Rhizoma panacis majoris glycosyl transferase PjvmUGT45 as well as mutant and application thereof
By screening and mutating bead ginseng glycosyltransferase PjvmUGT45 and its mutant K280A, the problem of low catalytic efficiency in the prior art was solved, and the biosynthesis of rare ginseng saponins Rh2 and F2 was achieved efficiently catalyzed.
Patent Information
- Application Number
- CN202411310882.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2024-12-13
AI Technical Summary
In the prior art, there are fewer types of glycosyltransferases that catalyze the formation of rare ginsenosides, and have low catalytic efficiency, making it difficult to achieve large-scale biosynthesis.
The UGT candidate gene with the PSPG box was screened from the rhizome of bead ginseng, identified as the bead ginseng glycosyltransferase PjvmUGT45, and the mutant K280A was obtained through site-directed mutation of the amino acid sequence, significantly improving the catalytic efficiency.
PjvmUGT45 and its mutants can catalyze the formation of rare ginseng saponins Rh2 and F2 using the progenitor ginseng diol saponins PPD and CK as substrates, and the catalytic efficiency is significantly higher than that of the existing glycosyltransferases UGTPg45 and PgUGT74AE2, especially the mutant K280A. The catalytic efficiency is higher, which is conducive to the in vitro synthesis of rare ginseng saponins.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and more specifically to a ginseng glycosyltransferase PjvmUGT45, a mutant thereof and an application thereof. Background Art
[0002] Ginsenosides are the main active ingredients extracted from Araliaceae plants (such as ginseng, American ginseng, Korean ginseng, Panax notoginseng, etc.), and have a variety of pharmacological activities and effects, such as anti-cancer, antioxidant, anti-inflammatory, etc. At present, nearly 100 kinds of prototype ginsenoside monomer active ingredients have been separated and identified, such as protopanaxandiol saponins (PPD), Ra, Rb series, Rc, Rd, Re, Rg1, etc. It was later found that the active ingredients formed after metabolic transformation of ginsenosides have better anti-cancer effects and stronger biological activities, so they were named rare ginsenosides. Rare ginsenosides are derivatives of prototype ginsenosides after metabolic transformation, such as Rg3, Rh1, CK, Rh2, Rg5, Rk1, Rh3, etc. However, the content of rare ginsenosides in Panax plants is extremely low, and it is very difficult to extract and prepare them directly from plants. For example, the rare ginsenoside Rh2 only exists in wild ginseng, red ginseng, and ginseng fruit, and the content is only one ten-thousandth level.
[0003] The biosynthetic pathway of ginsenosides can be divided into the upstream MVA pathway and the MEP pathway, as well as the downstream cyclization, hydroxylation and glycosylation steps, with 2,3-oxidosqualene as the center. Among them, the glycosylation reaction is the last step in the saponin synthesis pathway and is also a key step in determining the activity of monomeric saponins (Augustin JM et al., 2011). Glycosyltransferases (GTs) have been identified as key enzymes in the biosynthetic pathway of ginsenosides. Therefore, the study of glycosyltransferases in the biosynthetic pathway of ginsenosides is a current research hotspot.
[0004] In the carbohydrate-active enzyme database, GT is divided into 114 families (GT1-GT114), and those responsible for the glycosylation of plant natural products are generally classified as the GT1 family. The glycosyltransferases of the GT1 family mainly use uridine diphosphate (UDP)-sugar as a donor, so they are called UDP-glycosyltransferases (UGT). For example, the glycosyltransferase PjUGT10 and its mutants in patent CN 117645982 A can catalyze the formation of pseudo-ginsenoside RT5 in vitro using octilon-type ginsenosides as substrates; the glycosyltransferase PjmUGT in patent CN113088502B can catalyze the formation of ginsenoside Rb1 in vitro using ginsenoside Rd as a substrate. It can be seen that the specific glycosyltransferases corresponding to different saponin substrates and the reactants formed may be different.
[0005] With the continuous elucidation of the biosynthetic pathway of ginsenosides, it has become possible to use synthetic biology technology to mass-produce rare ginsenosides. For example, PgUGT74AE2 and UGTPg45 are specific glycosyltransferases that can catalyze the glycosylation reaction of the C-3 hydroxyl group of protopanaxadiol (PPD) to produce the rare ginsenoside Rh2. However, there are few types of glycosyltransferases that can catalyze the formation of rare ginsenosides from ginsenosides, and the existing catalytic efficiency is low. Therefore, it is urgent to extract and screen more or more glycosyltransferases with high catalytic efficiency, and clone their gene sequences through existing genetic engineering technology, which is conducive to the large-scale biosynthesis of ginsenosides, especially for the biosynthesis of rare ginsenosides. Summary of the invention
[0006] In view of the above defects or improvement needs of the prior art, the present invention provides a glycosyltransferase PjvmUGT45 of Panax notoginseng, its mutant and its application, the purpose of which is to screen out a UGT candidate gene with a PSPG box from the rhizome of Panax notoginseng. After functional verification, it was found that the protein encoded by the gene has the function of catalyzing the formation of rare ginsenosides Rh2 and F2 with protopanaxadiol saponins PPD and CK as substrates and UDP-Glc as sugar donor. The protein (named PjvmUGT45) was identified as a new glycosyltransferase, and the catalytic activity of the protein was The efficiency is significantly higher than that of the existing glycosyltransferases UGTPg45 and PgUGT74AE2 with the same catalytic function; further, site-directed mutagenesis of its amino acid sequence was performed, and the results showed that the catalytic efficiency of the mutants obtained with the mutation site K280A or S275A was also higher than that of UGTPg45 and PgUGT74AE2, especially the mutant with the mutation site K280A, whose catalytic efficiency was significantly higher than that of the wild-type PjvmUGT45, thereby solving the technical problem of low catalytic efficiency of existing glycosyltransferases using protopanaxadiol saponins PPD and CK as substrates.
[0007] To achieve the above object, according to one aspect of the present invention, a glycosyltransferase PjvmUGT45 of Panax notoginseng and a mutant thereof are provided, wherein the glycosyltransferase PjvmUGT45 has an amino acid sequence as shown in SEQ ID NO.2;
[0008] The amino acid sequence of the mutant is shown in SEQ ID NO.4 or SEQ ID NO.6.
[0009] Preferably, the glycosyltransferase PjvmUGT45 and its mutants, and the amino acid sequence of the mutants are shown in SEQ ID NO.4.
[0010] According to another aspect of the present invention, a gene encoding the glycosyltransferase PjvmUGT45 or a mutant thereof according to the present invention is also provided, wherein the gene encoding the glycosyltransferase PjvmUGT45 has a nucleotide sequence as shown in SEQ ID NO.1;
[0011] The gene encoding the glycosyltransferase PjvmUGT45 mutant has a nucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.5.
[0012] Preferably, the gene encoding the glycosyltransferase PjvmUGT45 mutant has a nucleotide sequence as shown in SEQ ID NO.3.
[0013] According to another aspect of the present invention, a biological material expressing the glycosyltransferase PjvmUGT45 or its mutant as described in the present invention is also provided, which includes an expression cassette, a recombinant vector, a transgenic cell line or a transgenic engineered bacteria carrying the target gene shown in SEQ ID NO.1, SEQ ID NO.3, or SEQ ID NO.5.
[0014] Preferably, the target gene carried by the biological material is as shown in SEQ ID NO.1 or SEQ ID NO.3.
[0015] According to another aspect of the present invention, there is provided a use of the glycosyltransferase PjvmUGT45 and its mutants or the biomaterials according to the present invention in the biosynthesis of rare ginsenosides, wherein the rare ginsenosides are Rh2 or F2.
[0016] Preferably, the application comprises the following steps:
[0017] Using protopanaxadiol saponin PPD or ginsenoside CK as a substrate and UDP-Glc as a glycosyl donor, the glycosyltransferase PjvmUGT45 or a mutant thereof as claimed in claim 1 is used as a catalytic enzyme to catalyze the substrate in vitro to generate rare ginsenoside Rh2 or F2.
[0018] Preferably, in the use, the glycosyltransferase PjvmUGT45 or its mutant catalyzes the substrate PPD to form the rare ginsenoside Rh2 in vitro, or catalyzes the substrate CK to form the rare ginsenoside F2 in vitro.
[0019] Preferably, in the application, the amino acid sequence of the mutant is as shown in SEQ ID NO.4.
[0020] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:
[0021] The present invention provides a glycosyltransferase PjvmUGT45 from Panax notoginseng and a mutant thereof. PjvmUGT45 is a glycosyltransferase extracted from Panax notoginseng. Purification and functional experiments show that the protein can catalyze the production of rare ginsenosides F2 and Rh2 using ginsenoside CK and PPD as substrates, belongs to a glycosyltransferase, and has a catalytic efficiency significantly superior to two known glycosyltransferases UGTPg45 and PgUGT74AE2 with the same catalytic function in the genus Panax. The protein can be used for in vitro catalytic synthesis of rare ginsenosides F2 and Rh2.
[0022] Further point mutations were performed on its amino acid sequence to obtain two mutants S275A and K280A with catalytic efficiency higher than that of UGTPg45 and PgUGT74AE2. In particular, the catalytic efficiency of mutant K280A was higher than that of the wild-type glycosyltransferase PjvmUGT45, which is beneficial to the in vitro synthesis of rare ginsenosides F2 and Rh2. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is the electropherogram of PCR amplification of gene PjvmUGT45;
[0024] Figure 2 This is the SDS-PAGE electrophoresis result after the purification of PjvmUGT45 protein;
[0025] Figure 3 It is the glycosyltransferase PjvmUGT45 that catalyzes the substrate CK to produce F2;
[0026] Figure 4 It is the glycosyltransferase PjvmUGT45 that catalyzes the substrate PPD to generate Rh2;
[0027] Figure 5 It is a comparison of the catalytic efficiency of glycosyltransferases PjvmUGT45, PgUGT74AE2 and UGTPg45 on substrates CK and PPD;
[0028] Figure 6 It is a comparison of the catalytic efficiency of glycosyltransferase PjvmUGT45 and its different mutants on substrates CK and PPD;
[0029] Figure 7 It is the mutants S275A and K280A that catalyze the substrate CK to form F2;
[0030] Figure 8 It is the mutants S275A and K280A that catalyze the substrate PPD to form Rh2. DETAILED DESCRIPTION
[0031] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.
[0032] Glycosylation modification determines the types and functional diversity of ginsenosides, and glycosyltransferase is the key rate-limiting enzyme in the biosynthesis of ginsenosides. Most of the amino acid sequences of the GT1 family in plants have a relatively conserved PSPG box at the C-terminus, which is composed of 44 amino acid residues and is a potential binding site for glycosyltransferase to recognize UDP sugar donors.
[0033] We have previously completed comparative transcriptome analysis of different parts of the rhizome of the bamboo ginseng variant Panax notoginseng, and screened a large number of UGT candidate genes with PSPG boxes. Functional verification revealed a UGT candidate gene (nucleotide sequence shown in SEQ ID NO.1), which encodes a protein with an amino acid sequence shown in SEQ ID NO.2. The protein can catalyze the production of rare ginsenosides F2 and Rh2 in vitro using ginsenosides CK and PPD as substrates, and its catalytic efficiency is significantly better than the two known glycosyltransferases with the same catalytic function in the genus Panax, UGTPg45 and PgUGT74AE2. After identification, the protein has a conserved PSPG conserved domain and is a typical plant GT45 protein, belonging to the UDP-glycosyltransferase of the GT45 family, and is named PjvmUGT45.
[0034] Furthermore, based on its amino acid sequence, site-directed mutagenesis of key amino acids was performed by alanine scanning mutagenesis and overlap extension PCR technology. Specifically, a total of 6 sites, including Ser275, Lys280, Gln334, His349, Ser354, and Asp373, were successfully mutated to alanine (Ala, A). The results showed that the enzyme activities of the 6 mutation points had significant changes, among which the conversion rates of the mutants with mutation points of Ser275 and Lys280 were higher than those of the existing UGTPg45 and PgUGT74AE2, and the catalytic activity of the mutant K280A with the mutation point of Lys280 for CK and PPD was higher than that of the unmutated glycosyltransferase PjvmUGT45, which were 1.32 times and 1.53 times higher than those of the unmutated one, respectively. The amino acid sequence of the mutant (K280A) of the glycosyltransferase PjvmUGT45 is shown in SEQ ID NO.4, and the gene encoding the mutant protein is shown in SEQ ID NO.3. The amino acid sequence of the mutant (S275A) of the glycosyltransferase PjvmUGT45 is shown in SEQ ID NO.6, and the gene encoding the mutant protein is shown in SEQ ID NO.5.
[0035] Based on this, the present invention provides a glycosyltransferase PjvmUGT45 of Panax notoginseng and a mutant thereof, wherein the glycosyltransferase PjvmUGT45 is derived from Panax notoginseng, and its amino acid sequence is shown in SEQ ID NO.2;
[0036] The mutant of the glycosyltransferase PjvmUGT45 has an amino acid sequence as shown in SEQ ID NO.4 or SEQ ID NO.6; the mutant of PjvmUGT45 is mutant S275A or K280A, which is obtained by site-directed mutagenesis of the amino acid sequence shown in SEQ ID NO.2, and the mutant S275A is obtained by mutating the serine (Ser, S) at position 275 of the amino acid sequence shown in SEQ ID NO.2 to alanine (Ala, A); the mutant K280A is obtained by mutating the lysine (Lys, K) at position 280 of the amino acid sequence shown in SEQ ID NO.2 to alanine (Ala, A).
[0037] The preferred mutant is K280A, which has higher enzyme activity than the wild-type PjvmUGT45, and its amino acid sequence is shown in SEQ ID NO.4.
[0038] In addition, the present invention also provides a gene encoding the glycosyltransferase PjvmUGT45 or a mutant thereof of the present invention; wherein the gene encoding the glycosyltransferase PjvmUGT45 has a nucleotide sequence as shown in SEQ ID NO.1;
[0039] The gene encoding the glycosyltransferase PjvmUGT45 mutant (K280A) has a nucleotide sequence as shown in SEQ ID NO.3;
[0040] The gene encoding the glycosyltransferase PjvmUGT45 mutant (S275A) is a nucleotide sequence shown in SEQ ID NO.5.
[0041] In addition, the present invention also provides a biological material for expressing the glycosyltransferase PjvmUGT45 or its mutant as described in the present invention, which includes an expression cassette, a recombinant vector, a transgenic cell line or a transgenic engineered bacterium carrying a target gene as shown in SEQ ID NO.1, SEQ ID NO.3, or SEQ ID NO.5, and is used to express the target gene to obtain the glycosyltransferase PjvmUGT45 or its mutant.
[0042] The biological material preferably carries an expression cassette, recombinant vector, transgenic cell line or transgenic engineering bacteria of the target gene as shown in SEQ ID NO.1 or SEQ ID NO.3; more preferably carries an expression cassette, recombinant vector, transgenic cell line or transgenic engineering bacteria of the target gene as shown in SEQ ID NO.3.
[0043] In addition, the present invention also provides an application of the glycosyltransferase PjvmUGT45 or its mutant or the biological material as described in the present invention in the biosynthesis of ginsenosides, especially in the in vitro biosynthesis of rare ginsenosides Rh2 or F2.
[0044] The application comprises the following steps:
[0045] Using protopanaxadiol saponin PPD or ginsenoside CK as a substrate and UDP-Glc as a glycosyl donor, the glycosyltransferase PjvmUGT45 or a mutant thereof as described in the present invention is used as a catalytic enzyme to catalyze the substrate in vitro to generate rare ginsenoside Rh2 or F2.
[0046] In some embodiments, protopanaxadiol saponin PPD is used as a substrate, UDP-Glc is used as a glycosyl donor, and the glycosyltransferase PjvmUGT45 described in the present invention or a mutant with an amino acid sequence as shown in SEQ ID NO.4 is used as a catalytic enzyme to catalyze the substrate in vitro to produce rare ginsenoside Rh2.
[0047] In some embodiments, ginsenoside CK is used as a substrate, UDP-Glc is used as a glycosyl donor, and the glycosyltransferase PjvmUGT45 described in the present invention or a mutant with an amino acid sequence as shown in SEQ ID NO.4 is used as a catalytic enzyme to catalyze the substrate in vitro to produce rare ginsenoside F2.
[0048] The mutant having the amino acid sequence as shown in SEQ ID NO. 4 is preferably used as the catalytic enzyme.
[0049] The following are examples
[0050] Example 1 Cloning of the full-length cDNA of the PjvmUGT45 gene
[0051] Based on the transcriptome data of Glehnia littoralis, primers were designed according to the unigene sequence of PvjmUGT45. The designed primer sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. According to the primers, cDNA was used as a template for PCR to amplify the full-length sequence of the target fragment. The PCR reaction system is shown in Table 1, and PCR amplification was performed according to the PCR reaction program in Table 2. After the PCR amplification reaction, 4 μL of the reaction solution was taken, 1 μL of 5×Loading Buffer was added to mix, and the mixture was spotted into the injection hole of 1% agarose gel, and 5 μL of Marker (DL 2,000) was added. Electrophoresis was performed at 120V for 15 minutes. After the electrophoresis was completed, the electrophoresis results were displayed in the agarose gel imaging system. If the band was around 1,400 bp, it can be preliminarily considered that the full-length amplification of the target gene was successful. The details are as follows:
[0052] Using Glehnia littoralis cDNA as a template, amplification was performed using a high-fidelity DNA polymerase, where the amplification primer sequences were:
[0053] F primer: 5'-CGGGATCCATGGAGAGAGAAATGTTGAGCAAAA-3', R primer: 5'-
[0054] ACGCGTCGACTCAGGAGGAAACAAGCTTTGAAAT-3',
[0055] The amplification system is shown in Table 1 , and the gene amplification PCR program is shown in Table 2 .
[0056] Table 1 Gene amplification system
[0057]
[0058]
[0059] Table 2 Gene amplification PCR program
[0060]
[0061] The amplified product was purified and recovered using a DNA purification kit. The electrophoresis results of the amplification conditions are shown in Figure 1 .
[0062] The amplified product was sequenced for the entire length of the gene, and the sequencing results showed that the gene contained an ORF (nucleotide sequence shown in SEQ ID NO.1) with a length of 1368 bp, and the gene encoded a protein containing 455 amino acids (amino acid sequence shown in SEQ ID NO.2), the molecular weight of the protein was 50.97 kDa, and the isoelectric point was 5.31. The FIMO (Find Individual Motif Occurrences) software analysis showed that the protein sequence had a PSPG box (motif: WAPQAKILGHSSTGGFVS HCGWSSVTESSSYGVPVIAIPMNFDQ (p = 4.49e-21)), and was a UGT candidate gene (recorded as gene PjvmUGT45), and the protein encoded by it was named PjvmUGT45.
[0063] Example 2 Construction of prokaryotic expression vector of PjvmUGT45 gene
[0064] After the sequence cloning operation is completed, the target fragment is further cut off by double enzyme digestion with two specific enzyme cutting sites for subsequent prokaryotic expression. The double enzyme cutting sites selected in this embodiment are BamHI and SalI, and the double enzyme cutting system is shown in Table 3. After the configuration is completed, the sample is placed in a 37°C constant temperature incubator for two hours to ensure that it is fully digested.
[0065] Table 3 Double enzyme digestion system
[0066] Reagents volume Target plasmid fragment 6ul BamHI endonuclease 1ul Sal I endonuclease 1ul 10×Buffer 3ul <![CDATA[RNase free ddH2O]]> 19ul total 30ul system
[0067] The expression vector pCold-TF was digested with restriction endonucleases BamHI and SalI. The digestion conditions were to stand in a constant temperature (37° C.) incubator for 2 hours. The digestion system is shown in Table 4.
[0068] Table 4 Enzyme digestion system
[0069] Reagents volume Expression vector 5.4ul BamHI endonuclease 1ul Xhol endonuclease 1ul 10×Buffer 2ul <![CDATA[RNase free ddH2O]]> 12.6ul
[0070] The PjvmUGT45 gene fragment that had been digested by the two endonucleases was ligated with the expression vector after digestion. The enzyme ligation system is shown in Table 5.
[0071] Table 5 Enzyme-linked system of expression vector
[0072] Reagents volume Target gene 6.8ul Expression vector 1.2ul T4 DNA Ligase 1.0ul 10×T4 DNA Ligase Buffer 1.0ul
[0073] The enzyme-linked system was placed in an ice bath at 16°C overnight for 18 hours to obtain the product recombinant expression vector pColdPjvmUGT45.
[0074] The obtained recombinant expression vector was transformed into JM109 competent cells, and then a single large colony was obtained through LB solid culture medium, and then spot culture was performed to obtain the target bacterial solution. In the clean bench, a sterile pipette tip was used to dip a larger single colony with good growth on the plate, and then one end of the pipette tip with the bacteria was dipped into 2mL of liquid LB culture medium (Amp resistance). Usually 4-6 colonies were picked, and then cultured in a 37°C shaker at 180rpm for 8-14 hours. Then, PCR identification of the bacterial solution and plasmid sequencing were performed to screen the positive clones that were successfully connected. Then, the plasmid was extracted, and the positive plasmid was transferred into the Escherichia coli expression strain BL21 cells, and a single large colony was obtained through LB solid culture medium to obtain the recombinant strain BL21-pColdTF-PjvmUGT45.
[0075] The expression vector, transformed cells, plasmid and expression strain used in this embodiment are not particularly limited. Any biological material that can express the target gene of the present invention and encode the PjvmUGT45 protein in the prior art can be used. For example, in this embodiment, the expression vector pColdTF can be replaced by pET-28a, and the transformed cells and expression strains can be replaced by Saccharomyces cerevisiae and Bacillus subtilis.
[0076] In the present invention, the preferred expression vector is pColdTF-UGT. The pColdTF-UGT vector is optimal in heterologous expression, and the target protein induced to be expressed is soluble.
[0077] Example 3 Induced expression of PjvmUGT45 gene and protein purification
[0078] (1) Protein induction expression
[0079] A single large colony was picked from the solid culture medium cultured in Example 2, inoculated into a 10 mL centrifuge tube containing 2 mL of LB liquid culture medium (Amp resistance), and placed in a 37 ° C constant temperature shaking incubator for overnight culture. The next day, the 2 mL of cultured bacterial solution was added to a conical flask containing 100 mL of LB liquid culture medium (Amp resistance), and placed in a 37 ° C constant temperature shaking incubator for culture for 3-4 hours. When the OD value was between 0.6 and 0.8, the conical flask was taken out and placed in a sterile refrigerator to cool to 4-6 ° C, and then 30 ul of IPTG (isopropyl-β-D-thiogalactoside) was added to induce expression at 16 ° C, 180 rpm for 18 hours.
[0080] (2) Protein extraction
[0081] The 100mL cultured bacterial solution was centrifuged at 8000rpm and 4℃ for 20 minutes. After discarding the supernatant, the precipitated bacteria were washed and lysed with the prepared Lysis Buffer, and PMSF solution was added to a final volume of 20ml. The dissolved bacterial solution was then subjected to low-temperature and high-pressure cell disruption. The disruption conditions were: power 800W. Wait until the bacterial solution becomes relatively clear and transparent. The remaining solution was subjected to ultra-high-speed centrifugation at 4℃ and 15000g. The supernatant was taken and the precipitate was discarded. The supernatant was the extracted protein, and 1mL of the supernatant was retained for electrophoresis verification.
[0082] (3) Protein purification
[0083] Filter the broken bacterial solution through a 0.45um filter membrane using a clean and sterile syringe. First, rinse out the storage buffer with 3-5 column volumes of deionized water. Then use at least 5 column volumes of Lysis Buffer to equilibrate the chromatographic column. After equilibration, load the extracted protein onto the Ni column, and the loading amount should not exceed the binding capacity of the column. A large sample volume may cause a large back pressure, resulting in failure to pass through the column smoothly. After all samples have passed through the injection, wash the column with Wash Buffer, and then use Elution Buffer to elute in one step or linear gradient. In one-step elution, 5 column volumes of eluent are usually sufficient. Gradient elution can use a small gradient, such as 20 column volumes or more, to separate proteins with different binding strengths.
[0084] (4) Protein concentration
[0085] The purified protein was poured into an ultracentrifugal filter tube and concentrated by ultrafiltration at 25°C and 4000 g for 30 minutes in a high-speed centrifuge. The protein concentration was measured using an ELISA instrument, as shown in Table 6.
[0086] Table 6 Absorbance of prokaryotic expression purified protein samples
[0087]
[0088] SDS-PAGE electrophoresis was performed on the supernatant samples after disruption and the samples after protein purification. The results were as follows: Figure 2 As shown, the vector used expressed the corresponding target protein, which was about 51KDa in size, which was basically consistent with the predicted target protein size of 50.97KDa. Except for the absence of obvious target protein bands in the supernatant samples, the expression levels of the other protein samples of the two vectors met the requirements of the subsequent enzyme activity experiments.
[0089] Example 4 In vitro enzyme activity detection of PjvmUGT45 gene expression protein
[0090] The candidate substrates of PjvmUGT45 protein are ginsenoside CK, ginsenoside PPD, ginsenoside Rg3, ginsenoside Rd, etc., and UDP-Glc and UDP-GlcA are used as sugar donors, and the corresponding enzyme activity experiments are carried out. The specific reaction groups are as follows:
[0091] Using UDP-Glc or UDP-GlcA as the sugar donor, ginsenoside CK, ginsenoside PPD, ginsenoside Rg3, and ginsenoside Rd as substrates, and PjvmUGT45 protein as the glycosyltransferase, the enzymatic reaction system was prepared according to the system shown in Table 7.
[0092] Table 7 Enzymatic reaction system
[0093]
[0094] After preparation and mixing, the above system was placed at 37°C for reaction for 12 hours. After the reaction was completed, it was placed at 100°C for heating to inactivate the residual enzyme. Each group of reaction products was centrifuged at 12000rpm for 3-5 minutes, and then extracted with chromatographic grade methanol. The organic phase was placed in a new RNase free centrifuge tube, placed in a vacuum dryer for evaporation and evaporated, and then dissolved with 150ul of chromatographic grade methanol. After filtering through a 0.22um organic filter membrane, it was injected into a liquid phase injection bottle for use.
[0095] The products after the reaction of each group were analyzed by high performance liquid chromatography to detect whether new products were generated, wherein the high performance liquid chromatography detection conditions were: using a chromatographic column model of TC-C18 (4.6×250 mm, 5 um), a detection column temperature of 35° C., a detection wavelength of 203 nm, an injection volume of 100 ul, a flow rate of 1.0 mL / min, and using ultrapure water and acetonitrile as mobile phases. The elution program was: 0-20 min (water: 60%-30%, acetonitrile: 40%-70%), 20-40 min (water: 30%-0%, acetonitrile: 70%-100%), 40-60 min (water: 0%-60%, acetonitrile: 100%-40%).
[0096] The results of HPLC after the above reactions showed that only when the sugar donor was UDP-Glc and the substrates were ginsenoside CK and PPD, the PjvmUGT45 protein of the present invention could catalyze ginsenoside CK to F2 and PPD to Rh2, and the glycosyl modification processes were as follows: Figure 3 and Figure 4 shown.
[0097] in Figure 3Figure a shows the glycosylation process of ginsenoside CK under the action of PjvmUGT45 protein; Figure b shows the peak time of ginsenoside standard CK under the same detection conditions, which is 22.383 minutes; Figure c shows the peak time of rare ginsenoside F2 standard, which is 12.6 minutes; Figure 3 Where d is the substance detected after the enzymatic reaction. Figure 3 As shown in Figure d, after the addition of PjvmUGT45 protein, the peak values of the sugar donor and the reaction substrate ginsenoside CK decreased significantly. At the same time, a group of new peaks appeared at 12.6 minutes. By comparing the peak graph of the F2 standard, it was determined that the new catalytic product formed was F2.
[0098] Figure 4 Figure a shows the glycosylation process of ginsenoside PPD under the action of PjvmUGT45 protein; b shows the peak time of ginsenoside PPD standard under the same detection conditions, which is 37.217 minutes; c shows the peak time of rare ginsenoside standard Rh2, which is 23.483 minutes; d shows the substance detected after enzymatic reaction.
[0099] Depend on Figure 4 As shown in Figure d, after the addition of PjvmUGT45 protein, the peak values of the sugar donor and the reaction substrate ginsenoside PPD decreased significantly, and a new group of peaks appeared at 23.483 minutes. The peak graph of the reference standard Rh2 confirmed that the new enzyme activity product was Rh2.
[0100] The results of HPLC detection showed that in the above reaction groups, the glycosyltransferase was Pjvm UGT45 protein. When the sugar donor was UDP-Glc and the substrates were ginsenoside CK and PPD, new products could be formed. The substrate ginsenoside CK could be catalyzed into F2 and PPD could be catalyzed into Rh2, among which F2 and Rh2 were rare ginsenosides.
[0101] Furthermore, the catalytic efficiency of glycosyltransferase PjvmUGT45 was compared with that of existing glycosyltransferases PgUGT74AE2 and UGTPg45, using glycosyltransferases PgUGT74AE2 and UGTPg45 as positive controls. The specific catalytic conditions were the same as those in Table 7 above, and the measurement results were as follows: Figure 5 shown.
[0102] Figure 5In the figure, a represents the production of F2 by glycosyltransferase PjvmUGT45 using substrate CK; b represents the production of F2 by glycosyltransferase PgUGT74AE2 using substrate CK; c represents the production of F2 by glycosyltransferase UGTPg45 using substrate CK; d represents the production of Rh2 by glycosyltransferase PjvmUGT45 using substrate PPD; e represents the production of Rh2 by glycosyltransferase PgUGT74AE2 using substrate PPD; f represents the production of Rh2 by glycosyltransferase UGTPg45 using substrate PPD; g represents the relative conversion rate of PjvmUGT45, PgUGT74AE2 and UGTPg45 using relative quantification method.
[0103] Figure 5 The results showed that, using the relative quantitative method, the relative conversion rate of PjvmUGT45 was 100%, and the conversion rates of glycosyltransferases PgUGT74AE2 and UGTPg45 from substrate CK to F2 were 30.3% and 11.9%, respectively, and the conversion rates of glycosyltransferases PgUGT74AE2 and UGTPg45 from substrate PPD to Rh2 were 66.4% and 35.4%, respectively. It can be seen that the conversion rates of glycosyltransferase PjvmUGT45 from CK to F2 were 3.30 times and 8.40 times of those of PgUGT74AE2 and UGTPg45, respectively; the conversion rates of glycosyltransferase PjvmUGT45 from PPD to Rh2 were 1.51 times and 2.82 times of those of PgUGT74AE2 and UGTPg45, respectively.
[0104] Compared with the catalytic efficiency of two known glycosyltransferases with the same catalytic function in Panax ginseng, UGTPg45 and PgUGT74AE2, we surprisingly found that the catalytic efficiency of the present glycosyltransferase PjvmUGT45 was significantly increased.
[0105] Example 5 Glycosyltransferase mutants
[0106] For glycosyltransferases, a 1% difference in their amino acid sequence may result in significant changes in their functions. We screened and identified mutations in important amino acid sites of the PjvmUGT45 protein and compared the differences in enzyme activity of different PjvmUGT45 mutants. The details are as follows:
[0107] Through molecular docking prediction analysis, the active sites that may exist on the surface of PjvmUGT45 protein were preliminarily determined to be Asn247, Tyr273, Ser275, Lys280, Cys332, Gln334, His349, Asn353, Ser354, Glu357, Asp373, and Gln374. Further, site-directed mutagenesis of key amino acids was performed by alanine scanning mutagenesis and overlap extension PCR (gene splicing by overlap extension PCR, SOE PCR). The results showed that a total of 6 sites, including Ser275, Lys280, Gln334, His349, Ser354, and Asp373, were successfully mutated to alanine (Ala, A), and 6 mutants (S275A, K280A, Q334A, H349A, S354A, and D373A) were obtained;
[0108] The unmutated PjvmUGT45 protein (wild type) was used as a positive control to determine the catalytic activity of each mutant on substrates CK and PPD. The specific catalytic reaction conditions were the same as those in Table 7 above. The enzyme activity determination results were as follows: Figure 6 .
[0109] The results of HPLC detection of protease activity of mutants S275A and K280A using ginsenoside CK as substrate are as follows: Figure 7 As shown, Figure 7 In the middle, a is the ginsenoside CK standard product, b is the rare ginsenoside F2 standard product, c is the mutant S275A experimental group, and d is the mutant K280A experimental group.
[0110] The results of HPLC detection of the protease activity of mutants S275A and K280A using ginsenoside PPD as substrate are as follows: Figure 8 As shown, Figure 8 In the middle, a is the standard product of ginsenoside PPD, b is the standard product of rare ginsenoside Rh2, c is the mutant S275A experimental group, and d is the mutant K280A experimental group.
[0111] Depend on Figure 6 It can be seen that compared with the wild-type PjvmUGT45 protein, the protease activity of the above mutants has changed significantly, among which the PjvmUGT45 mutants (Q334A, H349A, S354A, D373A) have low catalytic efficiency (less than 1%) for substrates CK and PPD, no obvious reaction products, and almost no catalytic effect.
[0112] Although the catalytic efficiency of the PjvmUGT45 mutant (S275A) for substrates CK and PPD is lower than that of the wild-type PjvmUGT45 protease activity, its catalytic efficiency is still higher than that of PgUGT74AE2 and UGTPg45. The catalytic efficiency of the PjvmUGT45 mutant (K280A) for substrates CK and PPD is significantly improved, which is 1.32 times and 1.53 times that of the wild-type glycosyltransferase PjvmUGT45, respectively. Through this experiment, we unexpectedly discovered a mutation site K280A that can further improve the catalytic efficiency of the wild-type PjvmUGT45. The catalytic efficiency of this mutant for substrates CK and PPD is significantly higher than that of the wild-type glycosyltransferase PjvmUGT45.
[0113] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.
Claims
1. A glycosyltransferase PjvmUGT45 from Panax notoginseng and its mutants, It is characterized in that The glycosyltransferase PjvmUGT45 has an amino acid sequence as shown in SEQ ID NO.2; The amino acid sequence of the mutant is shown in SEQ ID NO.4 or SEQ ID NO.
6.
2. The glycosyltransferase PjvmUGT45 and its mutants as claimed in claim 1, It is characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO.
4.
3. A gene encoding the glycosyltransferase PjvmUGT45 or a mutant thereof as claimed in claim 1, It is characterized in that The gene encoding the glycosyltransferase PjvmUGT45 has a nucleotide sequence as shown in SEQ ID NO.1; The gene encoding the glycosyltransferase PjvmUGT45 mutant has a nucleotide sequence as shown in SEQ ID NO.3 or SEQ ID NO.
5.
4. The gene according to claim 3, It is characterized in that The gene encoding the glycosyltransferase PjvmUGT45 mutant has a nucleotide sequence as shown in SEQ ID NO.
3.
5. A biological material expressing the glycosyltransferase PjvmUGT45 or a mutant thereof according to claim 1, It is characterized in that It includes an expression cassette, a recombinant vector, a transgenic cell line or a transgenic engineering bacterium carrying a target gene as shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.
5.
6. The biomaterial according to claim 5, It is characterized in that The carried target gene is shown as SEQ ID NO.1 or SEQ ID NO.
3.
7. Use of the glycosyltransferase PjvmUGT45 and its mutants as claimed in claim 1 or the biomaterial as claimed in claim 5 in the biosynthesis of rare ginsenosides, It is characterized in that The rare ginsenoside is Rh2 or F2.
8. The use according to claim 7, It is characterized in that The following steps are involved: Using protopanaxadiol saponin PPD or ginsenoside CK as a substrate and UDP-Glc as a glycosyl donor, the glycosyltransferase PjvmUGT45 or a mutant thereof as claimed in claim 1 is used as a catalytic enzyme to catalyze the substrate in vitro to generate rare ginsenoside Rh2 or F2.
9. The use as claimed in claim 8, It is characterized in that The glycosyltransferase PjvmUGT45 or its mutant catalyzes the substrate PPD to form the rare ginsenoside Rh2 in vitro, or catalyzes the substrate CK to form the rare ginsenoside F2 in vitro.
10. The use according to claim 8 or 9, It is characterized in that The amino acid sequence of the mutant is shown in SEQ ID NO.4.