Glycosyltransferase Pq3-O-UGT2 mutant with high yield of rare ginsenoside Rg3 and its application

Abstract

The present invention belongs to the field of enzyme genetic engineering and biotechnology, and discloses a glycosyltransferase Pq3-O-UGT2 mutant that produces a high yield of rare ginsenoside Rg3, the amino acid sequence of which is any one of the following: the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.3, and an amino acid sequence with a degree of identity of 50% or more to the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.3. The mutant of the present invention reduces the conversion efficiency of the original Pq3-O-UGT2 enzyme to ginsenoside F2 and improves the conversion efficiency to ginsenoside Rh2. That is, the reaction of adding a No. 2 glucose group to the C-2 hydroxyl group of the C3-O-glucose group of the ginsenoside Rh2 substrate is improved, catalyzing the production of a high-medicinal-value product, the rare ginsenoside Rg3, thereby improving the efficiency and selectivity of the enzymatic synthesis of the rare ginsenoside Rg3.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme genetic engineering and biotechnology, and in particular to a glycosyltransferase Pq3-O-UGT2 mutant capable of highly producing the rare ginsenoside Rg3, its encoding gene, its construction method and its application. Background Art

[0002] Ginsenosides are important active ingredients in plants such as ginseng, American ginseng, and Panax notoginseng. They are a series of triterpenoid glycoside compounds formed by aglycones and sugars, containing a 17-carbon tetracyclic sterane steroid nucleus. The active components formed after metabolic transformation of ginsenosides are more biologically active, with significant physiological effects such as anti-cancer, anti-aging, anti-fatigue, and anti-tumor effects, as well as immunostimulation and immunity enhancement. They have high application value in food, pharmaceutical, and health supplements. Ginsenosides include both standard ginsenosides and rare ginsenosides. Standard ginsenosides include Ra, Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, and Rf, while rare ginsenosides include Rg3, Rh1, CK, Rh2, Rg5, Rk1, Rh3, Rk2, Rh4, Rk3, aPPT, and aPPD. Standard ginsenosides have low anticancer activity and are poorly absorbed and utilized by the human body. Rare ginsenosides are metabolic derivatives of the original ginsenosides. Their natural concentrations are very low, making them difficult to extract directly from the plant. They can only be obtained through the conversion or metabolism of the original ginsenosides. Rare ginsenosides are readily absorbed by the human body and therefore possess higher biological activity. Among the many rare ginsenosides, Rg3 exhibits significant anti-cancer effects and can directly target drug-resistant tumor cells, reversing tumor resistance. Consequently, it is widely used in cancer treatment and holds significant health and economic value.

[0003] The development and application of rare ginsenoside products are significantly limited by the extremely low concentrations of rare ginsenosides in natural plants (<0.1%), the long raw material growth cycle, difficulty in extraction, low preparation efficiency, numerous byproducts, high production costs, and environmental pollution. With the identification of key enzymes in ginsenoside biosynthesis, genetic engineering and synthetic biology have become important approaches for the efficient production of rare ginsenosides. Enzyme-catalyzed synthesis mediated by glycosyltransferases (UGTs) offers numerous advantages, including regio- and stereo-selectivity, high catalytic efficiency, and simplified product purification. In the enzymatic conversion of ginsenosides, a glycosyltransferase Pq3-O-UGT2 derived from American ginseng can add glucosyl group 2 (glucosyl group 2 is a glucosyl group that forms a β(1-2) glycosidic bond with the C2 hydroxyl group of glucosyl group 1, and glucosyl group 1 specifically refers to a glucosyl group that directly forms a β-glycosidic bond with the C3 hydroxyl group of Rh2) to the C3 hydroxyl group of ginsenoside Rh2 to generate the rare ginsenoside Rg3 ( Figure 1a). Therefore, Pq3-O-UGT2 catalyzes the last step of the Rg3 synthesis pathway, which determines whether the rare ginsenoside Rg3 can be produced and its production efficiency. However, Pq3-O-UGT2 can not only catalyze the production of rare ginsenoside Rg3, but also catalyze the production of prototype ginsenoside Rd ( Figure 1 b) Under the same conditions, wild-type Pq3-O-UGT2 achieved a conversion rate of 69% for the reaction to produce rare ginsenoside Rg3, while it achieved a conversion rate of 92% for the reaction to produce prototype ginsenoside Rd. This indicates that wild-type Pq3-O-UGT2 exhibits higher selectivity for the reaction to produce prototype ginsenoside Rd, significantly hindering the biosynthesis of rare ginsenoside Rg3. Therefore, the development of Pq3-O-UGT2 mutants with significantly improved Rg3 synthesis efficiency and substrate selectivity is urgently needed to provide technical support for the efficient and large-scale biosynthesis of rare ginsenosides. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies in the prior art and provide a glycosyltransferase Pq3-O-UGT2 mutant, encoding gene, construction method and application for high-yield rare ginsenoside Rg3.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A glycosyltransferase Pq3-O-UGT2 mutant capable of producing a high yield of rare ginsenoside Rg3, wherein the amino acid sequence of the mutant is any one of the following a and b;

[0007] a. the amino acid sequence shown in SEQ ID NO. 2, and amino acid sequences that are 50% or more identical to the amino acid sequence shown in SEQ ID NO. 2;

[0008] b. The amino acid sequence shown in SEQ ID NO. 3, and amino acid sequences that are 50% or more identical to the amino acid sequence shown in SEQ ID NO. 3.

[0009] A gene encoding the glycosyltransferase Pq3-O-UGT2 mutant as described above.

[0010] An expression vector containing the encoding gene as described above.

[0011] The recombinant strain carries the expression vector described above.

[0012] The method for constructing the glycosyltransferase Pq3-O-UGT2 mutant as described above comprises the following steps:

[0013] First, specific PCR primers containing the predetermined mutation site were designed and synthesized. These primers were used to amplify the wild-type Pq3-O-UGT2 gene via overlap extension PCR, using the wild-type Pq3-O-UGT2 gene as a template. Subsequently, homologous recombination was used to efficiently insert the resulting PCR product into a selected expression vector. This vector was then transformed into host cells for further amplification. DNA sequencing was performed on the transformed positive clones to ensure successful mutation.

[0014] The application of the glycosyltransferase Pq3-O-UGT2 mutant described above in the production of rare ginsenoside Rg3.

[0015] Use of the glycosyltransferase Pq3-O-UGT2 mutant as described above in the enzymatic synthesis and / or conversion of rare ginsenoside Rg3.

[0016] Furthermore, a glucose group No. 2 is added to the C2-hydroxyl direction of the C3-glucose group of the rare ginsenoside Rh2, and the glucose group No. 2 is a glucose group that forms a β(1-2) glycosidic bond with the glucose group No. 1, and the glucose group No. 1 specifically refers to a glucose group that directly forms a β-glycosidic bond with the C3-hydroxyl of the protopanaxadiol saponin (PPD-type saponin).

[0017] The advantages and positive effects achieved by the present invention are:

[0018] 1. The glycosyltransferase Pq3-O-UGT2 mutant of the present invention reduces the conversion efficiency of the original Pq3-O-UGT2 enzyme for ginsenoside F2 (synthesizing the prototype ginsenoside Rd) and improves the conversion efficiency for ginsenoside Rh2 (synthesizing the rare ginsenoside Rg3). Specifically, the mutant enhances the reaction of adding a second glucosyl group to the C2-hydroxyl group of the C3-O-glucosyl group in the ginsenoside Rh2 substrate, catalyzing the production of the highly medicinally valuable product, the rare ginsenoside Rg3 (CAS 14197-60-5). This improves the efficiency and selectivity of the enzymatic synthesis of the rare ginsenoside Rg3.

[0019] 2. The existing technology urgently needs to solve how to improve the catalytic activity of glycosyltransferase Pq3-O-UGT2 in the process of adding glucose group No. 2 in the direction of the C3 hydroxyl group of ginsenoside Rh2, while reducing its catalytic activity in catalyzing the synthesis of prototype ginsenoside Rd from ginsenoside F2, and improving the selectivity of the enzyme.

[0020] Based on the three-dimensional structure of the glycosyltransferase Pq3-O-UGT2 independently discovered by the inventors, the original amino acid sequence of the glycosyltransferase Pq3-O-UGT2 was rationally optimized. The inventors mutated the leucine at position 181 to tryptophan (Leu181Trp) or simultaneously mutated the serine at position 175 and the leucine at position 181 to alanine and tryptophan (Ser175Ala / Leu181Trp). These two amino acid changes yielded two new enzymes, designated in this patent as New Enzyme 1 (Leu181Trp) and New Enzyme 2 (Ser175Ala / Leu181Trp). Both enzymes were purified and produced using an E. coli protein expression system. Both enzymes enhance the ability of Pq3-O-UGT2 to catalyze the addition of a 2-glucosyl group to the C2 hydroxyl group of the C3-O glucosyl group of ginsenoside Rh2, forming a β(1-2) glycosidic bond and synthesizing the rare ginsenoside Rg3. Both new enzymes reduce the activity of Pq3-O-UGT2 to add 2-glucosyl groups to the C2 hydroxyl group of the C3-O glucosyl group of ginsenoside F2, forming a β(1-2) glycosidic bond and synthesizing the prototype ginsenoside Rd. The activity data of new enzyme 1 (Leu 181Trp) (leucine at position 181 is replaced by tryptophan) for the enzymatic synthesis of rare ginsenoside Rg3 are shown in Figure 1. cat / K m It is 3.15 times that of wild-type Pq3-O-UGT2, and the enzymatic activity data for the enzymatic synthesis of prototype ginsenoside Rd is cat / K m The activity of the new enzyme 2 (Ser175Ala / Leu 181Trp) (serine at position 175 and leucine at position 181 were mutated to alanine and tryptophan) for the enzymatic synthesis of the rare ginsenoside Rg3 was 0.51 times that of the wild-type Pq3-O-UGT2. cat / K m It is 4.43 times that of wild-type Pq3-O-UGT2, and the enzymatic activity data for the enzymatic synthesis of prototype ginsenoside Rd is cat / K m The activity of these two novel enzymes towards Rh2 was significantly enhanced, while their activity towards F2 was significantly reduced. This modified Pq3-O-UGT2 enzyme has great potential for improving the production of the rare ginsenoside Rg3. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The structure of the ginsenosides in the present invention and the reaction diagram of Pq3-O-UGT2 participating in the glycosylation synthesis of ginsenosides are shown below:

[0022] Among them, a is a schematic diagram of the glycosyltransferase Pq3-O-UGT2 catalyzing the production of rare ginsenoside Rg3 using ginsenoside Rh2 as substrate. The number in the middle of each glucose represents the number of the glucose group, with glucose group No. 2 shown in blue;

[0023] b is a schematic diagram of the glycosyltransferase Pq3-O-UGT2 catalyzing the production of prototype ginsenoside Rd using ginsenoside F2 as substrate. The number between each glucose represents the number of the glucose group, with glucose group No. 2 shown in blue.

[0024] Figure 2 This is an electrophoresis diagram of the PCR amplification of the wild-type Pq3-O-UGT2 gene in Example 1 of the present invention;

[0025] Figure 3 This is a verification diagram of the recombinant plasmid pET28a-Pq3-O-UGT2 in Example 1 of the present invention;

[0026] Figure 4 This is an SDS-PAGE image of the purified protein samples of wild-type Pq3-O-UGT2 and new enzymes 1 and 2 in Example 3 of the present invention;

[0027] Figure 5 Graph showing the relative activity of wild-type Pq3-O-UGT2 catalyzing the substrates ginsenosides Rh2 and F2 in Example 4 of the present invention;

[0028] Figure 6 Graph showing the relative activity of the Pq3-O-UGT2 mutant catalyzing the substrates ginsenosides Rh2 and F2 in Example 4 of the present invention;

[0029] Figure 7 This is a fitting curve diagram of the enzyme kinetic parameters of the Pq3-O-UGT2 mutant catalyzing the substrates ginsenosides Rh2 and F2 in Example 4 of the present invention. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0031] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0032] A glycosyltransferase Pq3-O-UGT2 mutant capable of producing a high yield of rare ginsenoside Rg3, wherein the amino acid sequence of the mutant is any one of the following a and b;

[0033] a. The amino acid sequence shown in SEQ ID NO. 2, and amino acid sequences that are 50% or more identical to the amino acid sequence shown in SEQ ID NO. 2; the amino acid sequence shown in SEQ ID NO. 2 is based on the amino acid sequence of glycosyltransferase Pq3-O-UGT2, with Leu at position 181 mutated to Trp.

[0034] b. The amino acid sequence set forth in SEQ ID NO. 3, and amino acid sequences that are 50% or greater identical to the amino acid sequence set forth in SEQ ID NO. 3. The amino acid sequence set forth in SEQ ID NO. 3 is based on the amino acid sequence of glycosyltransferase Pq3-O-UGT2, with Ser at position 175 and Leu at position 181 simultaneously mutated to Ala and Trp.

[0035] A gene encoding the glycosyltransferase Pq3-O-UGT2 mutant as described above.

[0036] An expression vector containing the encoding gene as described above.

[0037] The recombinant strain carries the expression vector described above.

[0038] The method for constructing the glycosyltransferase Pq3-O-UGT2 mutant as described above comprises the following steps:

[0039] First, specific PCR primers containing the predetermined mutation site were designed and synthesized. Using the wild-type Pq3-O-UGT2 gene as a template, the gene was amplified by overlap extension PCR. Subsequently, homologous recombination was used to efficiently insert the resulting PCR product into the selected expression vector. The vector was then transformed into host cells for further amplification. DNA sequencing was performed on the transformed positive clones to ensure the successful mutation.

[0040] The application of the glycosyltransferase Pq3-O-UGT2 mutant described above in the production of rare ginsenoside Rg3.

[0041] Use of the glycosyltransferase Pq3-O-UGT2 mutant as described above in the enzymatic synthesis and / or conversion of rare ginsenoside Rg3.

[0042] Preferably, a glucose group No. 2 is added to the C2-hydroxyl direction of the C3-glucose group of the rare ginsenoside Rh2, and the glucose group No. 2 is a glucose group that forms a β(1-2) glycosidic bond with the glucose group No. 1, and the glucose group No. 1 specifically refers to a glucose group that directly forms a β-glycosidic bond with the C3-hydroxyl of the protopanaxadiol saponin (PPD-type saponin).

[0043] In the embodiment of the present invention, the relative activities of the two new enzymes were verified by liquid chromatography; the enzyme activity data k of the catalytic reaction of the two new enzymes were determined by liquid chromatography. cat / K m (Michaelis-Menten kinetic parameters), verifying the enzymatic activity data k of the new enzyme 1 for the enzymatic synthesis of rare ginsenoside Rg3 cat / K m It is 3.15 times that of wild-type Pq3-O-UGT2, and the enzymatic activity data for the enzymatic synthesis of prototype ginsenoside Rd is cat / K m The activity of the new enzyme 2 for the enzymatic synthesis of rare ginsenoside Rg3 was 0.51 times that of the wild-type Pq3-O-UGT2. cat / K m It is 4.43 times that of wild-type Pq3-O-UGT2, and the enzymatic activity data for the enzymatic synthesis of prototype ginsenoside Rd is cat / K m The activity of these two novel enzymes is 0.46 times that of wild-type Pq3-O-UGT2. These two novel enzymes can be used for the efficient production of the rare ginsenoside Rg3.

[0044] In addition to expressing, purifying, and preparing the natural state Pq3-O-UGT2 or the two new enzymes of the present invention in Escherichia coli, they can also be expressed and prepared in other biological systems, non-biological systems, cells, cell-free and other expression systems; these new enzymes that significantly promote the synthesis of the target product while weakening side reactions can be introduced into organisms, non-biological organisms, cells, and cell-free through transgenic or other gene introduction means for enzymatic conversion of ginsenosides, and in in vitro systems such as enzymes and enzyme immobilization, for enzymatic conversion of adding glucose group No. 2 to ginsenoside Rh2, including but not limited to the enzymatic conversion process of preparing ginsenoside Rg3.

[0045] Specifically, the relevant preparation and testing are as follows:

[0046] Example 1 Construction of wild-type Pq3-O-UGT2 protein expression vector

[0047] 1. Obtain the amino acid sequence of Pq3-O-UGT2 (NCBI GenBank: ALE15280.1).

[0048] To prevent the circumvention of the scope of protection of this patent by replacing non-essential amino acid sequences of Pq3-O-UGT2, as long as the translated protein amino acid sequence is 50% or more identical to the amino acid sequence of Pq3-O-UGT2, it is considered to belong to Pq3-O-UGT2.

[0049] Whether in the amino acid sequence or spatial structure of Pq3-O-UGT2, changing the sites equivalent to amino acids 175 and 181 or other sites (due to amino acid redundancy, by removing one or any amino acids at other positions, the amino acids 175 and 181 are not necessarily always numbered 175 and 181) to achieve the purpose of improving the catalytic activity and selectivity of Pq3-O-UGT2 for the rare ginsenoside Rg3 falls within the scope of protection of this patent.

[0050] The amino acid sequence of Pq3-O-UGT2 (the bold and italic amino acids are the amino acid sites modified by the present invention, and the underlined parts are the amino acid sequences artificially added, among which the S and L amino acids are the sites protected by this patent; the amino acid It is the protease cleavage site, in order to facilitate the elution of protein during purification; amino acid (6 histidine tags are for the convenience of purification):

[0051]

[0052] 2. Amplify the Pq3-O-UGT2 coding region using the primers listed in Table 1. The 5' and 3' ends of the amplified fragment will contain restriction enzyme sites for NcoI and BamHI, respectively. Amplify the Pq3-O-UGT2 gene (GenBank: ALE15280.1) by PCR using a cDNA sample from American ginseng roots as a template. Primers are designed as shown in Table 1.

[0053] Table 1 Primer sequences of pET28a-Pq3-O-UGT2

[0054]

[0055] (Add six histidine tags to the primers To facilitate subsequent purification, the underlined sequences are restriction enzyme sites for NcoI and BamHI).

[0056] 3. Double-digest the pET28a expression vector with NcoI and BamHI, and amplify the Pq3-O-UGT2 coding region by PCR. Then, seamlessly insert the Pq3-O-UGT2 coding region into the pET28a vector between the NcoI and BamHI restriction sites to construct the expression vector pET28a-Pq3-O-UGT2, which can express the glycosyltransferase Pq3-O-UGT2 in E. coli.

[0057] The reaction system for PCR amplification was 50 μL, as shown in Table 2. After PCR amplification, a 1406 bp Pq3-O-UGT2 gene fragment (including the target gene, restriction enzyme cutting site, 6×His tag, and homology arms) was obtained.

[0058] Table 2 PCR reaction system and procedure

[0059]

[0060] The PCR product was subjected to agarose gel electrophoresis, and a band of the wild-type Pq3-O-UGT2 encoding gene was observed, which was approximately 1500 bp (see Figure 2 ), and then the PCR product was recovered using a DNA gel recovery kit. At the same time, the vector plasmid pET-28a was double-digested (NcoI / BamHI) to obtain a linearized vector fragment, and then the linearized vector fragment was recovered using a DNA gel recovery kit. According to the requirements of the seamless cloning kit, the gene fragment and the vector fragment were mixed in proportion, and a circular recombinant plasmid was formed under the action of the seamless cloning reaction reagent. The reaction product was transformed into DH5α competent cells, and the correctness of the expression plasmid was verified by kanamycin plate culture medium screening, plasmid extraction and sequencing to complete the construction of the wild-type Pq3-O-UGT2 glycosyltransferase expression vector (see Figure 3 ).

[0061] Example 2 Construction of the expression vector for the mutant protein of glucose transferase Pq3-O-UGT2

[0062] 1. Using wild-type pET28a-Pq3-O-UGT2 as a template, the site-directed mutagenesis primers used to design two new enzymes, Leu181Trp and Ser175Ala / Leu181Trp, are shown in Table 3.

[0063] Table 3 Mutant primer sequences are as follows: (The underlined part indicates the mutation site)

[0064]

[0065] 2. Dissolve the primers in Table 3 with ddH2O and dilute the primer concentration to 10 μM. Use two pairs of mutation primers to perform PCR amplification using pET28a-Pq3-O-UGT2 as the template. The system and amplification procedure are the same as those in Table 2. After nucleic acid electrophoresis and gel excision recovery of the PCR product, the purified gene fragment is obtained. Under the action of seamless cloning reaction reagent, the gene fragment is connected to the linearized pET-28a vector to form a circular recombinant plasmid, which is then transformed into DH5α competent cells. After screening with kanamycin plate culture medium, plasmid extraction and sequencing verification, a recombinant plasmid containing the mutation site, namely pET28a-Pq3-O-UGT2, is obtained. L181W 、pET28a-Pq3-O-UGT2 S175A / L181W The amino acid sequences encoded by them are shown in SEQ ID No. 2 and SEQ ID No. 3 respectively.

[0066] The formula of the kanamycin plate culture medium used in the present invention is: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 15 g / L agar powder, and 50 mg / L kanamycin.

[0067] Example 3 Inducible Expression and Purification of Wild-Type Pq3-O-UGT2 and New Enzyme 1 (Leu181Trp) and New Enzyme 2 (Ser175Ala / Leu181Trp)

[0068] 1. Transform the corresponding expression plasmid into E. coli BL21 (DE3) competent cells for transformation. After ice bathing for 30 minutes, heat shock at 42°C water bath for 75 seconds, ice bath again for 2 minutes, add 1 ml of LB liquid medium, and culture at 37°C, 230 rpm for 45 minutes. Then, spread the bacterial liquid evenly on kanamycin plate medium. After culture at 37°C for 12-16 hours, pick a single clone and transfer it to 5 ml of kanamycin-resistant LB liquid medium. Culture at 37°C, 230 rpm for 12-16 hours, and then store the glycerol stock.

[0069] Among them, the formula of the kanamycin-resistant LB liquid culture medium used in the present invention is:

[0070] Tryptone 10g / L, sodium chloride 10g / L, yeast extract 5g / L, kanamycin 50mg / L, solvent is water.

[0071] 2. Pick the E. coli BL21 (DE3) glycerol bacteria saved in the previous step and inoculate it into 5 ml of kanamycin-resistant LB liquid medium, culture it at 37°C, 230 rpm for 12 h, inoculate it into 1 L of kanamycin-resistant LB liquid medium at a 1% inoculum volume, and culture it at 37°C, 230 rpm. When OD 600 When the pH reaches 0.6-0.8, cool the culture to 16°C and add the inducer IPTG (isopropyl β-D-1-thiogalactopyranoside) to a final concentration of 0.5 mM. Incubate at 16°C and 230 rpm for 16-18 hours. After induction, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, and collect the cells.

[0072] 3. After resuspending the cells in lysis buffer (50mM Tris-HCl pH 8.0, 150mM NaCl), the cells were ultrasonically disrupted to obtain a cell lysate. The supernatant was centrifuged at 15,000 rpm and 4°C for 30 minutes. The resulting supernatant was incubated with Ni-NTA at 4°C for 2 hours. After removing impurities with washing buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 30mM imidazole), the target protein was eluted with elution buffer (50mM Tris-HCl pH 8.0, 150mM NaCl, 500mM imidazole). The imidazole in the mixture was removed by dialysis. After quick freezing in liquid nitrogen, the supernatant can be stored at -80°C for a long time. The above steps can purify the wild-type Pq3-O-UGT2 and the new enzyme 1 (Leu181Trp) and the new enzyme 2 (Ser175Ala / Leu181Trp), and the purity is detected by SDS-PAGE. Figure 4 For wild-type Pq3-O-UGT2 and the two mutants, neoenzyme 1 and neoenzyme 2, SDS-PAGE results showed a clear, single, major band located between the molecular weight markers of 45kD and 65kD, close to 45kD, consistent with the expected molecular weight. This indicates that the purification step was highly efficient, resulting in highly purified target proteins.

[0073] In the present invention, the host bacteria is preferably Escherichia coli BL21, and more preferably includes Escherichia coli Rosetta. The preparation method provided by the present invention can efficiently purify a small amount of soluble protein.

[0074] Example 4 Enzyme Activity Determination of Wild-Type Pq3-O-UGT2, New Enzyme 1 (Leu181Trp) and New Enzyme 2 (Ser175Ala / Leu181Trp)

[0075] 1. Enzymatic conversion rate analysis

[0076] (1) First, the substrates of Pq3-O-UGT2, ginsenosides Rh2 and F2, are used as examples. Natural Pq3-O-UGT2 can catalyze the addition of glucose group 2 to substrates Rh2 and F2, respectively, to generate rare ginsenoside Rg3 and prototype ginsenoside Rd. The chemical reaction formula of the catalytic system is as follows: Figure 1 .

[0077] (2) Reaction Example. The reaction conditions were as follows: At room temperature, 100 μL of the reaction system included 0.5 mM UDPG, 0.5 mM ginsenoside Rh2 or F2, 50 mM Tris-HCl pH 8.0, and 10 μg of purified glucose transferase protein. The biotransformation was carried out in a 37°C water bath for 12 h. Finally, 100 μL of methanol was added, mixed, and vortexed to terminate the reaction. The mixture was centrifuged at 13,000 rpm for 10 min, and the supernatant was collected. The supernatant was used for HPLC analysis.

[0078] (3) Conversion rate = product peak area / (substrate peak area + product peak area).

[0079] (4) First, determine the conversion rate of wild-type Pq3-O-UGT2 to substrates Rh2 and F2. Figure 5 As shown, wild-type Pq3-O-UGT2 can catalyze ginsenosides Rh2 and F2 to produce rare ginsenoside Rg3 and prototype ginsenoside Rd ( Figure 5 a). Under the same conditions, the conversion rate of wild-type Pq3-O-UGT2 in synthesizing rare ginsenoside Rg3 was about 69%, while the conversion rate of wild-type Pq3-O-UGT2 in synthesizing prototype ginsenoside Rd was about 92% ( Figure 5 b) The determination of the product conversion rates of the new enzymes 1 and 2 showed that the conversion rates of the new enzymes 1 and 2 for the rare ginsenoside Rg3 were significantly increased, while the conversion rates for the prototype ginsenoside Rd were reduced. Figure 6 ).like Figure 6 As shown, compared with the wild type, the conversion rate (relative activity) of the new enzyme 1 to Rh2 increased by 1.22 times, while the conversion rate (relative activity) to F2 was 0.56 times that of the wild type; the conversion rate (relative activity) of the new enzyme 2 to Rh2 increased by 1.23 times, and the conversion rate (relative activity) to F2 was 0.69 times that of the wild type. These data show that the conversion rate of the new enzyme 1 and the new enzyme 2 to the rare ginsenoside Rg3 was significantly increased, while the synthesis efficiency of the prototype ginsenoside Rd was reduced. Both the new enzyme 1 and the new enzyme 2 increased the catalytic activity of the rare ginsenoside Rg3 and reduced the activity of the prototype ginsenoside Rd. The selectivity and catalytic activity of the new enzyme 1 and the new enzyme 2 for the synthesis of the rare ginsenoside Rg3 were significantly improved.

[0080] 2. Enzyme kinetic parameters (K cat / K m ) determination:

[0081] Under certain conditions (37°C, 50mM Tris-HCl pH 8.0), the concentration of substrate ginsenoside Rh2 or F2 was set at 20-500μM. Different concentration gradients of ginsenoside Rh2 or F2 were mixed with 100ng of purified wild-type glycosyltransferase Pq3-O-UGT2 and mutants respectively for a certain time and then terminated (Rh2: 15min, F2: 10min). The enzyme activity was determined according to the above-mentioned glycosyltransferase activity assay method. The K of the purified wild-type and mutants was calculated using the Michaelis-Menten equation. m , K cat and K cat / K m According to the determination of enzyme activity parameters in the present invention, the catalytic efficiency of the glycosyltransferase Pq3-O-UGT2 mutant in synthesizing rare ginsenoside Rg3 is improved compared with the wild type, as shown in Table 4. Figure 7 By measuring the enzyme kinetic parameters, the K of the new enzyme 1 for the substrate Rh2 was m was 72.17±10.49μM, k cat 6.51±0.26s -1 , k cat / K m 90.17±3.61s -1 ·mΜ -1 , and the K of the new enzyme 1 for substrate F2 is m was 83.61±8.43μM, k cat 5.04±0.23s -1 , k cat / K m 60.29±2.75s -1 ·mΜ -1 These data indicate that the affinity of the new enzyme 1 for the substrate Rh2 is improved (K m decreased), while the affinity for substrate F2 decreased (K m Increase), the catalytic efficiency of the new enzyme 1 to Rh2 (k cat / K m ) was 3.15 times higher than that of wild-type Pq3-O-UGT2. m was 45.43±3.61μM, k cat 5.74±0.13s -1 , k cat / K m 126.40±2.93s -1 ·mΜ -1 , while the K of the new enzyme 2 for substrate F2 is m 99.98±13.00μM, k cat 5.43±0.32s-1 , k cat / K m 54.29±2.05s -1 ·mΜ -1 These data show that the affinity of the new enzyme 2 for the substrate Rh2 is further improved (K m decreased), while the affinity for substrate F2 further decreased (K m Increase), the catalytic efficiency of the new enzyme 2 for Rh2 (k cat / K m ) was increased by 4.43 times compared to the wild-type Pq3-O-UGT2. In particular, the new enzyme 1 (Leu181Trp) and the new enzyme 2 (Ser175Ala / Leu181Trp) showed significant optimization in catalytic activity: their catalytic efficiency for Rh2 was greatly improved, while the catalytic activity for F2 was significantly reduced. This change not only enhances the catalytic efficiency for the rare ginsenoside Rg3, but also improves the selectivity. Specifically: the catalytic efficiency of the new enzyme 1 (Leu181Trp) for Rh2 is 1.5 times that of F2; the catalytic efficiency of the new enzyme 2 (Ser175Ala / Leu181Trp) for Rh2 is 2.3 times that of F2. These improvements indicate that the new enzymes 1 and 2 have successfully improved the selectivity and synthesis efficiency of glycosyltransferases for the target products, thereby providing strong support for the efficient production of the rare ginsenoside Rg3.

[0082] Table 4 Enzymatic properties of glycosyltransferase Pq3-O-UGT2 and its mutants (new enzyme 1, new enzyme 2)

[0083]

[0084] The HPLC detection conditions are as follows: the detector is an ultraviolet detector with a characteristic absorption peak of 203 nm, the chromatographic column is an Agilent Zorbax Eclipse Plus C18 reversed phase chromatography column (4.6×250 mm, 5 μm), the mobile phase A is an aqueous solution, the mobile phase B is an acetonitrile solution, and gradient elution is used. For the separation of ginsenosides Rh2, Rg3, F2, and Rd, 0 min-30 min, 30% B-90% B, the column temperature is 25°C, the injection volume is 20 μL, and the flow rate is 1.0 ml / min.

[0085] Several studies have attempted to engineer glycosyltransferases that synthesize ginsenosides, but the resulting mutants have only modestly improved activity. For example, a library of UGTPg45 mutants generated using error-prone PCR contains approximately 1500 mutants, and the catalytic efficiency (k cat / K m) is 1.4 times that of the wild type. The catalytic efficiency (k cat / K m ) is 1.3 times that of the wild type. This invention, through structural analysis of Pq3-O-UGT2, guided enzyme modification and engineering, resulting in two novel glycosyltransferase Pq3-O-UGT2 mutants. These mutants significantly enhance the synthesis of the rare ginsenoside Rg3, achieving higher selectivity for the substrate Rh2 over F2. These two mutant enzymes are expected to serve as biocatalysts for the production of ginsenoside Rg3 in biopharmaceuticals, health supplements, and functional foods.

[0086] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A glycosyltransferase Pq3-O-UGT2 mutant that produces a high yield of the rare ginsenoside Rg3, characterized by: The amino acid sequence of the mutant is any one of the following a and b; a. amino acid sequence shown in SEQ ID NO.2; b. Amino acid sequence shown in SEQ ID NO.

3.

2. A gene encoding the glycosyltransferase Pq3-O-UGT2 mutant according to claim 1.

3. An expression vector containing the gene according to claim 2.

4. A recombinant strain carrying the expression vector according to claim 3.

5. The method for constructing a glycosyltransferase Pq3-O-UGT2 mutant according to claim 1, wherein: The steps include: First, specific PCR primers containing the predetermined mutation site were designed and synthesized, and amplification was performed using the wild-type Pq3-O-UGT2 gene as a template via overlap extension PCR. Subsequently, the resulting PCR product was efficiently inserted into a selected expression vector using a homologous recombination strategy. The vector was then transformed into host cells for further amplification. DNA sequencing was performed on the transformed positive clones to confirm the successful mutation. The nucleotide sequence of the wild-type Pq3-O-UGT2 gene is shown in SEQ ID NO.

4.

6. Use of the glycosyltransferase Pq3-O-UGT2 mutant according to claim 1 in the production of rare ginsenoside Rg3.

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