β‐Amyrin synthase from Conyza blinii expressed in Saccharomyces cerevisiae

Conyza blinii H.Lév. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane‐type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from β‐amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a β‐amyrin synthase, which mediates cyclization of 2,3‐oxidosqualene to yield β‐amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative β‐amyrin synthase, named CbβAS. The open reading frame of CbβAS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of β‐amyrin synthases. Transcription of CbβAS was upregulated 4–24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of CbβAS in Saccharomyces cerevisiae successfully yielded β‐amyrin. The chemical structures and concentrations of β‐amyrin were confirmed by GC‐MS/MS. The target yeast ultimately produced 4.432 mg·L−1 β‐amyrin. Thus, CbβAS is an OSC involved in conyzasaponin biosynthesis.

Conyza blinii H.L ev. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane-type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from b-amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a b-amyrin synthase, which mediates cyclization of 2,3-oxidosqualene to yield b-amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative b-amyrin synthase, named CbbAS. The open reading frame of CbbAS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of b-amyrin synthases. Transcription of CbbAS was upregulated 4-24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of CbbAS in Saccharomyces cerevisiae successfully yielded bamyrin. The chemical structures and concentrations of b-amyrin were confirmed by GC-MS/MS. The target yeast ultimately produced 4.432 mgÁL À1 b-amyrin. Thus, CbbAS is an OSC involved in conyzasaponin biosynthesis.
Conyza blinii H.L ev. is a medicinal herb distributed in southwestern China (Sichuan, Yunnan, and Guizhou provinces). It is well known for its treatment of bronchitis cough and inflammatory diseases. The entirety of the plant can be medicinally prepared and the highest accumulate of its secondary metabolites are conyzasaponins (3.0% w/w, of dry weight). Seventeen conyzasaponins have been isolated from the ethanol extract of C. blinii, of which all are oleanane-type saponins [1][2][3].
The current studies suggest that the synthesis of saponins is divided into four stages: first, the biosynthesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate; second, the biosynthesis of 2, 3-oxidosqualene; third, the biosynthesis of the basic backbone; fourth, the modification of the backbone ring. The third step is a branch. This step is catalyzed by oxidosqualene cyclases (OSCs) and resulted in multiple saponins backbones, including oleanane type, lupeol type, ursane type. Many OSCs have been reported to have multifunctional activities that can biosynthesize more than one saponins backbone [4-6]. However, one of the OSCs, b-amyrin synthase, controls flux toward the oleanane-type backbone (b-amyrin).
b-Amyrin synthase (bAS) has been isolated and characterized from many high plants with abundant oleanane-type saponins. Jin et al.
[7] isolated a Polygala tenuifolia Willd. bAS (PtBS) that contained a 2289-bp reading frame. Expression of PtBS in the yeast led to the production of b-amyrin as the sole product. The bAS from Artemisia annua expressed in Saccharomyces cerevisiae with manipulation of 3hydroxyl-3-methylglutaryl-CoA (HMG-CoA) reductase and lanosterol synthase produced levels of 6 mgÁL À1 culture of b-amyrin [8]. Huang et al. [9] transformed Panax japonicus bAS into rice to produce 'ginseng rice', which was capable of producing oleanane-type sapogenin.
Saccharomyces cerevisiae was widely used as an excellent host for the production of medicinal terpenes because of its mevalonate pathway and safety. Paddon et al. [10] have semisynthesized artemisinin in S. cerevisiae. The production of artemisinic acid, a precursor of artemisinin, reached a level of 25 gÁL À1 . This technology may increase antimalarial treatments in the developing world. Engels et al. [11] produced 8.7 AE 0.85 mgÁL À1 taxadiene by using coexpression of codon-optimized taxadiene synthase, truncated HMG-CoA reductase, the UPC2-1 transcription factor gene, and geranylgeranyl diphosphate synthase in S. cerevisiae. Furthermore, Han et al. [12] combined biosynthesis of protopanaxadiol in S. cerevisiae via coexpression of dammarenediol synthase (DS) and cytochrome P450 monooxygenase. After 2-day induction, the engineering yeast yielded 17.32 lgÁg À1 (FW) protopanaxadiol. In this study, we express a b-amyrin synthase gene of C. blinii in S. cerevisiae to produce b-amyrin. The putative biosynthesis pathway for b-amyrin in native yeast is shown in Fig. 1.
Here, we cloned and characterized CbbAS, a b-amyrin synthase that catalyzes the cyclization of oxidosqualene in the biosynthesis of conyzasaponins. Ectopic expression of CbbAS in INVSc1 yeast successfully yielded b-amyrin. The results confirm that CbbAS is a b-amyrin synthase.

Plant material
Conyza blinii used for gene cloning were collected in 2014 from Panzhihua, Sichuan, China. C. blinii multiple shoots (differentiated by our laboratory) were induced in 1/2 MS culture medium, which containing 0.1 mgÁL À1 1-naphthylacetic acid to obtain aseptic seedling. Seedlings were grown with light and constant temperature at 24°C. Two months later, plants were treated with either the 100 lmolÁL À1 methyl jasmonate (MeJA) or the control ethanol by  spraying. Leaves were collected at 0, 2, 4, 8, 12, and 24 h after treatment and then stored at À80°C.

Cloning of CbbAS
Ten OSC genes were discovered from the C. blinii transcriptome annotation library [13]. The phylogenetic analysis was used to select the bAS gene. OSC protein sequences including bAS, DS, CAS, and LUS were retrieved from NCBI. The sequence alignments were performed using CLUSTALW program (http://clustalw.ddbj.nig.ac.jp). The MEGA 5.05 software [14] was used to build the phylogenetic tree with neighbor-joining method and 1000 bootstrap replications.
According to the selected sequence, specific primers BAS1 and BAS2 (Table 1) were designed. The 50 lL reaction system included 25 lL PrimeSTAR Max DNA Polymerase Premix (29) (TaKaRa, Kyoto, Japan), 10 pmol BAS1, 10 pmol BAS2, 100 ng cDNA, and ddH 2 O. According to the introduction of Max DNA Polymerase, the three-step PCR program was used to amplify the CbbAS gene. PCR products were then purified (TaKaRa MiniB-EST Agarose Gel DNA Extraction Kit Ver.4.0) and sequenced (Invitrogen Trading, Shanghai, China). Afterward, the nucleotide sequence and the deduced amino acid sequence were characterized by bioinformatics tools.

Quantitative RT-PCR analysis
Methyl jasmonate-treated leaves were used as samples for qRT-PCR analysis. The same amount of RNA from samples was used for reverse transcription into the singlestranded cDNA according to the PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa). The housekeeping gene previously published, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; GenBank ID: KF027475) [15], was used as the internal control. The qRT-PCR primers are in Table 1. A 25 lL reaction system with SYBR Premix Ex Taq II (TaKaRa) was used for quantification on a CFX96 Real-Time PCR Instrument (Bio-Rad, Hercules, CA, USA). The 2 ÀDDCT method [16] was used to calculate differences among gene expression. The experiments were replicated four times.

Expression of CbbAS in Saccharomyces cerevisiae INVSc1
The expression vector pYES2/NT B (provided by Zongyun Feng, Sichuan Agricultural University) and the S. cerevisiae strain INVSc1 (provided by Zongyun Feng, Sichuan Agricultural University) were used to examine CbbAS function. The open reading frame of CbbAS was amplified with primers BAS3 and BAS4 (Table 1). The PCR products were inserted into the NotI and XbaI restriction sites of the pYES2/NT B vector to construct pYES-CbbAS recombinant plasmid. The pYES-CbbAS plasmid was transformed into INVSc1 by electroporation (1.5 kV, 3 ms, 2.5 lF, 200 Ω) [17]. After 3 days of growth, single clones of INVSc1 containing pYES-CbbAS or pYES2/NT B were inoculated in 15 mL of SC minimal media lacking uracil (SC-U) medium containing 2% glucose. Precultures were grown overnight at 30°C with shaking at 200 r.p.m. To induce gene expression, the precultures were washed and inoculated into 50 mL of induction medium (SC-U medium containing 2% galactose) with a starting optical density of 0.4. The cultures were further incubated for 60 h to induce CbbAS expression.

Metabolite extraction for GC-MS/MS analysis
Extraction of metabolites followed the method previously described by Kirby et al.
[8] with some modifications. 50 mL of induction cells was centrifuged at 2739 g for 5 min to obtain a cell pellet. The cells were resuspended in 10 mL 20% KOH/50% EtOH (W/V), and the supernatant was discarded. The mixture was boiled for 10 min. After cooling, metabolites were extracted twice using hexane (15 mL  helium was 1.5 mLÁmin À1 . The column temperature program was performed using the same method described by Seki et al. [18]. For the quantification of b-amyrin, the secondary MS was used. The ion m/z 189 and m/z 203 were designated as quantitative ion and qualitative ion, respectively. The standard b-amyrin was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Phylogenetic analysis of OSCs and cloning of CbbAS
According to the transcriptome analysis, ten tags corresponded to OSC genes (Table 2). Annotation results showed that six tags were predicted to be b-amyrin synthase. To further determine the bAS gene, we performed the phylogenetic analysis between these tags and OSCs from other plants. The results revealed that tag Cb18076 was homologous to b-amyrin synthase from Aster sedifolius, which has been reported to only produce b-amyrin in yeast [19] (Fig. 2). The tags Cb54088, Cb70382, Cb827, and Cb874 were phylogenetically related to Ricinus communis LUS [20]. Cb72002 was similar to LUS from Kalanchoe daigremontiana, which produces lupeol and b-amyrin in a ratio of 13 : 1 [21]. In addition, another four tags Cb34533, Cb35585, Cb38895, and Cb46070 were homologous to DS from the Panax species, which is involved in the ginsenoside biosynthetic pathway [22,23]. Therefore, we selected the Cb18076 tag as a bamyrin synthase gene.

Expression of CbbAS gene following treatment by MeJA
Methyl jasmonate is used as an exogenous elicitor that can enhance the content of secondary metabolites such as saponins [26,27] and the transcription levels of genes involved in saponins biosynthesis [12,28]. Therefore, to identify whether CbbAS gene involved in conyzasaponins pathway, we investigated expression of CbbAS after elicitation by MeJA using qRT-PCR (Fig. 4). The transcript level of CbbAS at 24 h was 2.8-fold higher than at 0 h. Furthermore, MeJA-treated CbbAS transcript levels were six times higher than those of EtOH-treated CbbAS at 24 h. CbbAS expression was significantly upregulated by MeJA. The results preliminarily confirm that CbbAS is involved in conyzasaponins biosynthetic pathway.

Functional characterization of CbbAS
To detect the activity of CbbAS, the recombinant plasmid pYES-CbbAS was constructed. The pYES-CbbAS plasmid was then expressed in INVSc1 under the control of GAL1 promoter. To verify the function of CbbAS, the yeast extracts were examined by GC-MS. The GC retention time showed that at 19.5 min, pYES-CbbAS strain and standard b-amyrin appeared a peak, while the pYES strain did not (Fig. 5). The MS spectrum then confirmed that the peak detected in pYES-CbbAS transgenic strain was b-amyrin (Fig. 6).
GC-MS/MS is an advanced detection system that provides high sensitivity for achieving very low detection thresholds. The precursor ion 203 m/z and daughter ion 105.1 m/z were used to detect b-amyrin. Simultaneously precursor ion 189 m/z and daughter ion 119.1 m/z were used for quantification analysis (Fig. 7). The results showed that the pYES-CbbAS yeast yielded 4.432 mgÁL À1 b-amyrin after induction by galactose for 60 h in 50 mL medium.

Discussion
Currently, Chinese herbal medicine has become increasingly popular due to their abundant primary and secondary metabolites. These metabolites can be used to treat many diseases and have little side effects. However, the natural plants yield low contents of metabolites and require a long time to grow, which hampered the applications of the pharmacologically active compounds. Therefore, synthetic biology is an effective way to solve this contradiction [29]. For example, the popular anticancer drug taxol [30][31][32] and the antimalarial drug artemisinin [33][34][35] are both successfully biosynthesized by microorganisms. The major pharmacological compound of C. blinii to be used in Chinese traditional medicine is conyzasaponins. However, there is a lack of information on the biosynthetic pathways of a majority of pharmacologically active compounds in C. blinii, especially conyzasaponins. In this study, we investigated this specific pathway by cloning and characterizing a bAS gene involved in it. To our knowledge, this is first study on conyzasaponins pathway.
Previous reports indicated that the DCTAE motif is highly conserved in eukaryotic OSCs. This motif is responsible for initiating the polycyclization reaction of squalene epoxide [36]. The acidic carboxyl residue Asp in this motif releases protons to attack on the terminal epoxide ring of 1, which triggers a cascade of the ring-forming reaction. The sequence analysis results of CbbAS suggest that it is an OSC. Besides, the MWCYCR is a characteristic motif of b-amyrin synthase [37]. In this motif, the Trp residue controls b-amyrin formation by stabilization of oleanyl cation and the Tyr residue is involved in producing pentacyclic triterpenes. Therefore, the MWCYCR motif in CbbAS (Fig. 3) indicated that it is a special OSC, b-amyrin synthase.
The preliminary functional verification of CbbAS is carried out by qRT-PCR after the treatment of MeJA. Hayashi et al. [26] previously described that MeJA treatment can upregulate bAS mRNA levels and enhance the accumulation of soyasaponin (oleananetype triterpene saponin). Another report described by Liu et al. [38] also indicated that MeJA treatment upregulated the Gentiana straminea bAS expression levels and oleanolic acid accumulations. Conclusively, MeJA treatment can stimulate the accumulation of oleanane-type saponins or sapogenins and the expression level of bAS gene. Therefore, if CbbAS is involved in the conyzasaponins pathway, its expression level will be upregulated by MeJA treatment. The qRT-PCR results confirmed this conjecture that CbbAS is an enzyme involved in conyzasaponins formation.
We expressed CbbAS in S. cerevisiae to determine its function. GC-MS/MS analysis showed that genetically engineered yeast with CbbAS produced 4.432 mgÁL À1 b-amyrin. Currently, the highest b-amyrin titer achieved by microbial fermentation is 107.0 mgÁL À1 [39]. And the others indicated that by introducing bAS of A. annua [8] and Pisum sativum [40], the engineered S. cerevisiae produced 6 and 3.93 mgÁL À1 b-amyrin, respectively. The b-amyrin yield of CbbAS transgenic yeast compared with earlier is not high. Modification of promoter and coexpression of genes involved in b-amyrin pathway can be solutions to increase b-amyrin contents.
In addition, further research on cytochrome P450 genes and glycosyltransferase genes involved in the conyzasaponins biosynthetic pathway is required to expand upon our results to utilize synthetic biology to produce conyzasaponins.

Author contributions
HC and QW conceived and designed research. RS wrote the manuscript. SL provided C. blinii samples. ZZT and CLL contributed reagents or analytical tools. TRZ and TW performed the experiments. All authors read and approved the manuscript.