Molecular Cloning, Expression, Purification, and Functional Characterization of Dammarenediol Synthase from Panax ginseng

The objective of this study is to clone and charecterize the expression of dammarenediol synthase gene and then to determine the relationship between the expression of dammarenediol synthase gene that is involved in the ginsenoside biosynthetic pathway and the ginsenoside content. A cDNA phage library was constructed from a five-year-old ginseng root. The cDNA library was screened for the dammarenediol synthase gene by using its specific primers. It was further cloned and expressed in pET-30a vector. The recombinant plasmid pET-30a-DS was expressed in Rosetta E. coli. The recombinant DS protein was purified by affinity chromatography. The production of dammarenediol was detected by liquid chromatography-mass spectrometry (LC-MS). Results showed that dammarenediol synthase gene was cloned from the cDNA library and was expressed in Rosetta E. coli and the SDS-PAGE analysis showed the presence of purified DS protein. LS-MS showed the activity of DS protein, as the protein content increases the dammarenediol increases. Our results indicate that the recombinant dammarenediol synthase protein could increase the production of dammarenediol and the expression of DS played a vital role in the biosynthesis of ginsenosides in P. ginseng.


Introduction
Medicinal Panax ginseng C. A. Meyer belongs to Panax genus under Araliaceae family. As a traditional medicinal plant, it has been in use for thousands of years. Modern medicinal chemistry and pharmacology studies have con�rmed that various compositions of ginseng have a role in enhancing the immunity, neural protection, antiaging, antidepressant, antitumor, and other aspects [1][2][3][4][5][6] and also have good application prospect with considerable economic value.
In spite of these prospects, the difficulty of ginsenosides supplies in pure form at larger quantities has prevented in usage of them as clinical drugs. And ginsengs' quality and growth was severely restricted by its low growth rate, due to the impact of climate, geography, and cultivation conditions [7,8]. Isolation and puri�cation of each ginsenoside involves longer and complicated procedures. Alternatively, the expression of the genes for biosynthetic enzymes in heterologous host organisms, hopefully in fast growing microbes such as Escherichia coli, can lead to ginsenoside production in larger quantities. Genetic engineering was expected to become the most promising way to produce medicinal secondary metabolites of low content, and cloning of the relevant genes of ginsenoside biosynthesis is highly expected.
Ginsenoside belongs to triterpenoid, which could be classi�ed into dammarane type and oleanane type [9,10] according to the basic structure of aglycone, and most of the ginsenoside were of dammarane type. At present, the biosynthetic process can be divided into 3 processes: (1) synthesis of 3 isoprene pyrophosphate and two methyl allyl pyrophosphate; (2) synthesis of important intermediate precursors of 2,3-squalene oxide; and (3) cyclization of 2,3 oxidation of squalene [10,11]. e cyclization of oxidosqualene is a biosynthetic branching which directs not only for phytosterols and triterpenes, but also for dammarane and oleanane type ginsenosides. In previous study, the �rst committed step in ginsenoside synthesis is the cyclization of 2, 3-oxidosqualene to dammarenediol II, catalyzed by the DS of the oxidosqualene cyclase group [12]. It means that the oxidation of squalene was �rst synthesized into dammarenediol under the action of the dammarenediol synthase (DS), and then generated to be various types of dammarane saponins through a series of hydroxylation and glycosylation reactions [10].
In the recent years, many researches focus on developing effective biological systems for the ginsenoside production by using recombinant technology. Recently, a large number of pentacyclic triterpene synthases have been cloned and functionally characterized from various plant species [13][14][15]. However, the expression of the genes for biosynthetic enzymes in heterologous host organisms, such as Escherichia coli, has not been obtained. And whether there was a certain correlation of the DS activity and saponin content in vitro was still in need of experimental veri�cation. In our present study, we attempted to clone and express the dammarenediol synthase in prokaryotic system, the �rst committed enzyme for the biosynthesis of genuine sapogenin of dammarane type ginsenosides. It was shown that endogenous dammarenediol synthase protein was obtained by prokaryotic expression and analyzed the correlation of the DS activity and the accumulation of dammarenediol by LC-MS.

Experimental Material.
One-, three-, four-, and �veyear-old ginseng were taken from the Medicinal Botanical Garden, Jilin Agricultural University, and the �brous roots of ginsengs root tissues were used in experimental studies.

Reagents.
Trizol reagent was purchased from Invitrogen, Smart TM cDNA Library Construction kit was purchased from Clontech company, USA, GigapackIII Gold packing extract kit was purchased from the Stratagene company, Reverse transcriptase, Ex Taq DNA polymerase, pMD18-T vector, DL-2000 Marker were purchased from TaKaRa company, Real Master Mix was purchased from the Changchun company, Ni-agarose protein puri�cation kit was purchased from the Beijing Kangwei Century Company, Squalene was purchased from the Changchun DingGuo Biotechnology Co., Ltd., Dammarenediol Standard was purchased from the YunNan XiLi Biotechnology Co., Ltd., Squalene epoxidase enzyme was prepared in the laboratory, and E. coli Strain Rosetta and pET-30a Expression Vector were from our laboratory.

Construction and Detection cDNA Phage Library of
Ginseng Root Tissue. Total RNA from different year-old ginseng root tissues was extracted by using Trizol. cDNA was synthesized by using the total RNA as template, with AMV reverse transcriptase and oligo-dT primers. full-length cDNA library of �ve-year-old P. ginseng was constructed by using SmartTM cDNA Library Construction kit. selection of the plaque, the detection of the size of the inserted fragment, and calculation of the reorganization rate was performed by PCR (Sense: CCATTGTGTTGGTACC CGG, Antisense: ATACGACTCACTATAG GGCGAATT).

DS Gene Cloning and Sequence
Analysis. DS gene conservative primers (Sense: ATTAAGAATGTGGAAGCA-GAAGG, Antisense: CTTAAATTTTGAGCTGCTGGTG) were designed based on the related sequences of GenBank and used to screen the DS gene from the cDNA library. e targeted PCR fragment was recovered aer agarose gel electrophoresis and cloned into the pMD18-T vector. To identify the correct recombinant plasmid, it was sequenced (Shanghai Sheng Gong biological engineering, Shanghai), and the DS gene DNA sequences were submitted to GenBank (accession number: JN596111). e name of the correct construct is pMD-18T-DS.

2.�. Construction and �denti�cation of pET�3�a�DS. pET-30a
and DS gene fragments from pMD-18T-DS plasmid were digested by NcoI and NotI, and ligated with T4DNA ligase at 16 ∘ C, then transformed into Rosetta E. coli competent cell, and cultured overnight at 37 ∘ C. Positive clones were selected by double digestion and to identify the correct insert, it was sent for sequencing. e positive clone was named as pET-30a-DS.

2.�. �nduced Expression of Recombinant and Puri�cation of Fusion
Protein. E. coli cells harboring an expression construct were grown in 10 mL LB broth containing 30 g/mL kanamycin plus 34 g/mL chloramphenicol for Rosetta at 37 ∘ C overnight with constant shaking. e recombinant bacteria were inoculated in LB medium and cultured at 37 ∘ C to OD600 of 0.4 to 0.6, with different IPTG concentration (�nal concentrations of 0.1, 0.2, 0.6, 0.8, and 1.0 mmol/L) to induce expression, and induced for 4 h under the determined optimal concentration of IPTG. e bacterial lysate was collected 4 h later and detected by SDS-PAGE electrophoresis, with the noninduced recombinant Rosetta as the control.
Overnight cultures of bacterial cells were diluted 50-fold into 1 L LB broth, supplemented with appropriate antibiotics, cultured as above until an OD600 of 0.4-0.6 was reached, and then induced to express the recombinant proteins for 4 h by adding isopropyl -D-galactopyranoside (IPTG) to a �nal concentration of 0.6 mM. e cells were sonicated for 2 min, and the precipitate was dissolved by Soluble Binding Buffer (20 mmol/L Tris-HCl, 10 mmol/L, imidazole, 0.5 mol/L NaCl) solution. e precipitate obtained from 12000 g centrifugation for 20 min is the inclusion body. Inclusion body was dissolved in ice water bath for 1 h and centrifuged at 10000 g for 20 min, and supernatant, were �ltered by 0.45 m membrane �ltration. Aer the balanced Niagarose affinity chromatography, the His tagged recombinant proteins were solubilized by Soluble Binding Buffer solvent and puri�ed by Ni-NTA agarose chromatography� �nally the elution peaks were collected according to 280 nm ultraviolet absorption value.

Detection of the Activity of the Recombinant Protein.
In the present study, SEQ enzyme was expressed and puri-�ed using prokaryotic expression vector, which had been completed previously. e process of the catalytic reaction of DS enzyme involves two stages. Firstly, squalene was selected as a substrate. Under the same amount of substrate, squalene was catalyzed with the same SQE enzyme amount to produce the intermediate product of 2,3-oxidosqualene in different reactions systems. Subsequently, 2,3-oxidosqualene was further catalyzed with different DS enzyme amounts to produce dammarandiol. Aer the completion of enzymatic reaction, the enzyme was inactivated by heating at 100 ∘ C. Based on the characteristics of high solubility of the sapogenin dammarandiol in ethanol and low solubility in acetone, following extraction by ethanol, acetone was added into ethanol to separate out dammarandiol, which was then detected by LC-MS technique.
e experiment could be divided into �ve DS enzyme reaction systems. Substrate squalene (20 L) were added into a centrifugal tube which have Tris-HCl buffer (pH 6.0) in it, and then added the DS enzyme solution (0.65 g/mL) 5 L, 10 L, 15 L, 20 L, 25 L respectively, and added SQE enzyme solution (0.35 g/mL) 20 L in each of the centrifugal tube and they reacted at 37 ∘ C for 1 h, and then the temperature was raised to 100 ∘ C. Aer 10 min, the enzymatic reaction was stopped. e dammarnediol production was detected by liquid chromatography-tandem mass spectrometry.
Chromatographic column condition: Zorbax Extend-C18 column, 5 m particle size, 150 × 4.6 mmL⋅D, American Agilent company; mobile phase: water, acetonitrile, formic acid; �ow: 0.8 mL/min; column temperature: room temperature; sample size: 25 L. Mass spectrometric conditions: electrospray ionization source (ESI); ion injection voltage: 5500 V; temperature: 550 ∘ C; source gas 1 (GS1, N2) pressure: 70 psi; gas 2 (GS2, N2) pressure: 60 psi; positive ion mode monitoring: scanning mode for the positive ion monitoring; DP voltage: 70 V; EP voltage: 10 V; CXP: 4.0 V; disintegration energy: 40 V. e ion pair for the qualitative analysis is 391/93.1; the ion pair for quantitative analysis is 391/149. Products of different enzyme reaction systems are injected into the injection hole to observe the changes of elution peak area. e standard concentration of dammarenediol was 1 mg/L and the generation amount of dammarenediol was calculated by comparing with the peak area ratio of the standard.

Synthesis of Double-Strand cDNA and Quality Detection of Phage Library.
Aer detecting ultraviolet spectrophotometry, the A260/A280 of total RNA is 1.96. e electrophoresis results showed that the double-stranded cDNA is located between 0.3 and 2.5 kb, presenting an elongated waterfalllike band, which agreed well with the distribution of plant tissue DNA. According to the number of bacterial colony and bacteria liquid PCR identi�cation results, the recombination rate was 95.17%. e capacity of the original library is 8.38 × 10 6 pfu/mL −1 .

DS Gene Cloning and Sequence Analysis.
Aer electrophoresis, the PCR ampli�cation product of DS gene obtained a speci�c band about 2310 bp, which agreed well with the expected results. pMD-18T-DS plasmid was digested

Identi�cation of Recombinant Expression �ector and Induced Expression of Recombinant.
Speci�c band with the size about 2310 bp, consistent with expectation, was obtained by the application of NcoI and NotI double digestion pET-30a-DS. e results found that DS gene was inserted into pET-30a vector, and the recombinant plasmid pET-30a-DS was constructed. e recombinant plasmid pET-30a-DS was transformed into Rosetta E. coli to be induced for gene expression. e expressed product was identi�ed by SDS-PAGE, and speci�c protein bands at about 89 kDa were found, which matched the molecular weight size of the predicted His tagged recombinant protein (Figure 1). �hen the IPTG �nal concentration reached to 0.6 mmol/L, protein expression is highest, and the difference of protein expression was not signi�cant at different time intervals (1, 2, 3, 4, 5, 6, and 7 h). Puri�cation of recombinant proteins by SDS-PAGE analysis showed that there were protein bands at about 89 kDa, which had the same molecular weight with fusion protein pET-30a-DS, and highly purity (Figure 2).

Biological Activity Detection of Recombinant Proteins.
Dammarenediol was identi�ed by the extraction ion chromatography of LC-MS and the retention time of peaks was 12.0 min. e LC-MS detection showed that under the condition of similar amounts of the substrate squalene, with the increase in the amount of DS enzyme, compared with the dammarandiol standard peak area, the generation amount of damarindiol gradually increased. e experimental results indicated that the recombinant proteins expressed in E. coli had the activity, and it could generate squalene into dammarenediol. At the same time, a close correlation of dammarenediol synthetase and the generation amount of ginsenoside were found (Figure 3 and Table 1).

Discussion
e cDNA library constructed by SMART technology ensured the integrity of cDNA library information [16,17]. In addition, such kind of cDNA library construction of the �veyear-old ginseng root was rarely reported. SMART technology can improve expression probability and recombination efficiency of the insertion region gene, so that the lowabundance genes in the library were enriched, which was in line with the low abundance of the gene expression in ginseng root separation. e original storage capacity of the �ve-yearold ginseng root cDNA library constructed in this study is greater than 10 6 pfu/mL −1 , inserted fragments were mainly concentrated between 500 and 2500 bp, which has reached the standard value of library and has laid the foundation for screening the key genes in the ginsenoside biosynthetic pathway. e �rst committed step in ginsenoside synthesis is the cyclization of 2,3-oxidosqualene to dammarenediol, catalyzed by the DS of the oxidosqualene cyclase (OSC) group.
Dammarenediol synthase is the most important enzyme in the biosynthetic pathway, which had a great relevance to the ginsenoside content. Additionally, the content was extremely low in ginseng root [18]. Elder ginseng from exuberant growth and metabolism season, with relative high expression of all types of genes were, chosen in this study. And the DS gene of open reading frame encoding 769 amino acids was achieved by the constructed cDNA library, thus a reference for further understanding of the DS gene characteristics and functions was provided. Cloning of dammarenediol synthase cDNA in this study has provided a signi�cant step for the production of dammarane type ginsenosides by heterologous expression systems, since subsequent hydroxylation(s) and glycosylation(s) lead to a varieties of ginsenoside structures.
Recent years research on obtaining the active key enzyme protein by prokaryotic expression vector has rarely been reported. Morita et al. [15] have observed the production of dammarenediol II by the expression of PNA in an ERG7-de�cient yeast-mutant (GIL77), but its regulatory mechanism is unclear. We inserted the ORF of DS cDNA in the pET-30a   [19,20]. Based on the above research, the dammarenediol generation amount in different amounts of DS enzyme reaction was analyzed by LC-MS technology. e LC/MS method had high speci�city, sensitivity, accuracy, and other advantages, with the detection range of 2.0 ng⋅mL −1 [21,22]. LC-MS analysis revealed that under the similar amount of the substrate squalene, with a corresponding decrease in the amount of DS enzyme, the generation amount of dammarenediol progressively increased, which illustrated that the recombinant protein expressed in E. coli had activity. e experiments further proved evidence to the close correlation of dammarenediol synthetase and saponin production in vitro, but the speci�c catalytic activity was not clear yet. Han et al. [23] studied on DS ginseng gene by RNA interference and found that RNA interference of DS led to a reduction of ginsenoside production to 84.5% in P. ginseng roots, which was very similar to the �ndings of this study. ese results indicated that the overexpression of DS will be useful for the enhanced production of pharmacologically-important ginsenosides from P. ginseng or other plant species by genetic transformation.
In conclusion, this study explained further that DS gene plays a key role in ginsenoside biosynthesis pathway, ginsenoside accumulation was inseparable from the key genes expression levels changes, to a certain extent it could determine the trend of triterpene saponin biosynthesis, and DS was one of the key enzymes in control of ginsenoside synthesis. erefore, to improve the expression of ginseng DS gene by the genetic engineering technology was most likely an effective means to increase the amount of ginsenosides. erefore, our study provides a basic experimental data available for the study on DS genes and also provides a theoretical reference for the postdepth study of the DS gene function and regulatory mechanism on the biosynthetic pathway of ginsenoside.
�on��ct of �nterests e authors declare that they have no con�ict of interests.