Molecular Cloning and Characterisation of Farnesyl Pyrophosphate Synthase from Tripterygium wilfordii

Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.


Introduction
Tripterygium wilfordii is a traditional Chinese medicinal plant used to treat inflammatory diseases because of its analgesic and anti-microbial properties [1]. This plant has been widely used in the treatment of immune and tumour diseases [2][3][4]. Terpenoids are the primary active substances (Fig 1) of T.wilfordii and include sesquiterpene, diterpenoids andtriterpenoids. Triptolide [5], which has been recognised as one of the primary active constituents of T. wilfordii, is a class of 3 epoxygroups and an α,β-unsaturated five-membered lactone ringand is also a unique abietane diterpene. T. wilfordii has attracted much attention due to its architecture and significant activities. Tripterine [6], which was the first monomer isolated from T. wilfordii has important biological activity.
FPS belongs to the family of short-chain prenyltransferases [7]. FPS catalyzes the head-totail condensation reaction of dimethylallyl pyrophosphate (DMAPP) with two molecules of isopentenyl pyrophosphate (IPP) to form FPP [8][9], which is the precursor of all sesquiterpenes [10], such as artemisinin and wilfordine.FPS provides substrate FPP to squalene synthase and sesquiterpene synthase. Squalene synthase brings the synthesis into steroids and saponins, which involved in cell membrane systems building; sesquiterpene synthase put the synthesis on cyclic sesquiterpene compounds [11]. Upon treatment with elicitors, FPP, which is normally employed in sterol biosynthesis, is diverted from the sterol pathway as large amounts of antibiotic sesquiterpenesare produced [12][13][14]. This diversion is accomplished by the induction of sesquiterpene cyclases and coeval suppression of squalene synthesis, the initial committed step of phytosterol production [13][14].
Thus far, FPS has been isolated from sorghum [15], corn [16], sandalwood [17] and 41 other species of plants. Recently, FPS has been isolated from Panax ginseng [18], Salvia miltiorrhiza, Acanthopanax senticosus, Artemisia annua [19], Ginkgo biloba [20] and other medicinal plants. International and domestic academics have studied the gene expression patterns of farnesyl pyrophosphate synthase in plants and found that this gene has tissue-specific expression with increased isoprenoid derivative contents. Gene expression studies have demonstrated that gene expression is related to the isoprene content in plants.
The effective components in T. wilfordii are difficult to obtain by traditional chemical methods. In addition, the products of bioactive compounds cannot be synthesized through microbial methods. The current information regarding terpenoid biosynthesis genes is limited, and studies regarding key enzymes in T. wilfordii are few. Based on the above issues, we present the cloning and characterisation of full-length FPS cDNAs of T. wilfordii (TwFPSs) for the first time.

Plant materials
Fresh leaves and stems of T. wilfordii were sheared, cleaned and disinfected. Then, the leaves were cut into 1.0 cm×1.0 cm pieces, and the stems were cut into 1.0 cm lengths after rinsing with sterile water. These tissues were cultivated in MS agar medium containing 2,4-D hormone at 25°C in dark. After two weeks, calluses began to grow at the explant slits. The calluses which had a white lustre, were soft and grew well, were cultured in MS agar medium containing 0.5 mg/L of 2,4-D, 0.1 mg/L of KT, and 0.5 mg/L of IBA at 25°C in the dark.
The calluses were cultured in MS agar medium containing 0.2 mg/L of IAA, 0.5 mg/L of KT, and 1.5 mg/L of 6-BA at 25°C under 16 h light/8 h dark conditions. The aseptic seedlings were harvested after subculturing once a month.
After 3 subcultures, we chose the calluses that grew well and that had a loose texture and clipped these calluses into small pieces with tweezers. These calluses were cultured in MS medium containing 0.5 mg/L of 2,4-D, 0.1 mg/L of KT, 0.5 mg/L of IBA, and cell suspensions of 2.0 g/40 mL in the dark at 25°C with rotary shaking at 120 rpm. Elicitor treatment T. wilfordii cell suspensions displaying good growth were chosen as the experimental material. After 12 days, 1 mM [21] methyl jasmonate (MeJA) [22][23] was added to the treatment groups, and DMSO was added to the control groups. After treatment, the cell suspensions were harvested in liquid nitrogen at 0, 4, 12, 24 and 48 hand stored at -80°C.

Sequence and phylogenetic analyses
The nucleotide and protein sequences were compared using NCBI (http://www.ncbi.NLM. NIH.gov). The sequences were translated into amino acidsusing DNAMAN software. The ORF was searched using ORF Finder (www.ncbi.NLM.NIH.gov/Gorf/Gorf.html). The theoretical isoelectric point (pI) and molecular weight (Mw) were determined using the Compute pI/ Mw tool (http://Web.ExPASy.org/compute_pi/). Multiple sequence alignments were performed using DNAMAN and ClustalW software. Phylogenetic analysis was performed using MEGA5.1 software to build evolutionary trees.

Expression of TwFPS1 andTwFPS2 in E. coli and purification of recombinant protein
Based on the cloning vector, the primers for recombination were designed; an NcoI site and a HindIII site were introduced at the start and stop codons, respectively. TwFPS1: 5 0 -GAG GAGCCATGGCTATGAGCGACACCAAGTCCAAG-3 0 (forward) and 5 0 -GAGGAGAAGCTTC TACTTCTCTCGCTTGTATAT-3 0 (reverse). The primers for recombination were designed; a KpnI site and an EcoRI site were introduced at the start and stop codons, respectively. TwFPS2: 5 0 -GAGGAGGGTACCATGGCGGATCTCAAGTCAACG-3 0 (forward) and 5 0 -GAGGAGGAAT TCCTACTTCTGTCTCTTGTATATC-3 0 (reverse). The fragments were cloned into the expression vector pET-32a(+). E. coli BL21 (DE3) cells were used for recombinant plasmid expression and the induction was performed at 16°C for 20 h with the addition of 1 mM isopropyl thiogalactoside (IPTG). After collection, the cells were resuspended in 2 mL lysis buffer A (20 mM Tris-HCl(pH 8.0), 500 mM NaCl, 1 mM PMSF and bacteria protease inhibitor cocktail), lysed by sonication (lysed for 5s, paused for 5s) and centrifuged for 30 min at 13000 g. Thus, the supernatant protein was harvested.
This supernatant was loaded onto a Ni-NTA column and pre-equilibrated by lysis buffer A. The column was washed with 10 column volumes of lysis buffer A containing 20 mM imidazole. The target proteins were eluted by buffer B (20 mM Tris-HCl(pH 8.0) containing 500 mM NaCl and 50 mM imidazole) and buffer C (20 mM Tris-HCl(pH 8.0) containing 150 mM NaCl and 250 mM imidazole). The purified protein was examined by SDS-PAGE gel electrophoresis. The concentration of FPPS protein was determined by the Bradford method [25].

Assay for enzymatic activity
Scale assays (200μL) for the identification of enzymatic reaction were performed with purified FPPS proteins (200μg) using 100μM IPP and 100μM DMAPP in assay buffer (50 mM Tris-HCl, pH 7.6) containing 5 mM MgCl 2 , 25 mM DTT, and 10% [v/v] glycerol. The assays were incubated at 30°C for 2 hours. To stop the assay and hydrolyse all diphosphate esters, 200μL of solution containing 2 units of potato apyrase and 2 units of calfintestine alkaline phosphatasein 0.2 M Tris-HCl(pH 9.5)was added to all assays, followed by incubation for 8 h at 30°C. After enzymatic hydrolysis, the resulting isopropyl alcohols were extracted into 500μL hexane 3 times. The hexane phase was concentrated by passing N 2 at the opening of the tube, and then the products were dissolved in 100μL hexane and used for GC-MS measurements.
GC-MS analysis was detected on an Agilent 6890N gas chromatograph (splitless; injector temperature, 250°C) with a 5975imass spectrometer (GC-MS). One microliter of dissolved organic phase was injected. The separation was performed on an HP-5MS column (50 m×0.25 mm×0.5μm) with helium as the carrier gas (flow rate of 1 mL/min) on a temperature gradient from 60°C, at 10°C per min to 90°C (hold 1 min),and then 3°C per min to 220°C (hold 1 min). Mass spectra, 70eV (in EI mode), ion trap heating, 230°C; scan range, 30-500 amu. The products were identified viamass spectrometry profiles, and farnesol could be identified using standard chemicals and retention times. The assay was performed with empty vector as a control.

Real-time quantitative PCR analysis of TwFPS1 andTwFPS2 expression
Total RNA from different tissues (roots, stems, and leaves) and different inductive stages was extracted separately as described above. The primers for real-time quantitative PCR analysis were designed using Primer Premier 5.0 software. TwFPS1: 5 0 -GGGTGTATTTGCGGAGT-3 0 (forward) and 5 0 -CGGCAGAATCTAATGGAG-3 0 (reverse); TwFPS2: 5 0 -CAGACCCTCACC TTCCATT-3 0 (forward) and 5 0 -AAGAGTAACCATAAGCAGCAGAC-3 0 (reverse). The βtactin gene was used as an endogenous control to normalize expression. The PCR reaction conditions were as follows: an initial incubation at 95°C for 3 min and then cycling at 95°C for 3 s and 60°C for 30 s for 40 cycles. There were three samples in each group and each sample was repeated for three times to insure the credibility of the data. The relative quantification of the TwFPS transcript levels was achieved by the 2 -ΔΔCt [26] method using ABI 7500 Software v2.0.1 (PE Applied Biosystems).

Results
Molecular cloning of the full-length TwFPS1 andTwFPS2 cDNAs RT-PCR was performed with total RNA from T. wilfordii. TwFPS gene fragments were obtained by 3 0 rapid amplification of cDNA ends (3 0 -RACE-PCR) and 5 0 -RACE-PCR. The fulllength cDNA encoding the FPS protein was isolated from T. wilfordii. The full-length cDNA of TwFPS1was 1345bp, with a1029bp ORF, which encodes a 342 amino acid polypeptide, flanked by an 81bp 5 0 -untranslated region and a 235bp 3 0 -untranslated region including a 28bp poly (A) tail. The predicted TwFPS1 protein had a calculated molecular mass of 39.54 kDa and a theoretical pI of 5.59 (GenBank accession number KM058711). The full-length cDNA of TwFPS2is1312bp, with a1029bp ORF, which encodes a 342 amino acid polypeptide, flanked by a 68bp 5 0 -untranslated region and a 215bp 3 0 -untranslated region including a of 26bp poly (A) tail. The predicted TwFPS2protein had a calculated molecular mass of 39.54 kDa and a theoretical pI of 5.28 (GenBank accession number KM058712).
A BLAST search of the NCBI protein database showed that the deduced amino acid sequence of TwFPS1 had 77-85% identity to the FPSs from Mangifera indica, Panax quinquefolius, Panax ginseng, Panax notoginseng, Eucommia ulmoides, Aralia elata, Centella asiatica, Astragalus membranaceus and Malus domestica. The deduced amino acid sequence of TwFPS2 had 78-88% identity to those FPSs from Glycyrrhiza uralensis, Astragalus membranaceus, Hevea brasiliensis, Euphorbia pekinensis, Medicago sativa, Pyrus communis, Aquilaria sinensis, Gossypium hirsutum, and Gentiana lutea. The two proteins are highly conserved; the sequence identity was 77.26%, and the amino acid identity was 80.12%. Both TwFPS1 and TwFPS2had five conserved regions, which were numbered I to V [27]. The highly conserved aspartate-rich motif in region II with the sequence DDXX(XX)D is called FARM (first Asp-rich motif), which is highly conserved in all known prenyltransferases and which has been designated as the chain length determination region [28]. Region V with the sequence DDXXD is called SARM (second Asp-rich motif). These regions marked with lines are characteristic of prenyltransferases that can be used to synthesise isoprenoid diphosphates [29] (Fig 2).
A phylogenetic tree of isoprenyl diphosphate synthases including GPPS, FPPS and GGPPS from different organisms was constructed to investigate the evolutionary relations. All of the plant isoprenyl diphosphate synthases sequences were separated into three main groups. T. wil-fordii1 and T. wilfordii2 clustered with 28 FPPS sequences. A. grandis, G. rigescens GPPS and M. luteus, E. coli FPPS clustered with 11 GGPPS sequences. This tree showed that FPSs evolved from a common ancestor and that the two TwFPSs belong to the clade of the plants (Fig 3). T. wilfordii1 and Eucommia ulmoides were classified into one cluster, and T. wilfordii 2 and Malus domestica were classified into one cluster. These clusters mean that these plants had the closest evolutionary relations. Moreover, these plants all belong to Angiospermae Dicotyledoneae. However, the two genes are not close, which suggests to us that a certain difference exists between TwFPS1 and TwFPS2.

Characterisation of TwFPS1 andTwFPS2 recombinant protein
The entire reading frame of TwFPSs was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwFPS protein for characterising the farnesylpyrophosphate synthase activity. After induction by IPTG, the recombinant protein was expressed. The molecular mass of TwFPS1 (Fig 4A) fused with a Trx-tag and a His-tag on N-terminal is approximately 58 kDa, and the TwFPS2 (Fig 4B) fusion protein is approximately 58 kDa, as determined by SDS-PAGE. The Trx-tag is a fusion tag and advantageous to the soluble protein expression. His-tag is a purification tag. Highly purified preparation of the protein can be obtained through purification with Ni-column affinity chromatography.
The purified proteins were assayed for farnesylpyrophosphate synthase catalytic activity. When the purified enzyme was incubated with DMAPP and IPP, the products had the same retention time as the farnesol standards (Fig 5A-5D). The GC retention time (RT) of farnesol was 29.588 min; TwFPS1 samples of the product, 29.590 min; and TwFPS2 samples of the product, 29.584 min. The blank control sample was not detected in the corresponding characteristic peak. Under GC-MS analysis (Fig 5E-5G), the FPS sample product qualified as farnesol, which had the characteristic peaks, including m/z = 222.0 (Molecular ion: M+) and m/z = 69.10 (CH3(CH3) = CHCH2-). Thus, these results indicated that the coding regions of TwFPSs encode functional FPP synthase.

Tissue-specific and inducible expression of TwFPS1 andTwFPS2
When total RNAs were isolated from the roots, stems and leaves of T. wilfordii, the lowest level of FPPS mRNA expression was found in leaves. Its value was set up as 1. The other FPPS expression tissues were evaluated relative to the leaves level. We found that TwFPSs are preferentially expressed in the roots. Among different tissues of T. wilfordii, the highest transcript levels of TwFPS1 (Fig 6A) and TwFPS2 (Fig 6B) were observed in the roots, and the lowest levels of TwFPS1and TwFPS2expression were found in the leaves. The highest levels of TwFPS1  Moreover, real-time PCR analysis with cell suspensions at different developmental stages was also performed to examine the changes in the expression of TwFPS genes upon MeJA treatment. Specifically, MeJA caused a significant increase in TwFPS levels in T. wilfordii cell suspensions. Interestingly, the dynamic range of the induction varied. The levels of TwFPS1 ( Fig 7A) and TwFPS2 (Fig 7B) expression both increased at first, peaked at 12 h, then decreased gradually, and the expression levels reached almost the same levels that of the control group after 48 h. The value of 0h was set up as 1 and the TwFPSs mRNA expression in other stage was evaluated relative to the 0h. The TwFPS1 expression of MeJA group at 12h was 2.4-hold higher than control group. The TwFPS2 expression of MeJA group at 12h was 4-hold higher than control group. These results indicated that the expression of TwFPS2relative to 0 h was higher than that of TwFPS1.
In Arabidopsis thaliana, for example, FPS exists in the form of a small gene family. The FPS gene family encodes three isomers of FPS (FPS1S, FPS1L and FPS2). The primary difference between FPS1S and FPS1L is the N-terminus. FPS1S and FPS2 are found in plastids, and FPS1L is found in mitochondria [32]. The different metabolic channels determine their different functions and expression patterns. Thus, the secondary metabolism that FPS is involved in is complex, with great diversity and specificity. A study of rice shows that FPS exists in chloroplasts and in plastids [44]. FPS1 is expressed in different parts of the plants and participated in all life processes. FPS2 is primarily expressed in the flowers, as well as in the root tips of the lateral root and in the juncture with the primary root or secondary root [45]. The subcellular localisation prediction showed that the major localisation of TwFPS1 is the cytoplasm; however, the localisation of TwFPS2 is the cytoplasm or mitochondria. Therefore, a tentative inference of this result is that TwFPS1 and TwFPS2 participate in different metabolic channels.
These findings suggest that the expression of the native FPS genes in T. wilfordii result in the accumulation of the active components. The cloning and identification of key genes involved in the biosynthesis of active compounds from medicinal plants is important for the analysis of synthesis pathways. A recently established strategy in synthetic biology [46][47][48][49] is to use microorganisms such as E. coli to synthesize active ingredients found in medicinal plants, which will provide a new effective strategy and research approach for the sustainable utilisation of medicinal plant resources.

Accession Number
The encoded nucleotide sequence can be found in the GenBank/NCBI data libraries. The number of TwFPS1 is KM058711 and the number of TwFPS2 is KM058712.