Echinacea purpurea SCPL family acyltransferase EpCYS gene and application thereof

Abstract

The invention discloses an echinacea purpurea SCPL family acyltransferase EpCYS gene, the amino acid sequence coded by the EpCYS gene is shown as SEQ ID NO.2, the gene is subjected to heterologous expression in yeast or nicotiana benthamiana, and after an expression protein catalytic substrate is used in vitro, a catalytic product is identified; the result shows that the EpCYS protease can be used for catalyzing the generation of Cynarine; more resources are provided for application of plant acyltransferase in synthetic biology, biosynthesis production of Cynarine as a pharmaceutical ingredient is promoted, and a new contribution is made to human health.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to an acyltransferase EpCYS gene of Echinacea purpurea SCPL family, and also to an application of the gene. Background Art

[0002] Cynarine (1,3-Dicaffeoylquinic acid, Chinese name cynarin acid) is a caffeic acid derivative first identified from the perennial herb artichoke. It has multiple biological activities such as protecting the liver, regulating blood sugar, antibacterial and anti-inflammatory, and inhibiting cancer cells. It also has therapeutic effects on certain chronic diseases and cardiovascular diseases. Echinacea is a world-renowned medicinal plant. Commonly used source plants include Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida, which also contain a large amount of cynarine in their active ingredients. The biosynthesis of caffeic acid derivatives in plants originates from the phenylpropanoid metabolic pathway. Phenylalanine is converted to p-coumaric acid by phenylalanine ammonialyase (PAL) and cinnamate-4-hydroxylase (C4H). Coenzyme A is linked to p-coumaric acid by 4-coumarate:CoA ligase (4CL) to produce p-coumaroyl-CoA. p-Coumaroyl-CoA can enter the caffeic acid derivative synthesis pathway catalyzed by hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT) and p-coumaroyl-3'-hydroxylase (C3'H).In recent years, researchers have characterized a variety of enzymes that synthesize caffeic acid derivatives in Echinacea, including hydroxycinnamoyl-CoA: tartaric acidhydroxycinnamoyl transferase (HTT) for synthesizing caffeoyl-CoA, hydroxycinnamoyl-Coenzyme A: quinate hydroxycinnamoyl transferase (HQT) for synthesizing chlorogenic acid, hydroxycinnamoyl-Coenzyme A: shikimate / quinate hydroxycinnamoyl transferase (HCT) for synthesizing caffeoylmalate, and hydroxycinnamoyl-Coenzyme A: malic acid hydroxycinnamoyl transferase (HCT) for synthesizing chlorogenic acid. transferase (HMT), chicoric acid synthase (EpCAS) for synthesizing chicoric acid, and chlorogenic acid condensing enzyme (EpCCE1 / 2) for synthesizing 3,5-dicaffeoylquinic acid. Previous studies have shown that the plant BAHD acyltransferase family, SCPL acyltransferase family, and GDSL enzymes may provide acyl transfer functions, and all have been reported to catalyze the synthesis of dicaffeoylquinic acid.

[0003] In recent years, with the development of science and technology, the analysis of the biosynthetic pathway of natural products has ushered in new development opportunities. The comprehensive use of multiple candidate gene screening methods such as co-expression analysis, phylogenetic analysis, activity tracking, and gene cluster analysis is conducive to obtaining candidate genes efficiently and accurately. In this study, Echinacea purpurea, a plant of the genus Echinacea in the Asteraceae family that contains Cynarine, was used as the research object, and an acyltransferase EpCYS of the SCPL family was identified. This enzyme can synthesize Cynarine using Chlorogenic acid as a substrate. According to the gene function, it is named EpCYS (Cynarinesynthase). This gene is helpful for the production of Cynarine using metabolic engineering technology and the full utilization of Cynarine and its derivatives. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide a purple coneflower SCPL family acyltransferase EpCYS gene, a second object of the present invention is to provide a yeast expression vector containing the purple coneflower SCPL family acyltransferase EpCYS gene; a third object of the present invention is to provide a recombinant plant expression vector containing the purple coneflower SCPL family acyltransferase EpCYS gene; a fourth object of the present invention is to provide the use of the purple coneflower SCPL family acyltransferase EpCYS in catalyzing chlorogenic acid to produce cynarine.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. An acyltransferase EpCYS gene of the SCPL family of Echinacea purpurea, wherein the amino acid sequence encoded by the EpCYS gene is shown in SEQ ID NO.2.

[0007] Preferably, in the present invention, the nucleotide sequence of the EpCYS gene is as shown in SEQ ID NO.1.

[0008] 2. A yeast expression vector containing the Echinacea SCPL family acyltransferase EpCYS gene.

[0009] Preferably, the yeast expression vector is obtained by ligating the sequence shown in SEQ ID NO. 1 into the Sal I and Xho I restriction sites of the pESC-HIS vector.

[0010] 3. A recombinant plant expression vector containing the Echinacea SCPL family acyltransferase EpCYS gene.

[0011] Preferably, the nucleotide sequence shown in SEQ ID NO.1 is obtained by ligating into pDGB3_α according to the GoldenBraid principle.

[0012] 4. Application of Echinacea SCPL family acyltransferase EpCYS in catalyzing chlorogenic acid to produce cynarine.

[0013] The beneficial effects of the present invention are as follows: the present invention clones an SCPL family acyltransferase that can catalyze chlorogenic acid to produce cynarine from purple coneflower, and names it EpCYS. The gene is 1389 bp in length and encodes 462 amino acids. Further, pESC-HIS is used as a yeast protein expression vector, and Saccharomyces cerevisiae BY4741 is used as a host cell to realize heterologous expression of the protease, and its catalytic product is identified after using the expressed protein to catalyze the substrate in vitro; 2×35S:EpCYS constructed according to the GoldenBraid principle is used as a stable expression vector of Nicotiana benthamiana, and Agrobacterium GV3101 is used as a host cell to realize heterologous expression of the protease in Nicotiana benthamiana, and its catalytic product is separated and identified to show that EpCYS protease can catalyze the production of cynarine. That is, the present invention provides an enzyme of the SCPL family with acyl transfer function, and verifies its role in the biosynthesis of cynarine, which provides more resources for the application of plant acyltransferases in synthetic biology, promotes the biosynthetic production of cynarine as a drug ingredient, and will make new contributions to human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0015] Figure 1 The results of LC-MS analysis of cynarine formation catalyzed by yeast-expressed recombinant protein and standard (chromatogram; XIC);

[0016] Figure 2 The results of LC-MS analysis of cynarine formation catalyzed by yeast-expressed recombinant protein and standard products (mass spectrum);

[0017] Figure 3 The LC-MS analysis results of wild-type and transgenic tobacco compound Cynarine and standard substances (chromatogram; XIC);

[0018] Figure 4 This is the LC-MS analysis result (mass spectrum) of the transgenic tobacco compound Cynarine and the standard. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0020] Example 1: Cloning of the Echinacea purpurea EpCYS gene

[0021] (1) Extraction of total RNA from Echinacea purpurea genome

[0022] The total RNA of Echinacea purpurea was extracted using the Polysaccharide and Polyphenol Plant RNA Extraction Kit ( Plant Total RNA Isolation Kit), the specific steps are as follows: Take the purple coneflower seedlings and freeze them in liquid nitrogen and then grind them at low temperature. Take 50mg of the sample, add 500μL Buffer PRL preheated at 65℃, and immediately vortex and shake vigorously for 30-60sec. The lysate was placed in a 65℃ water bath for 5min, inverted 1-2 times during the period, and centrifuged at 13,400g for 10min. Transfer the supernatant to a new 1.5mL RNase-free centrifuge tube, add 0.5 times the volume of the supernatant with anhydrous ethanol, and immediately blow and mix. Transfer the above mixture to FastPure gDNA-Filter ColumnⅡ, centrifuge at 13,400g for 2min, and discard the filtrate. Put FastPure gDNA-Filter ColumnⅡ into a new Collection Tubes 2mL, add 500μL Buffer PRLPlus, centrifuge at 13,400g for 30sec, and collect the filtrate. Add 0.5 times the volume of the filtrate with anhydrous ethanol to the filtrate and immediately blow and mix. Transfer the above mixture to FastPure RNA Column Ⅳ, centrifuge at 13,400g for 2 minutes, and discard the filtrate. Add 700μL Buffer PRW1 to FastPure RNA Column Ⅳ, let it stand at room temperature for 1 minute, centrifuge at 13,400g for 30 seconds, and discard the filtrate. Add 500μL Buffer PRW2 to FastPure RNA Column Ⅳ, centrifuge at 13,400g for 30 seconds, and discard the filtrate. Repeat this step twice. Put the FastPure RNA Column Ⅳ adsorption column back into the collection tube, centrifuge at 13,400g for 2 minutes, and remove the residual Buffer PRW2 in FastPure RNA Column Ⅳ. Transfer FastPure RNA Column IV to a new RNase-free Collection Tubes 1.5mL centrifuge tube, add 30μL of RNase-free ddH2O to the center of the adsorption column, let it stand at room temperature for 2 minutes, and centrifuge at 13,400g for 1 minute to obtain RNA solution, which can be used directly in downstream experiments or stored at -85-65℃.

[0023] (2) Cloning of the Echinacea EpCYS gene

[0024] The total RNA of Echinacea purpurogena was reverse transcribed using the reverse transcription kit of TAKAR to obtain the first-strand cDNA. Primers were designed based on the nucleotide sequence information of EpCYS in the transcriptome data of the germination process of Echinacea purpurogena to amplify the complete coding region. The nucleotide sequence is shown in (SEQ ID NO.1), and the encoded amino acid sequence is shown in (SEQ ID NO.2). The amplification primers are as follows:

[0025] Upstream primer EpCYS-F: 5′-ATGGAGTGTGGATGGATGAC-3′ (SEQ ID NO. 3);

[0026] Downstream primer EpCYS-R: 5'-TTACATGACGCTTGAATAAGCTTCC-3' (SEQ ID NO. 4);

[0027] The first-strand cDNA obtained by reverse transcription was used as a template, and the upstream and downstream primers shown in (SEQ ID NO.3) (SEQ ID NO.4) were used as primer pairs. After PCR amplification, the resulting DNA was sequenced and verified by Sangon Biotech (Shanghai) Co., Ltd.

[0028] Example 2: Construction of yeast expression vector and engineered bacteria containing EpCYS gene

[0029] The EpCYS gene was amplified again using primer pairs pESC-EpCYS-F and pESC-EpCYS-R to obtain the EpCYS gene with homology arms of the pESC-HIS vector. The pESC-HIS vector was digested with Sal I and Xhol I and constructed into the plant expression vector pESC-HIS by homologous recombination.

[0030] The specific sequences of pESC-EpCYS-F and pESC-EpCYS-R are as follows:

[0031] pESC-EpCYS-F: 5'-ctatagggcccgggcgtcgacatggagtgtggatggatgac-3' (SEQ IDNO.5);

[0032] pESC-EpCYS-R: 5'-gcggtaccaagcttactcgagttaatgatggtgatggtgatgcatgacgcttgaataagc-3' (SEQ ID NO. 6);

[0033] E. coli DH5α was connected and transformed, positive clones were screened, and then plasmids were extracted for PCR detection and sequencing, and the correctness of the gene was confirmed by sequencing by Sangon Biotech (Shanghai) Co., Ltd. A recombinant expression vector containing the EpCYS gene was obtained and named pESC-EpCYS-HIS vector.

[0034] The pESC-EpCYS-HIS vector was transformed into Saccharomyces cerevisiae BY4741, and positive clones were screened to obtain an engineered bacterium containing the pESC-EpCYS-HIS vector, which was named BY4741-pESC-EpCYS-HIS. At the same time, the pESC-HIS vector was used to transform Saccharomyces cerevisiae BY4741 to obtain an engineered bacterium containing the BY4741-pESC-HIS vector, which was named BY4741-pESC-HIS.

[0035] Example 3: Protein expression and functional verification in yeast

[0036] The BY4741-pESC-EpCYS-HIS engineered bacteria and the control strain BY4741-pESC-HIS were inoculated into 5 mL of 2% glucose SD-His liquid medium and cultured overnight at 30°C and 200 rpm. The culture solution was added into 50 mL of 2% glucose SD-His liquid medium to make the OD 600 =0.2-0.3, 30℃, 200rpm, culture for 36h, transfer all cells to 2% galactose SD-His medium to induce protein expression. After 36h, centrifuge at 4℃, 1,000g for 5min to collect bacteria, discard supernatant. Resuspend cells in sterile water and centrifuge. Collect cell pellets, freeze in liquid nitrogen and store at -80℃.

[0037] The cells were treated with an equal volume of Ni 2+ -NTA lysis buffer was resuspended. An equal volume of acid-washed glass beads (425-600 μm) was added and the cells were broken using a grinder at 60 Hz, 60 sec, 5 times. The supernatant was collected by centrifugation at 4°C, 5,000 g for 10 min; the supernatant was collected again by centrifugation at 4°C, 20,000 g for 10 min; the supernatant was filtered using a 0.22 μm PES membrane and then filtered using Ni 2+ -NTA column for protein purification. Detect protein concentration, add 20% glycerol, and store the protein at -80°C.

[0038] The in vitro activity test used a 100 μL system: a 1 mM chlorogenic acid standard; the solvent was a PBS buffer (pH = 4.0) without NaCl and KCl; finally, 10 μL of purified protein solution was added; incubated at 30°C for 12 h. The reaction was terminated by adding 900 μL of pure methanol, and the reaction solution was centrifuged at 4°C, 20,000g for 10 min, and 500 μL of supernatant was taken for LC-HRMS analysis.

[0039] Example 4: Detection of Cynarine Production in an In Vitro Reaction System Using LC-HRMS

[0040] (1) LC-HRMS detection conditions and preparation of standard solutions

[0041] Determination of phenolic acids using AB SCIEX qTOF X500R liquid chromatography tandem mass spectrometry

[0042] Chromatographic column: Yuexu Technology (Shanghai) Co., Ltd. XB-Phenyl column (2.1×100 mm, 3 μm);

[0043] Mobile phase: 0.1% formic acid in ultrapure water (A) and 95% acetonitrile (B);

[0044] Flow rate: 0.4 mL / min;

[0045] Column temperature: 40°C;

[0046] Injection volume: 4 μL;

[0047] The gradient elution program is shown in Table 1:

[0048] Table 1. Gradient elution program

[0049] Time(min) ％A ％B 1 98 2 4 80 20 7 80 20 9 65 35 11 0 100 12 0 100

[0050] (2) Configuration of standard products

[0051] Weigh the standard products of Chlorogenic acid and Cynarine (Chengdu Ruifensi Biotechnology Co., Ltd.) respectively, prepare a stock solution with a concentration of 100mM, add methanol to dilute to 1mM, and then test the sample. The test results are as follows: Figure 1 and Figure 2 shown.

[0052] The results of in vitro enzyme activity reaction of yeast-expressed recombinant protein showed that EpCYS can catalyze the conversion of chlorogenic acid into cynarine.

[0053] Example 5: Construction of a recombinant plant expression vector and engineered bacteria containing the EpCYS gene

[0054] According to the GoldenBraid construction principle, a recombinant plant expression vector containing the EpCYS gene was constructed. First, the obtained EpCYSDNA fragment was constructed into the entry vector pUPD2, and the ligation product was transformed into Escherichia coli DH5α competent cells (Chl resistance), blue-white screening was performed, and white colonies were picked for PCR identification to obtain the plasmid pUPD2-EpCYS; the constructed pUPD2-EpCYS entry vector and the pDGB3_α vector were simultaneously subjected to restriction digestion-ligation reaction to obtain the ligation product 2

[0055] ×35S:EpCYS:α2-2×35S:EpCYS:T35S-P35S:EGFP:T35S-Pnos:NptII:Tnos, transformed into Escherichia coli DH5α, screened positive clones, and then extracted plasmids for PCR detection and sequencing to obtain a recombinant plant expression vector containing the EpCYS gene.

[0056] The recombinant vector was transformed into Agrobacterium tumefaciens GV3101, and positive clones were screened to obtain engineered bacteria containing the recombinant vector.

[0057] Example 6: Protein expression and function verification in Nicotiana benthamiana

[0058] The seeds of Nicotiana benthamiana were sterilized in 75% alcohol for 30 seconds, washed with sterile water for 1 minute, and then sterilized with 84 disinfectant for 3-5 minutes, and washed with sterile water for 3 times. The sterilized tobacco seeds were sown on germination medium (4.43g / L MS vitamin + 30g / L sucrose + 7g / L agar, pH = 5.8-5.85), and cultured at 23°C, 16h / 8h light / dark for 4-5 weeks.

[0059] The GV3101-recombinant vector engineered bacteria obtained above were inoculated on LB solid medium (containing 40mg / L Rif+50mg / L Gent+50mg / L Kan+10g / L agar) and cultured at 28°C for 48h. A single colony of Agrobacterium was picked and cultured in LB liquid medium (containing 40mg / L Rif+50mg / L Gent+50mg / L Kan) at 28°C and 200r / min shaking for 24-48h until OD 600 =0.2. Cut the sterile tobacco leaves into small pieces with a scalpel and inoculate them in the Agrobacterium suspension for 10-15 minutes. Inoculate the infected tobacco leaves on a co-culture medium (4.43g / L MS vitamin + 30g / L sucrose + 100μMAS + 0.2mg / L NAA + 2mg / L 6BA + 7g / L agar, pH = 5.8-5.85) with a layer of sterile filter paper and culture them in the dark for 2 days.

[0060] After 2 days of co-cultivation, the tobacco leaves were transferred to a screening medium containing resistance (4.43g / L MS vitamin + 100mg / L Kana + 300mg / L Cef + 0.2mg / L NAA + 2mg / L 6BA + 7g / L agar, pH = 5.8-5.85) to induce callus, cultured at 23°C, 16h / 8h light / dark, and subcultured every 7-9 days. After the resistant buds grow, the callus tissue is excised and transferred to a culture tank (with the same composition as the above screening medium) for further culture.

[0061] When the resistant buds are 2 cm high, they are transferred to rooting medium (4.43 g / L MS vitamin + 30 g / L sucrose + 100 mg / L Kana + 300 mg / L Cef + 0.1 mg / L IBA + 7 g / L agar, pH = 5.8-5.85) for rooting growth. During the rooting growth period, DNA is extracted from tobacco leaves and PCR reaction is used to detect the positive status of the plants.

[0062] After the positive plants to be tested grow forked resistant roots, move the plants into peat soil, cover to keep moisture, and culture at 25°C, 16h / 8h light / darkness for about a week, then remove the cover and continue to culture for 2-3 weeks.

[0063] After the positive plants were transplanted into the peat soil for 3 weeks, 100 mg of fresh tobacco leaf samples were weighed, 1 mL of methanol was added, ground into powder, centrifuged at 5000 g for 10 min at 4 °C, and 500 μL of supernatant was drawn into a sample injection bottle for HPLC-MS analysis. The results of the enzyme activity test in tobacco showed that EpCYS could catalyze chlorogenic acid to produce cynarine. Figure 3 and Figure 4 shown.

[0064] Example 7: Detection of Cynarine Production in Nicotiana benthamiana Using HPLC-MS

[0065] (1) HPLC-MS detection conditions and preparation of standard solutions

[0066] Determination of phenolic acids using AB SCIEX qTOF X500R liquid chromatography tandem mass spectrometry

[0067] Chromatographic column: Yuexu Technology (Shanghai) Co., Ltd. XB-Phenyl column (2.1×100mm, 3μm)

[0068] Mobile phase: 0.1% formic acid in ultrapure water (A) and 95% acetonitrile (B);

[0069] Flow rate: 0.4 mL / min;

[0070] Column temperature: 40°C;

[0071] Injection volume: 4 μL;

[0072] The gradient elution program is shown in Table 2:

[0073] Table 2. Gradient elution program

[0074] Time(min) ％A ％B 1 98 2 4 80 20 7 80 20 9 65 35 11 0 100 12 0 100

[0075] (2) Configuration of standard products

[0076] Weigh the standard products of Chlorogenic acid, 1,5-Dicaffeoylquinic acid, 3,4-Dicaffeoylquinic acid, 4,5-Dicaffeoylquinic acid, and Cynarine (Chengdu Ruifensi Biotechnology Co., Ltd.) respectively, prepare a mother solution with a concentration of 100mM, add 70% methanol to dilute to 1mM, and then test the sample. The test results are as follows: Figure 3 and Figure 4 shown.

[0077] The results showed that Cynarine was detected in Nicotiana benthamiana compounds overexpressing EpCYS, but not in wild-type Nicotiana benthamiana compounds, indicating that EpCYS can catalyze the conversion of chlorogenic acid into Cynarine.

[0078] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited to the following.

[0079] The equivalent substitutions or changes made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

[0080] The protection scope of the present invention shall be subject to the claims.

Claims

1. The EpCYS gene of the SCPL family of Echinacea purpurogena, characterized in that: The amino acid sequence encoded by the EpCYS gene is shown in SEQ ID NO.

2.

2. The Echinacea SCPL family acyltransferase EpCYS gene according to claim 1, characterized in that: The nucleotide sequence of the EpCYS gene is shown in SEQ ID NO.

1.

3. A yeast expression vector containing the Echinacea SCPL family acyltransferase EpCYS gene according to claim 1.

4. The yeast expression vector according to claim 3, characterized in that: The yeast expression vector is obtained by connecting the sequence shown in SEQ ID NO.1 into the Sal I and Xho I restriction sites of the pESC-HIS vector.

5. A recombinant plant expression vector containing the Echinacea SCPL family acyltransferase EpCYS gene of claim 1.

6. The recombinant plant expression vector according to claim 5, characterized in that: The nucleotide sequence shown in SEQ ID NO.1 was obtained by ligating into pDGB3_α according to the GoldenBraid principle.

7. Application of Echinacea SCPL family acyltransferase EpCYS in catalyzing the production of cynarine from chlorogenic acid.

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