A methyltransferase CitOMT mutant, preparation method, biomaterial and application

Abstract

The present invention relates to a methyltransferase (CitOMT) mutant, its preparation method, biomaterial, and application. The methyltransferase mutant is derived from a CitOMT enzyme with an amino acid sequence as shown in SEQ ID NO:1, wherein the mutations include: a mutation of phenylalanine at position 164 to alanine; and / or a mutation of glutamine at position 238 to alanine. The methyltransferase mutant of the present invention is capable of efficiently converting eriodictyol into homoeriodictyol.

Description

Technical Field

[0001] The present invention relates to the technical field of mutation or genetic engineering, and in particular to a methyltransferase CitOMT mutant, a preparation method, a biomaterial and an application. Background Art

[0002] There are many types of methylated modifications in natural products. Methylation can significantly improve the water solubility, stability, and bioactivity of natural products. It is also a key step in the production of various important natural products and a significant source of medicinal natural products.

[0003] Homoeriodictyol is primarily found in mistletoe, citrus fruits (such as lemon), and traditional Chinese medicinal herbs (such as Candida altissima). Traditionally, its production relied on plant extraction or chemical synthesis. However, recent microbial synthesis techniques (e.g., in Yarrowia lipolytica and engineered Saccharomyces cerevisiae) have enabled efficient production, with yields reaching 6.8 g / L, paving the way for industrial application. It exhibits a wide range of biological activities, including antioxidant, anti-inflammatory, hepatoprotective, and neuroprotective effects. For example, it alleviates oxidative stress by activating the Nrf2 / ARE pathway and ameliorates acute liver injury by regulating the PI3K / AKT pathway (significantly reducing ALT and AST levels in mouse models). Furthermore, homoeriodictyol has shown potential therapeutic effects against diabetic complications, Alzheimer's disease, and cancer. In the food industry, it is used as a natural antioxidant in beverages and alcoholic beverages; in the pharmaceutical field, it could be developed as a hepatoprotective or neuroprotective agent. Biologically catalyzed methylation modification is primarily carried out by methyltransferases, but the catalytic efficiency of methyltransferases is limited, resulting in low production efficiency. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a methyltransferase CitOMT mutant, a preparation method, a biomaterial and an application.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a methyltransferase CitOMT mutant, characterized in that the methyltransferase CitOMT mutant is a mutant F164A or a mutant Q238A; the mutant F164A is obtained by mutating the phenylalanine at position 164 of the CitOMT enzyme as shown in the amino acid sequence of SEQ ID NO.1 to alanine, and the mutant Q238A is obtained by mutating the glutamine at position 238 of the CitOMT enzyme as shown in the amino acid sequence of SEQ ID NO.1 to alanine.

[0007] Preferably, the amino acid sequence of the mutant F164A is shown in SEQ ID NO: 3.

[0008] Preferably, the amino acid sequence of the mutant Q238A is shown in SEQ ID NO:5.

[0009] In a second aspect, the present invention provides a biomaterial for the methyltransferase CitOMT mutant described in the first aspect, wherein the biomaterial comprises any one of the following B1) to B5):

[0010] B1) a nucleic acid molecule encoding the methyltransferase CitOMT mutant according to the first aspect;

[0011] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0012] B3) a recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2);

[0013] B4) a recombinant microorganism, the recombinant microorganism comprising the nucleic acid molecule described in B1), the expression cassette described in B2, and / or the recombinant vector described in B3;

[0014] B5) A recombinant cell, comprising the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3).

[0015] Preferably, the nucleic acid molecule described in B1) comprises any one of the following coding genes:

[0016] The nucleotide sequence encoding the mutant F164A gene is shown in SEQ ID NO: 4;

[0017] The nucleotide sequence encoding the mutant Q238A gene is shown in SEQ ID NO: 6.

[0018] In a third aspect, the present invention provides a method for preparing the methyltransferase CitOMT mutant according to the first aspect, characterized in that it comprises the following steps:

[0019] S1. Connect the CitOMT enzyme encoding gene to the vector plasmid to obtain a recombinant plasmid;

[0020] S2. Designing and using site-directed mutagenesis primers to amplify the full length of the recombinant plasmid using the recombinant plasmid as a template, and performing enzyme digestion to obtain a mutant product; wherein the site-directed mutagenesis primers are used to perform directed mutagenesis on at least one of the phenylalanine at position 164 and the glutamine at position 238 of the CitOMTase;

[0021] S3. Transform the mutant product into host cells, screen and induce the host cells to express the methyltransferase CitOMT mutant.

[0022] Preferably, the nucleotide sequence of the gene encoding the CitOMTase is shown as SEQ ID NO: 2.

[0023] Preferably, in step S2, the nucleotide sequence of the primer set for mutating phenylalanine at position 164 of the CitOMTase to alanine is as shown in SEQ ID NO: 7-8; and / or,

[0024] The nucleotide sequences of the primer set for mutating glutamine at position 238 of the CitOMTase to alanine are shown in SEQ ID NOs: 9-10.

[0025] Preferably, the vector plasmid is pET28a plasmid, and the host cell is Escherichia coli.

[0026] In a fourth aspect, the present invention provides use of the methyltransferase CitOMT mutant in the first aspect in the preparation of homoeriodictyol.

[0027] Preferably, the methyltransferase CitOMT mutant is used to catalyze the conversion of eriodictyol to homoeriodictyol.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] A methyltransferase mutant of the present invention is based on the CitOMT enzyme, and after the amino acids at positions 164 and / or 238 are mutated to alanine with a strongly hydrophobic side chain and weak steric hindrance, it can convert eriodictyol into homoeriodictyol, while significantly improving the catalytic efficiency of the methyltransferase. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the conversion of eriodictyol to hesperetin.

[0031] Figure 2 This is the docking simulation diagram of CitOMT enzyme with eriodictyol and SAM.

[0032] Figure 3 This is a comparison of the catalytic efficiency of wild-type and mutant CitOMT enzymes in converting eriodictyol to hesperetin in whole cells.

[0033] Figure 4 The HPLC results of wild-type and mutant whole cells of CitOMT enzyme catalyzing the conversion of eriodictyol to hesperetin are shown. DETAILED DESCRIPTION

[0034] The present invention is further described below with reference to the accompanying drawings and specific examples. Experimental methods in the following examples, where specific conditions are not specified, generally follow conventional experimental conditions or those recommended by the manufacturer. Unless otherwise specified, all reagents used in the examples are commercially available. For molecular biology experimental methods not specifically described in these examples, refer to the Molecular Cloning Experiment Guide.

[0035] Example 1

[0036] This example uses Alphafold3 to simulate the three-dimensional structure of the wild-type methyltransferase and perform molecular docking with the substrate. Through molecular docking, the conformational relationship between the enzyme and the substrate is accurately predicted, and relevant mutation targets are efficiently obtained. Pythia is used to predict the mutation free energy and screen out key mutants.

[0037] The glycosyl transfer mechanism of the conversion of eriodictyol to homoeriodictyol is shown in Scheme 1.

[0038] The three-dimensional structural model of CitOMT enzyme was constructed and molecular docking was performed with its substrates eriodictyol and S-adenosylmethionine. Figure 2 As shown. Both eriodictyol and S-adenosylmethionine (SAM) are located in the active pocket of the CitOMT enzyme. The methyl group of S-adenosylmethionine is close to eriodictyol, about 3 angstroms away. Among them, the phenylalanine at position 164 of the enzyme is located in the pocket of the eriodictyol substrate. Mutating it to alanine can reduce the contact steric hindrance between the substrate and the enzyme and increase the volume of the catalytic pocket, thereby improving the catalytic efficiency. At the same time, its glutamine at position 238 is located in the channel outside the pocket. Mutating it to alanine can increase the channel volume, increase hydrophobicity and thus improve the catalytic efficiency.

[0039] Example 2

[0040] Construction of plasmid pET28a-CitOMT

[0041] The steps for constructing a vector containing the methyltransferase sequence of citrus peel are as follows:

[0042] The methyltransferase gene with the nucleotide sequence of SEQ ID NO: 2 was ligated to the pET-28a plasmid to obtain the plasmid pET28a-CitOMT; and the plasmid was transformed into Escherichia coli BL21 (DE3).

[0043] The resulting recombinant strain was named BL21(DE3) / pET28a-CitOMT. The recombinant strain was used to express the CitOMT enzyme having the amino acid sequence shown in SEQ ID NO: 1.

[0044] Example 3

[0045] Construction of pET28a-CitOMT mutant strain

[0046] 1. Construction of mutant enzyme vector by whole plasmid PCR

[0047] Step 1) extracting a small amount of the plasmid pET28a-CitOMT obtained in Example 2;

[0048] Step 2) Design mutagenesis primers. The primers include a 15 bp overlap region and a 15 bp extension region. The mutation sites are designed in the overlap region. The mutation sites include single point mutations at Phe164 and Gln238.

[0049] Step 3) Perform PCR amplification of the entire plasmid using the plasmid pET28a-CitOMT as a template. The PCR system is shown in Table 1:

[0050]

[0051] The primers Primer-F and Primer-R are PCR upstream primers and downstream primers designed accordingly according to different mutation sites. The specific primer information is shown in Table 2.

[0052]

[0053] Among them, the primer pair F164A-F and F164A-R were used to obtain the mutant enzyme F164A. The amino acid sequence of the mutant F164A was SEQ ID NO: 3, and the nucleotide sequence was SEQ ID NO: 4.

[0054] Primer pairs Q238A-F and Q238A-R were used to obtain mutant Q238A, the amino acid sequence of mutant Q238A being SEQ ID NO: 5, and the nucleotide sequence being SEQ ID NO: 6.

[0055] PCR amplification program: pre-denaturation at 98°C for 5 min; cycle setting: denaturation at 98°C for 15 s, annealing at 62°C for 30 s, extension at 72°C for 1.5 min, 30 cycles; final extension at 72°C for 10 min; after the reaction, the PCR product was recovered using a kit.

[0056] Step 4) Enzyme digestion to remove the template DNA, and then perform enzyme digestion on the PCR product. The enzyme digestion system is shown in Table 3:

[0057]

[0058] The above enzyme digestion system was placed in a 37°C metal bath for digestion for 1 hour. After the reaction was completed, the enzyme digestion product was recovered using a kit.

[0059] 2. Sequencing to verify whether the mutant enzyme strain is successfully constructed

[0060] The enzyme-digested products were transformed into Escherichia coli BL21 (DE3) competent cells, cultured inverted at 37°C overnight, and the pseudo-positive transformants were picked for sequencing verification. The mutant enzyme-expressing strains BL21 (DE3) / pET-28a-CitOMT-F164A and BL21 (DE3) / pET-28a-CitOMT-Q238A were successfully obtained.

[0061] Example 4

[0062] Determination of catalytic efficiency of CitOMT and its mutant enzymes in whole-cell reactions

[0063] 1. Enzyme protein induction of CitOMT and its mutant enzyme recombinant strains

[0064] The strains BL21(DE3) / pET-28a-CitOMT-F164A and BL21(DE3) / pET-28a-CitOMT-Q238A obtained in Example 3 were streaked and activated on LB plates containing Kan (100 μg / mL). After inverted culture at 37°C overnight, a single colony was picked and inoculated into 1 mL of LB liquid medium containing Kan and cultured at 37°C, shaking at 200 rpm for 12-16 hours. The overnight culture seed solution was inoculated into 100 mL of fresh LB liquid medium containing Kan at a 1% inoculum size and cultured at 37°C, shaking at 200 rpm for 2-3 hours until the OD600 reached 0.6-0.8. IPTG was then added to a final concentration of 0.1 mM. The culture was then cooled to 20°C and cultured at 150 rpm for 18 hours to induce protein expression.

[0065] 2. Whole-cell reaction of CitOMT and its mutant enzymes

[0066] After protein induction, collect the cells by centrifugation at 8000 rpm for 5 min at 4°C, weigh the wet weight of the cells, and prepare the corresponding mother liquor concentrations as shown in the table below. At the same time, prepare the mother liquor concentrations of eriodictyol, Tris-HCl (pH 7.5), and SAM mother liquor.

[0067] The reaction system was configured according to Table 4 below, and the reaction conditions were: 30° C., 200 rpm, 2 h.

[0068] All samples were run in triplicate. After 24 h, 500 μL of DMSO solution was added to terminate the reaction. The supernatant was collected and filtered after 1 min at 12,000 rpm for detection by HPLC.

[0069]

[0070] 3. Detection method of homoeriodictyol

[0071] HPLC was used for quantitative analysis of homoeriodictyol. The chromatographic conditions were as follows:

[0072] High performance liquid chromatograph: Agilent 1100 Series.

[0073] Column: Diamonsil ® 5 μm C18 (250mm x 4.6mm x 5μm).

[0074] Detector: VWD detector, detection wavelength 290nm.

[0075] Mobile phase ratio and elution conditions: flow rate 1 mL / min; column temperature 30°C; injection volume 10 μL; gradient elution system as shown in Table 5:

[0076]

[0077] 4. Results Analysis

[0078] like Figure 3 and Figure 4 As shown, when eriodictyol was used as substrate for whole-cell catalysis, the whole-cell catalytic efficiency of BL21(DE3) / pET-28a-CitOMT-F164A was 1.88 times that of the wild-type strain (BL21(DE3) / pET-28a-CitOMT-WT);

[0079] The whole-cell catalytic efficiency of BL21(DE3) / pET-28a-CitOMT-Q238A was 2.26 times that of the wild-type strain.

[0080] According to the test results, mutants F164A and Q238A have a higher affinity for eriodictyol and can effectively bind to the substrate for catalysis, thereby improving the catalytic efficiency of the specific conversion of eriodictyol to homoeriodictyol.

[0081] In summary, the present invention synthesized mutants F164A and Q238A through genetic engineering, and then verified through experiments that they have a higher affinity for eriodictyol, can effectively bind to the substrate for catalysis, and improve the catalytic efficiency of the specific conversion of eriodictyol to homoeriodictyol, and can be used for the preparation of homoeriodictyol.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A methyltransferase CitOMT mutant, characterized in that: The methyltransferase CitOMT mutant is mutant F164A or mutant Q238A; the mutant F164A is obtained by mutating the phenylalanine at position 164 of the CitOMT enzyme as shown in the amino acid sequence of SEQ ID NO.1 to alanine, and the mutant Q238A is obtained by mutating the glutamine at position 238 of the CitOMT enzyme as shown in the amino acid sequence of SEQ ID NO.1 to alanine.

2. The methyltransferase CitOMT mutant according to claim 1, characterized in that The amino acid sequence of the mutant F164A is shown in SEQ ID NO:

3.

3. The methyltransferase CitOMT mutant according to claim 1, characterized in that The amino acid sequence of the mutant Q238A is shown in SEQ ID NO:

5.

4. A biomaterial for producing the methyltransferase CitOMT mutant according to any one of claims 1 to 3, characterized in that: The biological material includes any one of the following B1) to B5): B1) a nucleic acid molecule encoding the methyltransferase CitOMT mutant according to any one of claims 1 to 3; B2) an expression cassette, said expression cassette comprising the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3; B5) A recombinant cell containing the nucleic acid molecule described in B1), the expression cassette described in B2) or the recombinant vector described in B3).

5. The biomaterial according to claim 4, wherein B1) The nucleic acid molecule is any one of the following coding genes: The nucleotide sequence encoding the mutant F164A gene is shown in SEQ ID NO: 4; The nucleotide sequence encoding the mutant Q238A gene is shown in SEQ ID NO:

6.

6. A method for preparing the methyltransferase CitOMT mutant according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Connect the CitOMT enzyme encoding gene to the vector plasmid to obtain a recombinant plasmid; S2. Design and use site-directed mutagenesis primers to amplify the full length of the plasmid using the recombinant plasmid as a template, and obtain the mutant product by enzyme digestion; The site-directed mutagenesis primer is used to carry out directed mutagenesis of phenylalanine at position 164 or glutamine at position 238 of the CitOMT enzyme; S3. Transforming the mutant product into host cells, screening and inducing the host cells to express the methyltransferase CitOMT mutant; the nucleotide sequence of the gene encoding the CitOMT enzyme is shown in SEQ ID NO:

2.

7. The method for preparing a methyltransferase CitOMT mutant according to claim 6, wherein: In step S2, the nucleotide sequence of the primer set for mutating the phenylalanine at position 164 of the CitOMTase to alanine is shown in SEQ ID NO: 7-8; or, The nucleotide sequences of the primer set for mutating glutamine at position 238 of the CitOMTase to alanine are shown in SEQ ID NOs: 9-10.

8. Use of the methyltransferase CitOMT mutant according to any one of claims 1 to 3 in the preparation of homoeriodictyol, wherein the methyltransferase CitOMT mutant is used for catalyzing the conversion of eriodictyol into homoeriodictyol.

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