.annua, indicating that transgenicA.annua can be used for producing taxadiene in the stems and artemisinin in the leaves. It was also found that, in transgenicA.annua plants, taxadiene accumulated during growth and reached the highest level in the late growth stage, especially when the plant is blooming (Figure 4(e)). Therefore, harvesting transgenicA.annua in bloom is an efficient way to obtain the highest content of artemisinin and taxadiene at the same time.A strong growth retardation and photosynthesis inhibition resulted from high expression ofTXS in transgenicArabidopsis [14] and tomato [15], but this was not observed in transgenic ginseng roots [16]; it says that, in leaves and stems, most GGPPs could convert to taxadiene, so they will produce less gibberellin, which made the transgenic plants growth retardate, but for roots, they do not need gibberellins for grow. In our research, no phenotypic or growth differences were observed in transgenicA.annua. In the growth chamber and greenhouse, all the plants in pots grew with similar height and the leaves were green and healthy. After growing in the field for 2.5 months, the height, branching, and stem diameters of both wild and transgenicA. annua were measured (Figure 5(a)). All plants grew well, healthy, and strong, suggesting that expression of the introducedTXSdid not affectA.annua growth. Additionally, whole plants were dried and weighed after collecting seeds, but no significant difference was found between the masses in different plant lines (Figure 5(a)). One possibility is that there is a more effective biosynthesis system for terpenoid inA. annua. In this case, the terpenoid building blocks, such as FPP and GGPP, are abundant enough to produce important primary substances, such as chlorophylls, carotenoids, gibberellins, and tocopherols, in transgenicA.annua, even when some GGPP is consumed by the taxadiene synthase, so the biosynthesis of taxadiene does not strongly influence the content of terpenoid precursor inA.annua leaves. This is supported by the results that the taxadiene content is much higher in the stem than in the leaves in transgenic line 55 (Figure 4(e)).(a) Comparison of the height, number of twigs, stem diameter, and whole plant dry weight between transgenic lines 55 and 56, expressing high levels ofTXS, and the wildtype control lines ofA.annua(ck1–6). (b) The weight of a thousand seeds of transgenic and wildA.annua plants (mean ± SD, n
=
3). ck1–6: different wildtype plants; 55 and 56: independent transgenicA.annua. (c) The germination capacity (%) of different seeds (mean ± SD, n
=
3). ck1–3: three different wildtype lines ofA.annua; 55 and 56: lines of independent transgenicA.annua.
(a)
(b)
(c)The only disadvantage of highTXS expression inA.annua is that the seeds became smaller and lighter (Figure 5(b)). However, the reduction in seed size and weight did not influence germination (Figure 5(c)). It is unclear why the seed size of the transgenic plant is smaller.


## 4. Conclusion

Our study demonstrates that genetic engineering of isoprenoid biosynthesis to produce taxadiene inA.annua is feasible. The transformation ofA.annua with theTXS gene did not result in growth retardation or photosynthesis inhibition. It is notable that the content of taxadiene reached up to 129.7 μg/g dry weight in transgenicA.annua line 55 and mostly concentrates in stems, while artemisinin accumulates in leaves. Therefore, transgenicA.annua harboring theTXS gene can be used to produce both artemisinin and taxadiene: leaves for artemisinin and stems for taxadiene.In future studies, more Taxol biosynthesis genes, including taxane 2α-hydroxylase, 5α-hydroxylase, 7β-hydroxylase, and 10β-hydroxylase, will be transformed intoA.annua to produce more advanced taxoids that could be purified and used for semisynthesis of Taxol and Taxotere. Furthermore, novel taxanes arising as the result of transgenic genes inTaxus and those encoded byA.annua itself will be monitored.



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*Source: 504932-2015-02-01.xml*