Glycosyl transferase Pq3-O-UGT2 mutant with high yield of rare ginsenoside Rg3 and application of glycosyl transferase Pq3-O-UGT2 mutant

Abstract

The invention belongs to the technical field of enzyme gene engineering and biology, and discloses a glycosyl transferase Pq3-O-UGT2 mutant for high yield of rare ginsenoside Rg3, and the amino acid sequence of the glycosyl transferase Pq3-O-UGT2 mutant is any one of the following sequences; the amino acid sequences are as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the degree of identity of the amino acid sequences as shown in SEQ ID NO.2 and SEQ ID NO.3 is 50% or above. According to the mutant disclosed by the invention, the conversion efficiency of Pq3-O-UGT2 original enzyme to ginsenoside F2 is reduced, and the conversion efficiency of the Pq3-O-UGT2 original enzyme to ginsenoside Rh2 is improved. According to the method, the reaction of adding a No.2 glucosyl group in the C-2 hydroxyl direction of a ginsenoside Rh2 substrate C3-O-glucosyl group is improved, the rare ginsenoside Rg3 which is a high-medicinal-value product is generated through catalysis, and the enzymatic synthesis efficiency and selectivity of the rare ginsenoside Rg3 are improved.

Description

Technical Field

[0001] The present invention belongs to the field of enzyme genetic engineering and biotechnology, and particularly relates to a glycosyltransferase Pq3-O-UGT2 mutant for high-yield rare ginsenoside Rg3, a coding gene, a construction method and an application. Background Art

[0002] Ginsenosides are important active ingredients of plants such as ginseng, American ginseng, and Panax notoginseng. They are a series of triterpene glycoside compounds formed by aglycones and sugars, and contain a 4-ring sterane steroid core formed by 17 carbon atoms. The active ingredients formed by ginsenosides after metabolic transformation have stronger biological activity, and have significant physiological effects such as anti-cancer, anti-aging, anti-fatigue, anti-tumor, immune stimulation, and improving immunity. They have extremely high application value in the fields of food, medicine, and health products. Ginsenosides include prototype ginsenosides and rare ginsenosides. Prototype ginsenosides include Ra, Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, Rf, etc. Rare ginsenosides include Rg3, Rh1, CK, Rh2, Rg5, Rk1, Rh3, Rk2, Rh4, Rk3, aPPT, aPPD, etc. Prototype ginsenosides have low anti-cancer activity and are difficult to be absorbed and utilized by the human body. Rare ginsenosides are metabolic derivatives of prototype ginsenosides. The content in natural plants is very low and cannot be directly extracted from plants. They can only be obtained by transforming or metabolizing prototype ginsenosides. Rare ginsenosides are easily absorbed by the human body and therefore have higher biological activity. Among the many rare ginsenosides, Rg3 has a significant anti-cancer effect and can directly act on tumor cells that have developed drug resistance, exerting the effect of reversing tumor resistance. Therefore, it is widely used in tumor treatment and has important health and economic value.

[0003] Since the content of rare ginsenosides in natural plants is extremely low (<0.1%), and is affected by factors such as long growth cycle of raw materials, difficulty in extraction, low preparation efficiency, many by-products, high production cost, and environmental pollution, the development and application of rare ginsenoside products are greatly limited. With the identification of various key enzymes in the biosynthesis of ginsenosides, the use of genetic engineering and synthetic biology to produce ginsenosides has become an important method for the efficient production of rare ginsenosides. The "enzyme catalysis" synthesis mediated by glycosyltransferases UGTs has many characteristics such as regional and stereoselective specificity, high catalytic efficiency, and simple 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 generated and its generation efficiency. However, Pq3-O-UGT2 can not only catalyze the generation of rare ginsenoside Rg3, but also catalyze the generation of prototype ginsenoside Rd ( Figure 1 b). Under the same conditions, the conversion rate of wild-type Pq3-O-UGT2 for the reaction of generating rare ginsenoside Rg3 was 69%, while the conversion rate for the reaction of generating prototype ginsenoside Rd was 92%, indicating that it showed higher selectivity for the reaction of generating prototype ginsenoside Rd, which seriously hindered the biosynthesis of rare ginsenoside Rg3. Therefore, it is urgent to develop Pq3-O-UGT2 mutants with significantly improved Rg3 synthesis efficiency and substrate selectivity to provide technical support for high-efficiency and large-scale biosynthesis of rare ginsenosides. Summary of the invention

[0004] The purpose of the present invention is 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 that produces 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 an amino acid sequence having an identity of 50% or more 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 whose identity with the amino acid sequence shown in SEQ ID NO.3 is 50% or more.

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

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

[0011] The recombinant strain carries the expression vector as 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 by overlap extension PCR technology with the wild-type Pq3-O-UGT2 gene as a template. Subsequently, the obtained PCR product was efficiently inserted into the selected expression vector using the homologous recombination strategy. The vector was then transformed into host cells for further amplification. The transformed positive clones were verified by DNA sequencing to ensure the successful mutation.

[0014] Application of the glycosyltransferase Pq3-O-UGT2 mutant as 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 process 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 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 to ginsenoside F2 (synthesizing the prototype ginsenoside Rd), and improves the conversion efficiency to ginsenoside Rh2 (synthesizing the rare ginsenoside Rg3). That is, the reaction of adding the No. 2 glucose group to the C2-hydroxyl direction of the C3-O-glucose group of the ginsenoside Rh2 substrate is improved, and the high medicinal value product of the rare ginsenoside Rg3 (Ginsenoside Rg3, CAS14197-60-5) can be catalyzed, thereby improving 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 to the C3 hydroxyl direction of ginsenoside Rh2, while reducing its catalytic activity in catalyzing the synthesis of prototype ginsenoside Rd from ginsenoside F2, so as to improve the selectivity of the enzyme.

[0020] Based on the three-dimensional spatial structure of glycosyltransferase Pq3-O-UGT2 independently discovered by the inventors, the original amino acid sequence of glycosyltransferase Pq3-O-UGT2 was rationally optimized. The inventors changed the leucine at position 181 to tryptophan (Leu181Trp), or mutated the serine at position 175 and the leucine at position 181 to alanine and tryptophan (Ser175Ala / Leu181Trp) at the same time. By changing the above two amino acids, two new enzymes can be obtained, which are named new enzyme 1 (Leu181Trp) and new enzyme 2 (Ser175Ala / Leu 181Trp) in this patent. The two new enzymes are purified and prepared by the E. coli protein expression system, and both of them can improve the catalytic addition of Pq3-O-UGT2 to the C2 hydroxyl of the C3-O glucose group of ginsenoside Rh2 to form a β(1-2) glycosidic bond and synthesize the rare ginsenoside Rg3. Both of these 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 to form a β(1-2) glycosidic bond and synthesize the prototype ginsenoside Rd. The activity data of the new enzyme 1 (Ile181Trp) (the isoleucine at position 181 was changed to tryptophan) for the enzymatic synthesis of the rare ginsenoside Rg3 were 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 data of the new enzyme 2 (Ser175Ala / Ile181Trp) (serine at position 175 and isoleucine at position 181 were mutated to alanine and tryptophan) for the enzymatic synthesis of the rare ginsenoside Rg3 were 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 new enzymes towards Rh2 was significantly enhanced, while the activity towards F2 was significantly reduced. This modified Pq3-O-UGT2 enzyme has great potential in 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 schematic diagram of the reaction of Pq3-O-UGT2 participating in the glycosylation synthesis of ginsenosides:

[0022] Among them, a is a schematic diagram of 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, where glucose group No. 2 is 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 in the middle of each glucose represents the number of the glucose group, where glucose group No. 2 is shown in blue;

[0024] Figure 2 This is the 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 It is the SDS-PAGE image of the protein purification samples of wild-type Pq3-O-UGT2 and new enzymes 1 and 2 in Example 3 of the present invention;

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

[0028] Figure 6 The relative activity diagram of the Pq3-O-UGT2 mutant catalyzing the substrates ginsenoside 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 in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

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

[0032] A glycosyltransferase Pq3-O-UGT2 mutant that produces 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 the amino acid sequence with a degree of identity of 50% or more 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 shown in SEQ ID NO.3 and the amino acid sequence with a degree of identity of 50% or more to the amino acid sequence shown in SEQ ID NO.3. The amino acid sequence shown 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 a glycosyltransferase Pq3-O-UGT2 mutant as described above.

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

[0037] The recombinant strain carries the expression vector as 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, and the wild-type Pq3-O-UGT2 gene was used as a template for amplification by overlap extension PCR technology; then, the obtained PCR product was efficiently inserted into the selected expression vector using a homologous recombination strategy; the vector was then transformed into host cells for further amplification; and DNA sequencing was performed on the transformed positive clones to ensure the success of the mutation.

[0040] Application of the glycosyltransferase Pq3-O-UGT2 mutant as 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 process 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 the 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 of the two new enzyme catalytic reactions 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 It is 0.51 times that of wild-type Pq3-O-UGT2; the enzymatic activity data of the new enzyme 2 for the enzymatic synthesis of rare ginsenoside Rg3 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 rare ginsenoside Rg3.

[0044] In addition to expressing, purifying, and preparing 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, non-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-organisms, cells, non-cells (cell-free) through transgenic or other gene introduction methods for enzymatic conversion of ginsenosides, and in in vitro systems such as enzymes and enzyme immobilization, for enzymatic conversion of adding No. 2 glucose group to ginsenoside Rh2, including the preparation of but not limited to the enzymatic conversion process of ginsenoside Rg3.

[0045] Specifically, the relevant preparation and detection 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] In order to prevent the purpose of circumventing the protection scope of this patent by replacing the non-important amino acid sequence of Pq3-O-UGT2, as long as the amino acid sequence of the translated protein 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 the 175th and 181st amino acids or other sites (due to the redundancy of amino acids, by removing one or any amino acids at other positions, the 175th and 181st amino acids are not necessarily always numbered as the 175th and 181st amino acids) to achieve the purpose of improving the catalytic activity and selectivity of Pq3-O-UGT2 for the rare ginsenoside Rg3, all fall 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 added artificially, among which the S and L amino acids are the sites protected by this patent; the amino acids ENLYFQG It is the protease cleavage site, which is used to facilitate protein elution during purification; amino acid HHHHHH (6 histidine tags) are for the convenience of purification operation):

[0051] MDNQKGRISIALLPFLAHGHISPFFELAKQLAKRNCNVFLCSTPINLSSIKDKDSSASIKLVEL

[0052] HLPSSPDLPPHYHTTNGLPSHLMLPLRNAFETAGPTFSEILKTLNPDLLIYDFNPSWAPEIASS

[0053] HNIPAVYFLTTAAASSSIGLHAFKNPGEKYPFPDFYDNSNIIPEPPSADNMKLLHDFIACFERS

[0054] CDIILIKSFRELEGKYIDLLSTLSDKTLVPVGPLVQDPMGHNEDPKTEQIINWLDKRAESTVV

[0055] FVCCFGSEYFLSNEELEEVAIGLEISTVNFIWAVRLIEGEKKGILPEGFVQRVGDRGLVVEGWA

[0056] PQARILGHSSTGGFVSHCGWSSIAESMKFGVPVIAMARHLDQPLNGKLAAEVGVGMEVVR

[0057] DENGKYKREGIAEVIRKVVVEKSGEVIRRKARELSEKMKEKGEQEIDRAVEELVQICKKKKDEQ ENLY FQGHHHHHH*

[0058] 2. The coding region of Pq3-O-UGT2 was amplified using the primers listed in Table 1. The 5' and 3' ends of the amplified fragment will carry restriction enzyme sites of NcoI and BamHI, respectively. The cDNA sample from American ginseng root was used as a template to amplify the gene of Pq3-O-UGT2 (GenBank: ALE15280.1) by PCR method, and the primers were designed as shown in Table 1.

[0059] Table 1 Primer sequences of pET28a-Pq3-O-UGT2

[0060]

[0061] (Add six histidine tags to the primers To facilitate subsequent purification, the underlined sequences are restriction enzyme sites for NcoI and BamHI).

[0062] 3. Double digest the pET28a expression vector with NcoI and BamHI, and PCR amplify the Pq3-O-UGT2 coding region. Then insert the Pq3-O-UGT2 coding region into the NcoI and BamHI restriction sites of the pET28a vector by seamless cloning to form the expression vector pET28a-Pq3-O-UGT2 that can express the glycosyltransferase Pq3-O-UGT2 in Escherichia coli.

[0063] The reaction system of 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 site, 6×His tag, and homology arm) was obtained.

[0064] Table 2 PCR reaction system and procedure

[0065]

[0066] 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 use a DNA gel recovery kit to recover the PCR product. 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, completing the construction of the wild-type Pq3-O-UGT2 glycosyltransferase expression vector (see Figure 3 ).

[0067] Example 2 Construction of the expression vector for the mutant protein of glucose transferase Pq3-O-UGT2

[0068] 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.

[0069] Table 3 The mutant primer sequences are as follows: (the underlined part indicates the mutation site)

[0070]

[0071] 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 with pET28a-Pq3-O-UGT2 as templates. The system and amplification procedure are the same as Table 1. After nucleic acid electrophoresis and gel cutting and recovery of the PCR product, the purified gene fragment is obtained. Under the action of seamless cloning reaction reagent, the gene fragment is connected with the linearized pET-28a vector to form a circular recombinant plasmid, which is then transformed into DH5α competent cells. After kanamycin plate culture medium screening, plasmid extraction and sequencing verification, a recombinant plasmid containing the mutation site, i.e., 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.

[0072] 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.

[0073] Example 3 Inducible expression and purification of wild-type Pq3-O-UGT2 and novel enzyme 1 (Leu181Trp) and novel enzyme 2 (Ser175Ala / Leu181Trp)

[0074] 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℃ water bath for 75 seconds, ice bath for 2 minutes, add 1ml LB liquid medium, culture at 37℃, 230rpm for 45 minutes, evenly spread the bacterial solution on the kanamycin plate medium, culture at 37℃ for 12-16 hours, pick out the monoclonal colony and transfer it to 5ml kanamycin-resistant LB liquid medium, culture at 37℃, 230rpm for 12-16 hours, and then store the glycerol bacteria.

[0075] Among them, the formula of the kanamycin-resistant LB liquid culture medium used in the present invention is:

[0076] Tryptone 10g / L, sodium chloride 10g / L, yeast extract 5g / L, kanamycin 50mg / L, the solvent is water.

[0077] 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, and inoculate it into 1 L of kanamycin-resistant LB liquid medium at 1% inoculum, culture it at 37°C, 230 rpm. When OD 600 When the pH reaches 0.6-0.8, cool the bacterial solution to 16°C and add the inducer IPTG (isopropyl β-D-1-thiogalactopyranoside) to a final concentration of 0.5 mM, then culture at 16°C and 230 rpm for 16-18 hours. After the induction expression is completed, centrifuge at 5000 rpm for 10 minutes, discard the supernatant, and collect the bacteria.

[0078] 3. After the cells were resuspended in lysis buffer (50mM Tris-HCl pH 8.0, 150mM NaCl), the cells were ultrasonically broken to obtain cell lysis buffer, centrifuged at 15000rpm and 4℃ for 30min, the obtained supernatant was incubated with Ni-NTA at 4℃ for 2 hours, and the impurities were removed by 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), and the imidazole in the mixed solution was removed by dialysis. After quick freezing in liquid nitrogen, it can be stored at -80℃ 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, see Figure 4 For the wild-type Pq3-O-UGT2 and the two mutants, new enzyme 1 and new enzyme 2, the SDS-PAGE results showed a clear and single main band, located between the molecular weight markers 45kD and 65kD and close to 45kD, which was consistent with the expected molecular weight. This shows that the purification step is very efficient and a highly purified target protein is obtained.

[0079] In the present invention, the host bacteria is preferably Escherichia coli BL21; more preferably, it includes Escherichia coli Rosetta. The preparation method provided by the present invention can efficiently purify a small amount of soluble protein.

[0080] Example 4 Enzyme activity determination of wild-type Pq3-O-UGT2, novel enzyme 1 (Leu181Trp) and novel enzyme 2 (Ser175Ala / Leu181Trp)

[0081] 1. Analysis of enzymatic conversion rate

[0082] (1) First, the substrates of Pq3-O-UGT2, ginsenosides Rh2 and F2, are used as examples. The natural Pq3-O-UGT2 can catalyze the addition of glucose group 2 to the 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 .

[0083] (2) Example of reaction. The reaction conditions are as follows: at room temperature, 100 μL of the reaction system includes 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. Incubate in a 37°C water bath for 12 h for biotransformation, and finally add 100 μL of methanol, mix and vortex to terminate the reaction. Centrifuge the mixture at 13,000 rpm for 10 min, and collect the supernatant. The supernatant is used for HPLC detection.

[0084] (3) Conversion rate = product peak area / (substrate peak area + product peak area).

[0085] (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 the wild-type Pq3-O-UGT2 in synthesizing the rare ginsenoside Rg3 was about 69%, while the conversion rate of the catalytic synthesis of the prototype ginsenoside Rd was about 92% ( Figure 5 b). The determination of the product conversion rates of the new enzyme 1 and the new enzyme 2 showed that the conversion rates of the new enzyme 1 and the new enzyme 2 for the rare ginsenoside Rg3 were significantly increased, while the conversion rates for the prototype ginsenoside Rd were reduced ( Figure 6 ).like Figure 6As shown, compared with the wild type, the conversion rate (relative activity) of the new enzyme 1 to Rh2 increased by 1.22 times, and 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 rates of the new enzymes 1 and 2 for the rare ginsenoside Rg3 were significantly increased, while the synthesis efficiency of the prototype ginsenoside Rd was reduced. Both the new enzymes 1 and 2 increased the catalytic activity for the rare ginsenoside Rg3 and reduced the activity for the prototype ginsenoside Rd. The selectivity and catalytic activity of the new enzymes 1 and 2 for the synthesis of the rare ginsenoside Rg3 were significantly improved.

[0086] 2. Enzyme kinetic parameters (K cat / K m ) determination:

[0087] Under certain conditions (37°C, 50 mM Tris-HCl pH 8.0), the concentration of substrate ginsenoside Rh2 or F2 was set to 20-500 μM, and ginsenoside Rh2 or F2 with different concentration gradients were mixed with 100 ng of purified wild-type glycosyltransferase Pq3-O-UGT2 and mutants for a certain period of time and then the reaction was terminated (Rh2: 15 min, F2: 10 min), and the enzyme activity was determined according to the above-mentioned glycosyltransferase enzyme activity determination method. The Michaelis Menten equation was used to calculate the K of the purified wild-type and mutants. m , K cat and K cat / K m According to the determination of the enzyme activity parameters in the present invention, the catalytic efficiency of the glycosyltransferase Pq3-O-UGT2 mutant in synthesizing the 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 m 72.17±10.49μM, k cat 6.51±0.26s -1 , is k cat / K m 90.17±3.61s -1 ·mΜ -1 , while the K of the new enzyme 1 for substrate F2 is m 83.61±8.43μM, k cat 5.04±0.23s -1 , is k cat / K m 60.29±2.75s -1 ·mΜ -1These data show 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 The catalytic efficiency of the new enzyme 1 for Rh2 (k cat / K m ) was 3.15 times higher than that of wild-type Pq3-O-UGT2. m 45.43±3.61μM, k cat 5.74±0.13s -1 , is k cat / K m 126.40±2.93s -1 ·mΜ -1 , while the K of the new enzyme 1 for substrate F2 is m 99.98±13.00μM, k cat 5.43±0.32s -1 , is 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 The catalytic efficiency of the new enzyme 2 for Rh2 (k cat / K m ) was 4.43 times higher than the wild-type Pq3-O-UGT2. In particular, the new enzymes 1 (Leu181Trp) and 2 (Ser175Ala / Leu181Trp) showed significant optimization in catalytic activity: their catalytic efficiency for Rh2 was greatly improved, while their catalytic activity for F2 was significantly reduced. This change not only enhanced the catalytic efficiency for the rare ginsenoside Rg3, but also improved 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.

[0088] Table 4 Enzymatic properties of glycosyltransferase Pq3-O-UGT2 and its mutants (new enzyme 1, new enzyme 2)

[0089]

[0090] The detection conditions of the HPLC are as follows: the detector is an ultraviolet detector, the characteristic absorption peak is 203 nm, the chromatographic column is an Agilent Zorbax Eclipse Plus C18 reverse chromatographic 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.

[0091] The relevant sequences in the present invention are as follows:

[0092] Wild-type Pq3-O-UGT2 protein sequence:

[0093] SEQ ID NO.1

[0094]

[0095]

[0096] Mutant Pq3-O-UGT2 L181W Protein sequence:

[0097] SEQ ID NO.2

[0098]

[0099]

[0100] Mutant Pq3-O-UGT2 S175A / L181W Protein sequence:

[0101] SEQ ID NO.3

[0102]

[0103]

[0104] Gene sequence encoding wild-type Pq3-O-UGT2:

[0105] SEQ ID NO.4

[0106] 1 atggataacc aaaaaggtag aatcagtata gcgttgctac catttttagc ccatggtcac

[0107] 61 atatctccct tctttgagct agccaaacaa ctcgcaaaaa gaaattgcaa tgttttcctc

[0108] 121 tgttctaccc ctatcaatct tagctccatc aggattaagg attcctctgc ttctataaaa

[0109] 181 ctagttgagc ttcatcttcc atcttccct gatctccctc ctcactatca cacaacaaat

[0110] 241 ggcctccctt cccatctcat gctccccactc agaaacgcct ttgaaaccgc aggccccacc

[0111] 301 ttctctgaaa tccttaaaac cttaaacccc gatttgcttatttatgattt caatccctca

[0112] 361 tgggcaccgg agatcgcttc gtctcacaat attccggcag tttatttcct aaccacggca

[0113] 421 gcagccagct cttccatgg cctacatgct ttcaaaaacc caggtgaaaa atacccattt

[0114] 481 ccagattttt atgataacag taatattatc cctgaacctc cttctgcaga taacatgaag

[0115] 541 ctacttcatg attttatcgc ttgtttcgaa cgatcttgcg acattatttt gatcaagagt

[0116] 601 tttagagaac tagaagggaa atatattgat ttgctttcca ctttatctga taaaactttg

[0117] 661 gttcctgttg gtccactcgt tcaagatcct atgggccata atgaagatcc aaaaacagag

[0118] 721 cagattataa actggcttga caaagggct gatctacag tggtgttgt ctgctttgga

[0119] 781 agtgagtatt ttctccaatgaggaattg gagaagtag caatgggct agagattagc

[0120] 841 acggttaatt tcatatgggc tgtgagatta attgaggag agaaaaaagg gatttacca

[0121] 901 gaggggttg tcaagggt aggacagg gaggttggtg tggaggttg ggctccacag

[0122] 961 gcaagaattt taggacattc aagcaccggt gggtttgtga gccattgtgg gtggattct

[0123] 1021 attgcggaga gtatgaagtt tggttcca gtattgcca tggccaggcatctgattcag

[0124] 1081 ccttgaatg gtaagctggc ggcggaggtt ggtgtgggca tggaggttgtgagagatgag

[0125] 1141 Atgggagt attagagaga agggattgca gaggtaataa gaaagtggttgtgagaaa

[0126] 1201 agtgggggg tattcaggag gaagcagg gagttgagtg agaaatgaagaaagga

[0127] 1261 gaggagaga ttgatgaggc agtgaggag ctagtacaa ttgtagagagaaagat

[0128] 1321 summary

[0129] Several studies have attempted to engineer glycosyltransferases for the synthesis of ginsenosides, but the resulting mutants have only modestly improved activity. For example, a mutant library of UGTPg45 generated using error-prone PCR contained 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. The present invention obtains two new glycosyltransferase Pq3-O-UGT2 mutants by guiding the modification of the enzyme through structural analysis of Pq3-O-UGT2. The mutants can significantly improve the synthesis performance of the rare ginsenoside Rg3 and achieve higher selectivity for the substrate Rh2 than F2. These two mutant enzymes are expected to be used as biocatalysts to promote the production of ginsenoside Rg3, and can be used for the production of ginsenoside Rg3 in the fields of biopharmaceuticals, health products and functional foods.

[0130] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate 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 rare ginsenoside Rg3, characterized in that: The amino acid sequence of the mutant is any one of the following a and b; a. the amino acid sequence shown in SEQ ID NO.2 and an amino acid sequence having an identity of 50% or more to the amino acid sequence shown in SEQ ID NO.2; b. The amino acid sequence shown in SEQ ID NO.3 and amino acid sequences whose identity with the amino acid sequence shown in SEQ ID NO.3 is 50% or more.

2. A gene encoding the glycosyltransferase Pq3-O-UGT2 mutant as described in claim 1.

3. An expression vector containing the coding gene as claimed in claim 2.

4. A recombinant strain carrying the expression vector as claimed in claim 3.

5. The method for constructing a glycosyltransferase Pq3-O-UGT2 mutant according to claim 1, characterized in that: The steps include: First, specific PCR primers containing the predetermined mutation site were designed and synthesized, and the wild-type Pq3-O-UGT2 gene was used as a template for amplification by overlap extension PCR technology; then, the obtained PCR product was efficiently inserted into the selected expression vector using a homologous recombination strategy; the vector was then transformed into host cells for further amplification; and DNA sequencing was performed on the transformed positive clones to ensure successful mutation.

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

7. Use of the glycosyltransferase Pq3-O-UGT2 mutant as claimed in claim 1 in the enzymatic synthesis and / or conversion of rare ginsenoside Rg3.

8. The use according to claim 7, characterized in that: A No. 2 glucose group is added to the C2-hydroxyl direction of the C3-glucose group of the rare ginsenoside Rh2, and the No. 2 glucose group is a glucose group that forms a β(1-2) glycosidic bond with the No. 1 glucose group, and the No. 1 glucose group specifically refers to a glucose group that directly forms a β-glycosidic bond with the C3-hydroxyl group of the protopanaxadiol saponin.

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