Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase gene, protein and application thereof

Abstract

The invention relates to the technical field of synthetic biology, in particular to a polygala tenuifolia phenylpropanoid sucrose ester acyltransferase gene, protein and application thereof. The invention provides a phenylpropanoid sucrose ester acyltransferase gene PtPSE1, a phenylpropanoid sucrose ester acyltransferase gene PtPSE2, a phenylpropanoid sucrose ester acyltransferase gene PtPSE3 and a phenylpropanoid sucrose ester acyltransferase gene PtPSE4, and proteins coded by According to the present invention, the protein encoded by the gene is utilized, sucrose and phenylpropanoyl-CoA are adopted as substrates, and the synthesis of the phenylpropanoyl sucrose monoester is regulated and controlled in a PBS buffer solution; the catalytic reaction system is high in safety, the reaction substrate is simple, the system product is relatively single, and subsequent separation and utilization are easy. Therefore, the PtPSE1 gene, the PtPSE2 gene, the PtPSE3 gene and the PtPSE4 gene and the encoding protein of the PtPSE1 gene, the PtPSE2 gene, the PtPSE3 gene and the PtPSE4 gene have better application value and prospect in biosynthesis of the phenylpropanoid sucrose monoester.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a polygala phenylpropanoid sucrose ester acyltransferase gene, a protein and applications thereof. Background Art

[0002] Phenylpropanoid sucrose esters (PSEs) are phenylpropanoid glycoside ester compounds obtained by esterification reaction of hydroxyl groups of sucrose with carboxyl groups of phenylpropanoid phenolic acid, including sucrose ester products modified with 1 phenylpropanoid group, 2 phenylpropanoid groups, 3 phenylpropanoid groups or more phenylpropanoid groups. The phenylpropanoid groups in phenylpropanoid sucrose esters can be composed of different phenylpropanoid acyl groups, including benzoyl, p-hydroxybenzoyl, 4-methoxybenzoy, cinnamoyl, caffeoyl, feruloyl, sinapoyl, 3,4,5-trimethoxycinnamoyl, etc. PSEs compounds are widely distributed in the medicinal tissues of traditional Chinese medicinal materials. They have multiple pharmacological activities such as anti-tumor, antibacterial, antioxidant, antiviral, anti-inflammatory, anti-depression, anti-Alzheimer's, brain protection and inhibition of glycosidase. PSEs compounds have become an important precursor for the development of safe and effective new drugs, and have become one of the focuses of research in the field of traditional Chinese medicine for many years, and are increasingly attracting the attention of scholars at home and abroad.

[0003] The traditional Chinese medicine Polygala is the dried root of Polygala tenuifolia Willd. or Polygala sibirica L. of the Polygalaceae family. It is mainly distributed in Northeast my country, North China, Northwest China, Central China and Sichuan, with abundant resources. It has the effects of calming the mind and improving intelligence, connecting the heart and kidney, and removing phlegm and reducing swelling. It is often used to treat insomnia, forgetfulness, palpitations, confusion, coughing and expectoration, sores and swelling caused by disharmony between the heart and kidney. PSEs sugar esters are important indicator components of Polygala medicinal materials. The 2020 edition of the "Pharmacopoeia of the People's Republic of China" records that the content of 3,6'-dicorinoyl sucrose ester (hydroxycinnamoyl sucrose ester) is 0.50% or more as a quality evaluation indicator for medicinal materials.

[0004] PSEs are phenylpropanoid glycosides, which are secondary metabolites of plants. Their content in plants is often very low. The chemical synthesis of PSEs and the enzymatic production of lipase in vitro have been developed. However, there are few studies on the key enzymes in their biosynthesis pathway in plants, which seriously limits their in-depth development and application. Summary of the invention

[0005] The invention aims to propose a polygala phenylpropanoid sucrose ester acyltransferase (PhenylpropanoidSucroseEstersTransferases, PSEs) gene PtPSEs and its encoded protein and its application in the biosynthesis of phenylpropanoid sucrose esters. The encoded protein can regulate the synthesis of phenylpropanoid sucrose monoester sibiricose A5 (3-O-Feruloyl sucrose) or sibiricose A6 (3-O-Sinapyl sucrose) in a safe 1×PBS buffer (0.01 mol / L, pH 7.2-7.4).

[0006] The first aspect of the present invention provides a polygala phenylpropanoid sucrose ester acyltransferase protein, wherein the amino acid sequence of the polygala phenylpropanoid sucrose ester acyltransferase protein comprises any of the following groups of amino acid sequences:

[0007] (a1) the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:5 and has the same protein activity;

[0008] (a2) the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:6 and has the same protein activity;

[0009] (a3) the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:7 and has the same protein activity;

[0010] (a4) the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:8 and has the same protein activity.

[0011] The second aspect of the present invention provides a gene encoding the above-mentioned Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase protein.

[0012] Furthermore, the nucleotide sequence of the gene includes any of the following groups of nucleotide sequences:

[0013] (b1) the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to SEQ ID NO: 1;

[0014] (b2) the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence that is completely complementary to SEQ ID NO: 2;

[0015] (b3) the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence that is completely complementary to SEQ ID NO: 3;

[0016] (b4) the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence that is completely complementary to SEQ ID NO:4.

[0017] The third aspect of the present invention provides a recombinant vector, which is a recombinant vector containing the gene encoding the above-mentioned Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase protein.

[0018] The fourth aspect of the present invention provides a recombinant bacterium, which is a recombinant bacterium containing the gene encoding the above-mentioned Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase protein; or a recombinant bacterium containing a recombinant vector of the gene encoding the above-mentioned Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase protein.

[0019] The fifth aspect of the present invention provides a kit, which contains the above-mentioned Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase protein; or contains the above-mentioned gene encoding Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase protein; or contains the above-mentioned recombinant vector; or contains the above-mentioned recombinant bacteria.

[0020] The sixth aspect of the present invention provides a polygala phenylpropanoid sucrose ester acyltransferase protein, or a gene encoding a polygala phenylpropanoid sucrose ester acyltransferase protein, or the above-mentioned recombinant vector, or the use of any one of the above-mentioned recombinant bacteria in the synthesis of phenylpropanoid sucrose monoester.

[0021] Furthermore, the application requires the addition of substrates to synthesize phenylpropanoid sucrose monoester, and the substrates are sucrose and phenylpropanoid acyl-CoA.

[0022] Furthermore, the phenylpropanoid sucrose monoester is sibiricose A5 or sibiricose A6.

[0023] The seventh aspect of the present invention provides a method for synthesizing phenylpropanoid sucrose monoester, wherein sucrose is used as a substrate, and under the action of polygala phenylpropanoid sucrose ester acyltransferase protein, phenylpropanoid acyl-CoA is catalyzed to synthesize phenylpropanoid sucrose monoester, wherein the phenylpropanoid sucrose monoester is sibiricose A5 or sibiricose A6.

[0024] The beneficial effects of the present invention are as follows: 4 phenylpropanoid sucrose ester acyltransferase genes PtPSEs (PtPSE1, PtPSE2, PtPSE3 and PtPSE4), proteins encoded by PtPSEs, and applications of proteins encoded by PtPSEs in the biosynthesis of phenylpropanoid sucrose esters are provided. The application uses the proteins encoded by the PtPSEs genes to carry out an enzymatic reaction system, which only uses sucrose and phenylpropanoid acyl-CoA as substrates and PBS as a buffer to achieve the biosynthesis of phenylpropanoid sucrose esters. The system has high safety, simple reaction substrates, relatively single system products, and is easy to separate and utilize subsequently. Therefore, the PtPSEs gene and its encoded protein have good application value and prospects in the biosynthesis of phenylpropanoid sucrose monoesters. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is sibiricose A5 (3-O-Feruloyl sucrose).

[0026] Figure 2 It is sibiricose A6 (3-O-Sinapyl sucrose).

[0027] Figure 3 This is a graph showing the PCR results of PtPSE1, PtPSE2, PtPSE3 and PtPSE4 of the present invention, with the marker being DL5000.

[0028] Figure 4 Schematic diagram of the pCold-PtPSE1 recombinant vector.

[0029] Figure 5 Schematic diagram of the pCold-PtPSE2 recombinant vector.

[0030] Figure 6 Schematic diagram of the pCold-PtPSE3 recombinant vector.

[0031] Figure 7 Schematic diagram of the pCold-PtPSE4 recombinant vector.

[0032] Figure 8 These are the purification results of PtPSE1, PtPSE2, PtPSE3 and PtPSE4 proteins; L1 is BSA (1 mg / mL); L2 is pCold-TF; L3 is pCold-PtPSE1; L4 is pCold-PtPSE2; L5 is pCold-PtPSE3; and L6 is pCold-PtPSE4.

[0033] Fig. 9The HPLC results of the catalytic reaction of sucrose and Feruloyl-Coenzyme A to generate phenylpropanoid sucrose monoesters by PtPSE1, PtPSE2, PtPSE3 and PtPSE4 are shown in Figure 2. Fig. 9 A is the HPLC result of the formation of phenylpropanoid sucrose monoester sibiricose A5 (3-O-Feruloylsucrose); Fig. 9 B is the HPLC result of the formation of phenylpropanoid sucrose monoester sibiricose A6 (3-O-Sinapyl sucrose).

[0034] Fig.10 Calculation of enzyme kinetic parameters for PtPSE1 product abundance in response to substrate concentration gradient.

[0035] Fig.11 Calculation of enzyme kinetic parameters for PtPSE2 product abundance in response to substrate concentration gradient.

[0036] Fig.12 Calculation of enzyme kinetic parameters for PtPSE3 product abundance in response to substrate concentration gradient.

[0037] Fig.13 Calculation of enzyme kinetic parameters for PtPSE4 product abundance in response to substrate concentration gradient. DETAILED DESCRIPTION

[0038] The present invention provides four polygala tenuifolia phenylpropanoid sucrose ester acyltransferase genes, namely PtPSE1 (SEQ ID NO: 1), PtPSE2 (SEQ ID NO: 2), PtPSE3 (SEQ ID NO: 3) and PtPSE4 (SEQ ID NO: 4).

[0039] The present invention provides four proteins encoded by polygala tenuifolia phenylpropanoid sucrose ester acyltransferase genes, which are respectively a protein encoded by PtPSE1 (SEQ ID NO: 5), a protein encoded by PtPSE2 (SEQ ID NO: 6), a protein encoded by PtPSE3 (SEQ ID NO: 7) and a protein encoded by PtPSE4 (SEQ ID NO: 8).

[0040] In some embodiments of the present invention, primer sequences for cloning the above-mentioned Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase gene are provided, wherein the primers for cloning the PtPSE1 gene are PtPSE1_F (SEQ ID NO:9) and PtPSE1_R (SEQ ID NO:10), the primers for cloning the PtPSE2 gene are PtPSE2_F (SEQ ID NO:11) and PtPSE2_R (SEQ ID NO:12), the primers for cloning the PtPSE3 gene are PtPSE3_F (SEQ ID NO:13) and PtPSE3_R (SEQ ID NO:14), and the primers for cloning the PtPSE4 gene are PtPSE4_F (SEQ ID NO:15) and PtPSE4_R (SEQ ID NO:16).

[0041] In some embodiments of the present invention, primers for constructing a recombinant expression vector pCold-PtPSEs are provided, wherein the primers used to construct the recombinant expression vector pCold-PtPSE1 are pCold-PtPSE1_F (SEQ ID NO: 17) and pCold-PtPSE1_R (SEQ ID NO: 18), the primers used to construct the recombinant expression vector pCold-PtPSE2 are pCold-PtPSE2_F (SEQ ID NO: 19) and pCold-PtPSE2_R (SEQ ID NO: 20), the primers used to construct the recombinant expression vector pCold-PtPSE3 are pCold-PtPSE3_F (SEQ ID NO: 21) and pCold-PtPSE3_R (SEQ ID NO: 22), the primers used to construct the recombinant expression vector pCold-PtPSE4 are pCold-PtPSE4_F (SEQ ID NO: 23) and pCold-PtPSE4_R (SEQ ID NO: 24), and the primers used to construct the recombinant expression vector pCold-PtPSE5 are pCold-PtPSE5_F (SEQ ID NO: 25) and pCold-PtPSE5_R (SEQ ID NO: 26). NO:24).

[0042] In some embodiments of the present invention, a method for constructing a pCold-TF recombinant expression vector of genes PtPSE1, PtPSE2, PtPSE3 and PtPSE4 is provided, and the specific steps are as follows: the obtained PCR recovery products of PtPSE1, PtPSE2, PtPSE3 and PtPSE4 are used as templates, pCold-PtPSEs_F and pCold-PtPSEs_R are used as primers for amplification, and the target gene fragments are recovered, the target gene fragments and pCold-TF enzyme digestion products are mixed with homologous recombinase, and the ligation products are transformed into competent E.coli Turbo, cultured, 3 to 5 clones are randomly selected for sequencing, the single colony strain with correct sequencing is cultured overnight, the bacteria are collected by centrifugation, and the plasmid is extracted.

[0043] In some embodiments of the present invention, a protein expression method of genes PtPSE1, PtPSE2, PtPSE3 and PtPSE4 is provided, and the specific steps are as follows: transforming the pCold-PtPSE1, pCold-PtPSE2, pCold-PtPSE3 and pCold-PtPSE4 recombinant plasmids into E. coli BL21 (DE3) competent cells, picking recombinant monoclonal clones, selecting appropriate induction expression conditions, amplifying the expression in a large system and purifying the proteins.

[0044] In some embodiments of the present invention, there is provided an application of a protein encoded by the polygala tenuifolia phenylpropanoid sucrose ester acyltransferase gene PtPSEs in regulating the synthesis of phenylpropanoid sucrose esters. The application achieves the synthesis of phenylpropanoid sucrose monoesters with a specific substrate by utilizing the enzymatic reaction of the PtPSEs protein.

[0045] Furthermore, the specific substrate uses sucrose as an acceptor substrate and phenylpropanol acyl-CoA as a donor substrate.

[0046] In some embodiments of the present invention, a method for regulating the synthesis of phenylpropanoid sucrose esters using proteins encoded by polygala tenuifolia phenylpropanoid sucrose ester acyltransferase genes PtPSE1, PtPSE2, PtPSE3 and PtPSE4 is provided. The specific steps of the method are as follows: PtPSEs protein, Feruloyl-Coenzyme A or Sinapyl-Coenzyme A, and sucrose are added to 1×PBS buffer (0.01 mol / L, pH 7.2-7.4) to react for a period of time, and then methanol is added to terminate the reaction, centrifuged, and the supernatant is filtered through an organic filter membrane to obtain phenylpropanoid sucrose monoester sibiricose A5 (3-O-Feruloyl sucrose) or sibiricose A6 (3-O-Sinapyl sucrose).

[0047] Terminology explanation:

[0048] In the present invention, the phenylpropanoid sucrose monoester "sibiricose A5" refers to 3-O-Feruloyl sucrose; the phenylpropanoid sucrose monoester "sibiricose A6" refers to 3-O-Sinapyl sucrose.

[0049] In the present invention, "PtPSEs gene" is selected from one or more of the following: PtPSE1 gene (SEQ ID NO: 1), PtPSE2 gene (SEQ ID NO: 2), PtPSE3 gene (SEQ ID NO: 3) and PtPSE4 gene (SEQ ID NO: 4).

[0050] In the present invention, "protein expressed by PtPSEs", "protein encoded by PtPSEs" or "PtPSEs protein" is selected from one or more of the following: protein encoded by PtPSE1 gene (SEQ ID NO: 5), protein encoded by PtPSE2 gene (SEQ ID NO: 6), protein encoded by PtPSE3 gene (SEQ ID NO: 7) and protein encoded by PtPSE4 gene (SEQ ID NO: 8).

[0051] Below in conjunction with embodiment, technical scheme of the present invention is clearly and completely described, obviously, described embodiment is only a part of embodiment of the present invention, rather than all embodiments. Embodiment is carried out according to conventional experimental conditions or according to the condition suggested by manufacturer's specification. Unless otherwise defined, all professional terms used in the present invention have the same meaning as those skilled in the art are generally understood. In addition, any method and material similar or identical to the described content can be used in the inventive method, based on the embodiment in the present invention, all other embodiments obtained by those skilled in the art under the premise of not paying creative work, all belong to the scope of protection of the present invention.

[0052] The experimental methods used in the following examples were carried out according to conventional methods or those recommended by the manufacturer unless otherwise specified. The materials, reagents and consumables used were all commercially available unless otherwise specified.

[0053] Example 1: Cloning of PtPSEs genes

[0054] Gene cloning method: Using the cDNA of Polygala root as a template, PCR amplification was performed using cloning primers of genes PtPSE1, PtPSE2, PtPSE3, and PtPSE4. The gene cloning primer sequences used in this example are shown in Table 1. The PCR reaction system was 30 μL, including 1 μL of cDNA template, 1.5 μL of F and R primers, Max DNA Polymerase (Takara, R047A) 15 μL, ddH2O 11 μL.

[0055] Set the PCR program: 98°C, 3 min; 98°C, 10 sec, 60°C, 15 sec, 72°C, 60 sec, 35 cycles; 72°C, 8 min, store at 16°C.

[0056] After PCR was completed, the gene fragments were detected by 1% agarose gel electrophoresis (eg Figure 3 The target gene band was recovered using an agarose gel recovery kit.

[0057] Table 1 Gene cloning primer sequence information

[0058]

[0059] Example 2: Construction of PtPSEs gene expression vector

[0060] 1. Obtain PCR fragments:

[0061] Using the gene clone PCR recovery product as a template, PCR amplification was performed using the homologous recombination primers of genes pCold-PtPSE1, pCold-PtPSE2, pCold-PtPSE3, and pCold-PtPSE4. The sequences of the homologous recombination primers used in this example are shown in Table 2. The PCR reaction system was 30 μL, including 1 μL of PCR recovery product, 1.5 μL of F and R primers, Max DNA Polymerase (Takara, R047A) 15 μL, ddH2O 11 μL.

[0062] Set the PCR program: 98℃, 2min; 98℃, 10sec, 61℃, 15sec, 72℃, 30sec, 35 cycles; 72℃, 8min, store at 16℃.

[0063] After the PCR was completed, the gene fragment was detected by 1% agarose gel electrophoresis, and the target gene band was recovered using an agarose gel recovery kit.

[0064] Table 2 Homologous recombination primer sequence information of pCold-PtPSEs

[0065]

[0066] 2. Connection and reorganization:

[0067] The target band of the gene was recovered using an agarose gel recovery kit. After the concentration was detected, the gene fragment was connected to the cloning vector pCold-TF Vector (enzyme sites Sal I and Kpn I). The vector connection reaction system was 10 μL, including 2 μL of homologous recombination PCR product, 3 μL of pCold-TF double enzyme gel recovery product, and 5 μL of 2×ClonExpress Mix. After the reaction system was added, it was connected in a PCR instrument at 50°C for 25 minutes.

[0068] 3. Conversion:

[0069] 1) Add the ligation product to 100 μL of freshly thawed E. coli Turbo competent medium, then gently pipette to mix, and place on ice for 30 minutes.

[0070] 2) After the ice bath, heat shock the mixture in a 42°C water bath for 90 seconds, and then immediately ice bathe the mixture for 2 minutes.

[0071] 3) Add 900 μL of LB liquid medium without antibiotics, place on a shaker at 37°C, 220 rpm, and incubate for 1 h.

[0072] 4) Centrifuge at 6000 rpm for 3 min, discard part of the supernatant, and mix the remaining about 200 μL by pipetting. Use a spreading stick to spread the bacterial solution on a solid plate of LB (containing 100 mg / L Amp), and invert it in a 37°C incubator for overnight culture.

[0073] 5) Randomly select monoclonal bacteria for sequencing, obtain the monoclonal bacterial strain with correct sequencing and carrying the gene PtPSEs, culture overnight, collect the bacteria by centrifugation, and extract the plasmid. The schematic diagram of the PtPSEs gene vector is shown in the figure Figures 4 to 7 shown.

[0074] 4. Obtaining the PtPSEs gene expression vector and identifying its gene sequence:

[0075] By sequencing, the sequences of PtPSE1, PtPSE2, PtPSE3 and PtPSE4 were identified and obtained. Among them, the sequence of PtPSE1 is shown in SEQ ID NO.1; the sequence of PtPSE2 is shown in SEQ ID NO.2; the sequence of PtPSE3 is shown in SEQ ID NO.3; and the sequence of PtPSE4 is shown in SEQ ID NO.4.

[0076] Example 3: Protein expression of PtPSEs gene

[0077] 1. Transform pCold-PtPSEs plasmid into E.coli BL21 (DE3) competent cells:

[0078] 1) Take 100 μL of E. coli BL21 (DE3) competent cells melted in an ice bath, add 5 μL of the extracted plasmid, mix gently, and place in an ice bath for 30 minutes.

[0079] 2) Heat shock in a 42°C water bath for 60 seconds, then quickly transfer the tube to an ice bath for 2 minutes. Do not shake the centrifuge tube during this process.

[0080] 3) Add 900 μL of sterile LB medium (without antibiotics) to the centrifuge tube, mix well, and culture at 37°C, 200 rpm for 90 min to allow the cells to recover.

[0081] 4) Pipette 200 μL of the transformed competent cells onto LB agar medium containing ampicillin (100 ug / ml), and spread the cells evenly. Place the plate at 37°C until the liquid is absorbed, invert the plate, and culture at 37°C overnight.

[0082] 2. Induced expression and protein purification:

[0083] 1) Pick a single colony containing the recombinant plasmid from the plate, inoculate it into 5 mL (15 mL sterile centrifuge tube) LB medium containing ampicillin (100 ug / ml), and culture it at 37°C overnight.

[0084] 2) The next day, 4 mL of the culture medium was added to 400 mL LB (1 L Erlenmeyer flask) for expansion (1% inoculation), and cultured at 37°C with shaking until OD600 = 0.6-0.8. The culture solution was immediately transferred to 15°C and cultured with shaking at 180 rpm for 30 min. IPTG was added to the culture solution to a final concentration of 0.3 mM, and the culture was continued at 15°C and 180 rpm with shaking.

[0085] 3) After overnight shaking induction at 15°C, collect the cells by centrifugation. Add 10mL / g Binding buffer (20mMPhosphate Buffer, 500mM NaCl, 10mM Imidazole, pH7.4), sonicate, 45% power, 5s for 5s, 5s interval, for a total of 60min. Collect the supernatant after low-temperature centrifugation at 13000rpm and start protein purification. The specific steps refer to HisSep Ni-NTA Agarose Resin (Catalog#: 20502ES50, YEASEN, China) and make appropriate modifications as follows:

[0086] 4) Agarose Resin pretreatment: wash Agarose with Binding Buffer 3 times, centrifuge at 12000 rpm and discard the supernatant.

[0087] 5) Add the pretreated Agarose to the crude protein sample collected after high-speed low-temperature centrifugation and rotate it on a rotating mixer at 4°C for 12 hours.

[0088] 6) Add the sample to the affinity chromatography column, let the waste liquid flow out automatically by gravity, and the agarose and bound protein are retained. Wash the agarose and bound protein three times with Washing Buffer (20mM Phosphate Buffer, 500mM NaCl, 30mM Imidazole, pH7.4).

[0089] 7) Finally, use 10 mL Elution Buffer (20 mM Phosphate Buffer, 500 mM NaCl, 400 mM Imidazole, pH 7.4) to elute the target protein, and collect the eluate as the target protein sample.

[0090] 3. Concentration and impurity removal:

[0091] Place the purified protein sample into a 100kDa ultrafiltration tube (Millipore), add an appropriate volume of protein concentration buffer (Tris 50mM, NaCl 300mM, pH 7.3), centrifuge at 1500rpm for 30min. Discard the filtrate, add an appropriate volume of protein concentration buffer, and continue centrifugation to concentrate the protein. Repeat three times to concentrate the protein to a suitable concentration.

[0092] 4. Protein identification:

[0093] The retained final protein sample was packaged and quantitatively analyzed by BCA method. Further (10 μl) 10% SDS-PAGE was used to detect the protein purification expression. Figure 8 shown.

[0094] 5. Obtaining proteins expressed by PtPSEs:

[0095] By comparing with the marker and empty vector, the proteins expressed by PtPSE1, PtPSE2, PtPSE3 and PtPSE4 were obtained. Among them, the sequence of PtPSE1 protein is shown in SEQ ID NO.5; the sequence of PtPSE2 protein is shown in SEQ ID NO.6; the sequence of PtPSE3 protein is shown in SEQ ID NO.7; and the sequence of PtPSE4 protein is shown in SEQ ID NO.8.

[0096] Example 4: PtPSEs protein regulates the synthesis of sucrose esters Phenylpropanoid sucrose monoester

[0097] In this example, the regulatory effect of PtPSEs protein on the synthesis of phenylpropanoid sucrose monoesters (sibiricose A5 and sibiricose A6) was investigated.

[0098] In this embodiment, a 200uL reaction system is used as an example. PtPSEs protein (200ug) uses 10mM sucrose as the acceptor substrate and 100μM Feruloyl-Coenzyme A or Sinapyl-Coenzyme A as the donor substrate, and reacts in 1×PBS buffer (0.01mol / L, pH7.2-7.4). React for a period of time under suitable temperature conditions. The reaction temperature in this embodiment is 30°C for 4 hours. In other embodiments, other suitable temperature conditions can also be used to react for a period of time. The reaction is terminated by adding an equal volume of methanol, centrifuged to remove impurities, and filtered through an organic filter membrane to obtain the final product.

[0099] By analyzing the HPLC spectrum of the product, it was found that compared with the control group (pCold-TF), the PtPSEs proteins all produced an obvious substance peak sibiricoseA5 ( Fig. 9 A) and sibiricose A6 ( Fig. 9 B). This indicates that the PtPSEs protein successfully catalyzed the synthesis of phenylpropanoid sucrose monoester in the reaction system, and obtained phenylpropanoid sucrose monoester sibiricose A5 (3-O-Feruloyl sucrose) ( Figure 1 ) and sibiricoseA6(3-O-Sinapyl sucrose)( Figure 2 ).

[0100] Example 5. PtPSEs enzyme kinetic parameters

[0101] In this embodiment, a series of substrate concentration gradient experiments were designed to determine the enzyme kinetic parameters of four enzyme proteins of PtPSEs for feruloyl-CoA and sinapoyl-CoA. The reaction system includes different concentrations of feruloyl-CoA or sinapoyl-CoA substrates (10 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM, 600 μM), 10 mM sucrose, enzyme concentration (1 ug / uL), and the reaction is carried out in 1×PBS buffer (pH 7.2-7.4) to ensure that the reaction is in the initial velocity stage. The reaction conditions are controlled at 30°C and the reaction volume is 200 μL. The maximum reaction velocity Vmax, Michaelis constant Km and catalytic efficiency Kcat of the enzyme were calculated by nonlinear fitting of the Michaelis-Menten equation. The catalytic efficiency Kcat / Km of the enzyme at low substrate concentration was further calculated to evaluate the catalytic efficiency and substrate specificity.

[0102] The results are as follows Figure 10 to Figure 13 As shown in Table 3, Pt3G0003260 (named PtPSE1) has the highest maximum reaction rate, the smallest Michaelis constant and the highest catalytic efficiency for feruloyl-CoA. In contrast, the kinetic parameters of Pt4G0013510 and Pt9G0004120 (named PtPSE2 and PtPSE3, respectively) for feruloyl-CoA and sinapoyl-CoA are similar, although the maximum reaction rate of PtPSE2 for both substrates is one order of magnitude higher than that of PtPSE3. Compared with other PtPSEs, Pt3G0017700 (named PtPSE4) has the lowest maximum reaction rate for both feruloyl-CoA and sinapoyl-CoA.

[0103] In summary, PtPSE1 showed the highest specificity and catalytic ability for feruloyl-CoA. The above results provide an important reference for further optimization and selection of enzymes for the biosynthesis of phenylpropanoid metabolites.

[0104] Table 3 Determination of PtPSEs enzyme kinetic constants

[0105]

[0106] From the above examples of this study, it can be seen that the entire biocatalytic reaction of phenylpropanoid sucrose monoesters was carried out in 1×PBS buffer (0.01mol / L, pH7.2-7.4), and no other substances were added except sucrose, phenylpropanoid acyl-COA and catalytic PtPSEs protein. Compared with the substrates or solvents in most previous sucrose ester synthesis methods that require toxic reagents, the entire catalytic reaction system has high safety, simple reaction substrates, relatively single system products, and all products are phenylpropanoid sucrose esters, which are easy to separate and utilize later. Therefore, PtPSEs genes and their proteins have good application value and prospects in the biosynthesis of phenylpropanoid sucrose esters.

[0107] It should be noted that the specific parameters or some reagents in the above embodiments are specific embodiments or preferred embodiments of the present invention, rather than limiting the present invention; those skilled in the art can make adaptive adjustments within the scope of the present invention.

Claims

1. Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase protein, characterized in that The amino acid sequence of the polygala phenylpropanoid sucrose ester acyltransferase protein includes the amino acid sequence of any of the following groups: (a1) the amino acid sequence shown in SEQ ID NO:5, or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:5 and has the same protein activity; (a2) the amino acid sequence shown in SEQ ID NO:6, or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:6 and has the same protein activity; (a3) the amino acid sequence shown in SEQ ID NO:7, or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:7 and has the same protein activity; (a4) the amino acid sequence shown in SEQ ID NO:8, or an amino acid sequence in which one or more amino acids are added, deleted or substituted in the amino acid sequence shown in SEQ ID NO:8 and has the same protein activity.

2. A gene encoding the Polygala tenuifolia phenylpropanoid sucrose ester acyltransferase protein according to claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene includes any of the following groups of nucleotide sequences: (b1) the nucleotide sequence shown in SEQ ID NO: 1, or a nucleotide sequence that is completely complementary to SEQ ID NO: 1; (b2) the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence that is completely complementary to SEQ ID NO: 2; (b3) the nucleotide sequence shown in SEQ ID NO: 3, or a nucleotide sequence that is completely complementary to SEQ ID NO: 3; (b4) the nucleotide sequence shown in SEQ ID NO:4, or a nucleotide sequence that is completely complementary to SEQ ID NO:

4.

4. A recombinant vector, characterized in that: The recombinant vector is a recombinant vector containing the gene according to claim 2 or 3.

5. A recombinant bacterium, characterized in that: The recombinant bacteria are recombinant bacteria containing the gene according to claim 2 or 3 or the recombinant vector according to claim 4.

6. A kit, characterized in that: It comprises the polygala phenylpropanoid sucrose ester acyltransferase protein according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4 or the recombinant bacteria according to claim 5.

7. Use of any one of the polygala phenylpropanoid sucrose ester acyltransferase protein according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4, and the recombinant bacteria according to claim 5 in synthesizing phenylpropanoid sucrose monoester.

8. The use according to claim 7, characterized in that: The application requires the addition of substrates to synthesize phenylpropanoid sucrose monoesters, and the substrates are sucrose and phenylpropanoid acyl-CoA.

9. The use according to claim 7, characterized in that: The phenylpropanoid sucrose monoester is sibiricose A5 or sibiricose A6.

10. A method for synthesizing phenylpropanoid sucrose monoester, characterized in that: With sucrose as substrate, under the action of the polygala phenylpropanoid sucrose ester acyltransferase protein described in claim 1, phenylpropanoid acyl-CoA is catalyzed to synthesize phenylpropanoid sucrose monoester, and the phenylpropanoid sucrose monoester is sibiricose A5 or sibiricose A6.

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