3 '-O-methyltransferase mutant and application thereof

Abstract

The invention relates to a 3 '-O-methyltransferase mutant and application thereof. The 3 '-O-methyltransferase mutant is obtained by mutating tryptophan W at the 284th position of wild type plant oxygen methyltransferase derived from rice into alanine A, the amino acid sequence of the wild type plant oxygen methyltransferase is as shown in SEQ ID NO: 1, and the amino acid sequence of the 3'-O-methyltransferase mutant is as shown in SEQ ID NO: 2. According to the application, a key amino acid site of the catalytic activity of the flavonoid 3 '-O-methyltransferase is determined through a semi-rationally designed enzyme engineering strategy, a dominant mutant is obtained, and the dominant effect of oriented modification on the synthesis of methylated flavonoid compounds by catalyzing flavonoid compounds through the flavonoid 3'-O-methyltransferase is defined; the obtained 3 '-O-methyltransferase mutant has excellent catalytic performance, and meanwhile, the yield of methylated flavonoid compounds is further increased by optimizing and strengthening a metabolic pathway.

Description

[0001] This application is a divisional application of the application with application date of October 31, 2022, application number 202211350194.9, and invention name “A 3′-O-methyltransferase mutant and its application”. Technical Field

[0002] The invention belongs to the field of biotechnology, in particular to a mutant of a 3'-O-methyltransferase, and relates to a 3'-O-methyltransferase mutant and an application thereof. Background Art

[0003] In recent years, with the improvement of economic conditions and living standards, market and societal requirements for the food, pharmaceutical, health product, and cosmetic industries have become increasingly stringent, leading to a growing trend toward "natural" and "green" products. Naturally occurring methylated flavonoids have attracted widespread consumer attention due to their anti-inflammatory, anti-cancer, antioxidant, free radical scavenging, and metabolic-stimulating properties. Methylated flavonoids significantly enhance their absorption and oral availability, enhancing their ability to enter cells and preventing degradation. This, combined with the introduction of new biological activities, makes them more suitable as chemopreventive agents than unmethylated flavonoids.

[0004] Existing methylated flavonoids are primarily obtained through plant extraction, chemical synthesis, and in vitro enzymatic synthesis. While my country has made some progress in employing emerging microwave-assisted and ultrasound-assisted extraction methods, methylation reagents, and enzymatic reactions to supplement methyl donors, these methods still fall short of production requirements, primarily due to high costs, environmental concerns, low extraction yields, and complex production processes. Therefore, improving the catalytic efficiency of the key enzyme O-methyltransferase (O-MT) and utilizing biotransformation to synthesize methylated flavonoids holds significant market potential. Obtaining O-MTase mutants with superior catalytic performance and optimizing and enhancing metabolic pathways are key technical challenges in increasing the yield of methylated flavonoids. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a 3′-O-methyltransferase mutant and its application. The key amino acid sites of the catalytic activity of flavonoid 3′-O-methyltransferase are determined through a semi-rational design enzyme engineering strategy to obtain a superior mutant. The superior effect of targeted modification on the catalytic synthesis of methylated flavonoid compounds from flavonoid compounds by flavonoid-3′-O-methyltransferase is clarified. The obtained 3′-O-methyltransferase mutant has significantly improved substrate affinity and excellent catalytic performance. By optimizing and strengthening the metabolic pathway, the yield of methylated flavonoid compounds is further increased.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a 3′-O-methyltransferase mutant, obtained by mutating the tryptophan W at position 284 of a wild-type plant oxygen methyltransferase to an alanine A, wherein the amino acid sequence of the wild-type plant oxygen methyltransferase is as shown in SEQ ID NO: 1, and the amino acid sequence of the 3′-O-methyltransferase mutant is as shown in SEQ ID NO: 2.

[0007] Furthermore, amino acid site mutations were performed on the wild-type plant oxygen methyltransferase from rice.

[0008] Furthermore, the present invention also provides a gene encoding the aforementioned 3′-O-methyltransferase mutant, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO: 3.

[0009] Furthermore, the present invention also provides a plasmid containing the above-mentioned coding gene, wherein the plasmid also contains a coding gene for overexpressing S-adenosyl-L-methionine synthetase.

[0010] Furthermore, the present invention also provides a mutant strain containing the above-mentioned encoding gene, wherein the mutant strain also contains a gene encoding overexpression of S-adenosyl-L-methionine synthetase.

[0011] Furthermore, the present invention also provides a plasmid pACYCDuet-1 comprising the above-mentioned gene encoding overexpression of S-adenosyl-L-methionine synthetase.

[0012] Furthermore, the present invention also provides a mutant strain, wherein the mutant strain is transformed with a recombinant plasmid containing the above-mentioned 3′-O-methyltransferase mutant encoding gene, or

[0013] A recombinant plasmid overexpressing S-adenosyl-L-methionine synthetase and a recombinant plasmid containing the gene encoding the above-mentioned 3′-O-methyltransferase mutant were co-transformed.

[0014] Furthermore, the mutant strain includes Corynebacterium glutamicum, Escherichia coli or Bacillus subtilis.

[0015] Furthermore, the present invention also provides the use of one or more of the 3′-O-methyltransferase mutant, the encoding gene, the plasmid, and the mutant strain in producing methylated flavonoid compounds.

[0016] Furthermore, the present invention also provides a product comprising one or more of the 3′-O-methyltransferase mutant, encoding gene, plasmid, and mutant strain.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] The present invention provides a 3'-O-methyltransferase mutant, obtained by mutating the 284th amino acid of a flavonoid 3'-O-methyltransferase from tryptophan W to alanine A. Based on amino acid sequence alignment of the flavonoid-3'-O-methyltransferase domain, a methyltransferase-substrate docking model, catalytic kinetic parameters, fermentation testing of point mutant strains, and optimization of methyl supply in the synthetic pathway, the present invention clarifies the positive mutagenic effect of the mutation site of the rice-derived 3'-O-methyltransferase and the advantageous effect of targeted modification on the catalytic synthesis of methylated flavonoid compounds from flavonoid-3'-O-methyltransferase, providing a basis for utilizing this site to further increase the yield of methylated flavonoid compounds.

[0019] Furthermore, the present invention determines the key amino acid sites of the catalytic activity of flavonoid 3′-O-methyltransferase through a semi-rational enzyme engineering strategy. The steps are simple, the screening of mutant strains is relatively fast, and it is easier to obtain advantageous mutants. The present invention mainly includes screening the key amino acids of the rice-derived flavonoid 3′-O-methyltransferase (ROMT) domain that specifically transfers substrates, and mutating the obtained key amino acid tryptophan W at position 284 to alanine A to obtain a 3′-O-methyltransferase mutant with improved catalytic activity. Through the activity assay analysis of 3′-O-methyltransferase, it was found that compared with the wild-type 3′-O-methyltransferase, the 3′-O-methyltransferase mutant of the present invention exhibited significantly improved enzyme activity, and the yield of the methylated flavonoid compound luteolin catalyzed by luteolin as a substrate was increased by about 3.5 times compared with the wild-type.

[0020] Furthermore, the present invention uses metabolic engineering to introduce the endogenous S-adenosyl-L-methionine (SAM) synthase gene metk of Escherichia coli to rebuild the SAM supply pathway and improve the shortcomings of low catalytic activity and expression level of flavonoid 3'-O-methyltransferase. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HPLC profile of the mutant strain synthesizing chrysanthemum yellow;

[0022] Figure 2 The yield of methylated products of the dominant site mutant W284A and the wild type;

[0023] Figure 3 The amount of chlorophyll synthesized by the recombinant strain overexpressing the metk gene and exogenously added methionine. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is fully described below in conjunction with the drawings in the embodiments of the present invention. The described embodiments are only part of the embodiments of the present invention, which is intended to enable those skilled in the art to better understand the present invention, but does not limit the present invention in any way.

[0025] The present invention provides a 3′-O-methyltransferase mutant, which is obtained by mutating the amino acid at position 284 of a wild-type plant 3′-O-methyltransferase from rice from tryptophan W to alanine A. The amino acid sequence of the wild-type plant 3′-O-methyltransferase is shown in SEQ ID NO: 1:

[0026] MGSSHHHHHHSSGMGSTAADMAAAADEEACMYALQLASSSILPMTLKNAIELGLLETLQSAAVAGGGGGKAALLTPAEVADKLPSKANPAAADMVDRMLRLLASYNVVRCEMEEGADGKLSRRYAAAPVCKWLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMTAFEYHGTDARFN RVFNEGMKNHSVIITKKLLDLYTGFDAASTVVDVGGGVGATVAAVVSRHPHIRGINYDLPHVISEAPPFPGVEHVGGDMFASVPRGGDAILMKWILHDWSDEHCARLLKNCYDALPEHGKVVVVECVLPESSDATAREQGVFHVDMIMMLAHNPGGKERYEREFRELARAAGFTGFKATYIYANAWAIEFTK

[0027] The amino acid sequence of the 3′-O-methyltransferase mutant is shown in SEQ ID NO: 2:

[0028] MGSSHHHHHHSSGMGSTAADMAAAADEEACMYALQLASSSILPMTLKNAIELGLLETLQSAAVAGGGGGKAALLTPAEVADKLPSKANPAAADMVDRMLRLLASYNVVRCEMEEGADGKLSRRYAAAPVCKWLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMTAFEYHGTDARFN RVFNEGMKNHSVIITKKLLDLYTGFDAASTVVDVGGGVGATVAAVVSRHPHIRGINYDLPHVISEAPPFPGVEHVGGDMFASVPRGGDAILMKAILHDWSDEHCARLLKNCYDALPEHGKVVVVECVLPESSDATAREQGVFHVDMIMMLAHNPGGKERYEREFRELARAAGFTGFKATYIYANAWAIEFTK

[0029] The nucleotide sequence encoding the 3′-O-methyltransferase mutant is shown in SEQ ID NO: 3:

[0030]

[0031] Note: The yellow-marked sites are mutation sites.

[0032] Example 1:

[0033] 1. Construction of mutant strains and fermentation testing:

[0034] O-methyltransferases are a class of enzymes used to modify the structure of secondary metabolites, transferring methyl groups to groups such as the hydroxyl group of an acceptor molecule to synthesize a variety of methylated derivatives. Flavonoid O-methyltransferases use flavonoids as substrates to transfer the methyl group provided by S-adenosyl-L-methionine (SAM) to the hydroxyl group of the acceptor, catalyzing the synthesis of the corresponding methylated products. Rice-derived flavonoid 3′-O-methyltransferase (ROMT, amino acid sequence shown in SEQ ID NO: 1) is a Class I O-methyltransferase that specifically acts at the 3′ position of the B ring of the flavonoid backbone.

[0035] 1. Determination of mutation targets:

[0036] The caffeic acid-O-methyltransferase protein from ryegrass (COMT PDB ID: 3P9K, amino acid sequence shown in SEQ ID NO: 5) with a known structure and 78% homology to the rice-derived flavonoid 3′-O-methyltransferase protein was synthesized.

[0037] GSHGMGSTAADMAASADEDA CMFALQLASSSVLPMTLKNA IELGLLEILV

[0038] AAGGKSLTPT

[0039] EVAAKLPSAA NPEAPDMVDR ILRLLASYNV VTCLVEEGKD GRLSRSYGAA

[0040] PVCKFLTPNE

[0041] DGVSMAALAL MNQDKVLMES WYYLKDAVLD GGIPFNKAYG MSAFEYHGTD

[0042] PRFNRVFNEG

[0043] MKNHSIIITK KLLELYHGFE GLGTLVDVGG GVGATVAAIA AHYPTIKGVN

[0044] FDLPHVISEA

[0045] PQFPGVTHVG GDMFKEVPSG DTILMKWILH DWSDQHCATL LKNCYDALPA

[0046] HGKVVLVQCI

[0047] LPVNPEANPS SQGVFHVDMI MLAHNPGGRE RYEREFQALA RGAGFTGVKSTYIYANAWAI

[0048] EFTK

[0049] As a template, the protein skeleton model is obtained, and the active center of the enzyme Mutation sites were selected within a wide range, and attempts were made at multiple amino acid residue mutation sites: Met31, Leu34, Trp284, Cys316, Gly330, Val331, Ala372, and Asn373. The principles for screening mutants were to enhance the binding ability of the enzyme to the substrate and to increase the synthesis of methylated products. Ultimately, the optimal mutation target was determined to be the mutation of amino acid 284 in SEQ NO: 1 from tryptophan W to alanine A.

[0050] 2. Expression Vector Construction

[0051] The expression vector selected in this example is the pGS21a plasmid, which is an inducible expression vector. The plasmid itself contains a strong promoter T7-related sequence (including the operator sequence lacO). When an inducer such as IPTG is added, the repressor protein will be prompted to leave the operator sequence to initiate gene expression.

[0052] The pGS21a-OMT plasmid containing the wild-type 3′-O-methyltransferase coding sequence was used as a template. The whole plasmid PCR was performed using the recommended system of Max DNA Polymerase (Takara). The PCR amplification program was set as follows: denaturation at 95°C for 30 seconds, annealing at 65°C for 15 seconds, extension at 72°C for 7 minutes, 19 thermal cycles; extension at 72°C for 5 minutes, and storage at 25°C to obtain a PCR product containing a point mutation.

[0053] The PCR product containing the point mutation was recovered by 1% agarose gel electrophoresis and ligated to the vector plasmid pGS21a. The vector plasmid pGS21a was then transformed into Escherichia coli BL21 (DE3) competent cells and coated on LB screening plates containing ampicillin to obtain a recombinant plasmid containing a specific mutation site. After sequencing analysis, the mutant plasmid was named pGS21a-OMT-W284A.

[0054] The mutant in which tryptophan W was mutated to alanine A showed a positive mutation compared with the wild type.

[0055] The upstream and downstream primers designed to contain the mutation site are as follows:

[0056]

[0057] (Note: The darkened bases are the amino acids after mutation)

[0058] 3. Determination of 3′-O-methyltransferase mutant enzyme activity parameters:

[0059] The recombinant E. coli containing the pGS21a-OMT-W284A mutant plasmid was induced to express and purified by the following method:

[0060] Pick a mutant glycerol strain stored at -80°C and streak it onto an LB plate containing Amp resistance for activation; pick a single activated colony and culture it overnight in 3 mL of LB liquid medium containing 3 μL of Amp (80 mg / mL); transfer 1 mL of the overnight culture to 50 mL of fresh LB liquid medium containing 50 μL of Amp (80 mg / mL) at a ratio of 1:50, and culture at 37°C, 200 rpm to an OD600 of 0.6-0.7. Take 50 μL of the pre-induced cells and mark them as (-);

[0061] Add IPTG as an inducer at a final concentration of 0.5 mM and culture in a constant temperature shaker at 30°C and 200 rpm for 4 h. Take 50 μL of the induced cells and mark them as (+);

[0062] The remaining bacterial suspension was divided into clean 50 mL centrifuge tubes and centrifuged at 8000 rpm for 10 min at 4°C. The supernatant was discarded and the cells were resuspended in 10 mL of 1×NPB and frozen at -80°C for more than 3 h. The cells were thawed at room temperature and disrupted in an ice bath by adding PMSF at a final concentration of 0.05 mM. Ultrasonic disruption conditions were as follows: power 60 W; total duration 3 min (ultrasound on 3.0 s, off 3.0 s). 50 μL of the disrupted fraction was collected and labeled as Total (T).

[0063] The disrupted cells were centrifuged at 4°C and 6000 rpm for 10 min, and 50 μL of the supernatant was collected and labeled as Soluble (S);

[0064] The remaining supernatant was added to the equilibrated Ni-NTA affinity chromatography column and incubated at 4°C and 90 rpm for 3 h. The effluent was collected and 50 μL of the effluent fraction was taken and labeled as Unbound (U).

[0065] The column was washed with 50 times the volume of 1×NWB, and 50 μL of the effluent fraction was taken and marked as Wash (W);

[0066] Eluted with 3 mL of 1×NEB and collected 50 μL of the effluent fraction, labeled as Eluted (E);

[0067] The product was dialyzed in 2 L of pre-cooled 50 mM Tris-HCl for 8 h, the dialysate was replaced, and this step was repeated until the imidazole concentration was dialyzed to below 60 nM. After dialysis, the components were collected and sterilized using a 0.22 μm filter membrane to obtain the purified 3′-O-methyltransferase mutant.

[0068] Each fraction collected during the purification process was subjected to SDS-PAGE analysis using equal volumes of sample. Both the mutant and wild-type enzymes were expressed intracellularly and soluble. The protein was successfully expressed after induction, and the position of the protein band was consistent with the theoretically calculated protein molecular weight of 41.2 kDa. The separation gel concentration was 12%, and the preparation steps are shown in Table 1. The stacking gel concentration was 4%, and the preparation steps are shown in Table 2.

[0069] Table 1

[0070]

[0071] Table 2

[0072]

[0073] The concentration of 3′-O-methyltransferase after mutation was determined by protein quantification kit (BCA). The concentration of the purified 3′-O-methyltransferase mutant was 4 μg / mL, ignoring Ni. 2+ There was a slight loss of the target protein during the purification process, indicating that a relatively pure 3′-O-methyltransferase mutant was obtained.

[0074] Kinetic constants were determined to illustrate the difference in substrate affinity between the mutant strain and the wild-type strain, as well as the catalytic efficiency of the 3′-O-methyltransferase mutant. The specific method is as follows:

[0075] The preferred supply concentration of the immobilized methyl donor SAM is 200 μM. Luteolin is used as the substrate, and the assay concentration range is 0-60 μM, with luteolin concentration as a variable. The pure enzyme concentration is 4 μg / mL, and 2 mM DTT (dithiothreitol) is used. The assay is performed in 50 mM Tris-HCl, pH 8.0, at 37°C and 200 rpm, with an optimal reaction time of 30 min. Km and Vmax values are calculated using the Lineweaver-Burk method:

[0076]

[0077] Where: V is the reaction rate (μmol / (min·L)); Vmax is the maximum reaction rate (μmol / (min·L)); Km is the Michaelis constant (μM); [S] = substrate concentration (μM).

[0078] The experimental results are shown in Table 3: The affinity of the 3′-O-methyltransferase mutant (W284A) for the substrates luteolin and SAM was greatly improved, and the catalytic efficiency was comparable to that of the WT (wild-type 3′-O-methyltransferase). The catalytic ability was slightly weakened, but the affinity of the enzyme with the substrate was greatly improved after the mutation, thereby increasing the synthesis of the methylated flavonoid compound luteolin;

[0079] Table 3 Kinetic parameters of luteolin by 3′-O-methyltransferase mutants

[0080]

[0081] The Km of mutant W284A is 21.28 μM, which is lower than the Km value of the wild type (71.43 μM), indicating that the affinity of the mutant for the substrate luteolin is significantly improved compared with the wild type; however, its catalytic constant Kcat is 0.05S -1 Lower than the wild type Kcat value of 0.17S -1 , indicating that the catalytic ability of W284A for luteolin was not well improved. After the tryptophan at position 284 was mutated to alanine, the amount of substrate molecules converted by the enzyme per unit time was reduced. In addition, the kinetic parameter analysis of SAM showed that the Km value of W284A was 14.43μM, which was lower than the Km value of the wild type of 47.62μM, indicating that the mutant had a better affinity for SAM than the wild type. The Kcat value of W284A was 0.05S -1 It is lower than the wild type, indicating that its SAM catalytic ability is weaker than the wild type. Similarly, the mutation also reduces the enzyme's ability to convert SAM per unit time; but the Kcat / Km value of W284A is 3.47mM -1 S -1 Higher than the wild type Kcat / Km value of 1.89mM -1 S -1 , indicating that the catalytic efficiency of SAM has been improved after mutation.

[0082] 2. Construction of a recombinant strain co-expressing 3′-O-methyltransferase and methyl donor SAM synthase

[0083] 1. Construction of recombinant plasmid of methyl donor SAM synthase

[0084] Using the Escherichia coli genome as a template, primer metk-F (metk-F:

[0085] ATAAGAAGGAGATATACATATGGCAAAACACCTTTTTACGTC) and metk-R (metk-R: CGGTTTCTTTACCAGACTCGAGTTACTTCAGACCGGCAGCA), a specific fragment of the metk gene (1155 bp) was amplified from the genome, and the nucleotide sequence of the S-adenosyl-L-methionine (SAM) synthetase gene metk is shown in SEQ ID NO: 4:

[0086] atggcaaaac acctttttac gtccgagtcc gtctctgaag ggcatcctga caaaattgct

[0087] gaccaaattt ctgatgccgt tttagacgcg atcctcgaac aggatccgaa agcacgcgtt

[0088] gcttgcgaaa cctacgtaaa aaccggcatg gttttagttg gcggcgaaat caccaccagc

[0089] gcctgggtag acatcgaaga gatcacccgt aacaccgttc gcgaaattgg ctatgtgcat

[0090] tccgacatgg gctttgacgc taactcctgt gcggttctga gcgctatcgg caaacagtct

[0091] cctgacatca accagggcgt tgaccgtgcc gatccgctgg aacagggcgc gggtgaccag

[0092] ggtctgatgt ttggctacgc aactaatgaa accgacgtgc tgatgccagc acctatcacc

[0093] tatgcacacc gtctggtaca gcgtcaggct gaagtgcgta aaaacggcac tctgccgtgg

[0094] ctgcgcccgg acgcgaaaag ccaggtgact tttcagtatg acgacggcaa aatcgttggt

[0095] atcgatgctg tcgtgctttc cactcagcac tctgaagaga tcgaccagaa atcgctgcaa

[0096] gaagcggtaa tggaagagat catcaagcca attctgcccg ctgaatggct gacttctgcc

[0097] accaaattct tcatcaaccc gaccggtcgt ttcgttatcg gtggcccaat gggtgactgc

[0098] ggtctgactg gtcgtaaaat tatcgttgat acctacggcg gcatggcgcg tcacggtggc

[0099] ggtgcattct ctggtaaaga tccatcaaaa gtggaccgtt ccgcagccta cgcagcacgt

[0100] tatgtcgcga aaaacatcgt tgctgctggc ctggccgatc gttgtgaaat tcaggtttcc

[0101] tacgcaatcg gcgtggctga accgacctcc atcatggtag aaactttcgg tactgagaaa

[0102] gtgccttctg aacaactgac cctgctggta cgtgagttct tcgacctgcg cccatacggt

[0103] ctgattcaga tgctggatct gctgcacccg atctacaaag aaaccgcagc atacggtcac

[0104] tttggtcgtg aacatttccc gtggg.

[0105] The cloning vector pACYCDuet-1 was selected, and two restriction enzyme cutting sites, Xho I and Nde I, were selected in the multiple cloning site of the vector for double enzyme digestion of the vector.

[0106] The target gene fragment and vector fragment were subjected to electrophoresis on a 1.2% agarose gel. The amplified specific fragments were recovered from the gel and ligated using homologous recombination. The ligated products were transformed into competent E. coli DH5α cells. Single colonies were verified by enzyme digestion and recombinant plasmid sequencing, confirming the successful cloning of the metk gene and completing the construction of the recombinant plasmid pACYCDuet-1-metk.

[0107] 2. The recombinant plasmid pACYCDuet-1-metk and the 3′-O-methyltransferase mutant plasmid were co-expressed in the Escherichia coli BL21 (DE3) strain, and the recombinant strain containing the co-expression of the two enzymes was screened and obtained for the production of the methylated flavonoid compound chrysanthemum yellow.

[0108] 3. Fermentation and Condition Optimization of Recombinant Strains

[0109] The mutant strain was tested in a 48-well plate with a fermentation volume of 1 mL and a substrate addition amount of 200 mg / L luteolin. The OD600 value of the strain was measured after 24 h of fermentation. The content of the methylated flavonoid compound luteolin in the fermentation supernatant was analyzed by high performance liquid chromatography (HPLC). The peaks of the liquid chromatogram of the substrate luteolin and the methylated flavonoid product luteolin were as shown in FIG. Figure 1 As shown, the retention time of the methylated flavonoid compound chrysanthemum luteolin is about 17 minutes and its polarity is weaker than that of the substrate luteolin, wherein the UV detection wavelength is set to 280 nm; the mobile phase flow rate is 1 mL / min; the acquisition time is 20 minutes; the sample volume is 10 μL; the mobile phase A is: acetonitrile containing 0.1% formic acid; the mobile phase B is: water containing 0.1% formic acid; the program is: 10%-40% A (0-10 minutes), 40%-60% A (10-15 minutes), 60%-10% A (15-20 minutes); the chromatographic column is: Eclipse XDB-C18 column (5 μm, 150×4.6 mm).

[0110] like Figure 2 As shown, the yield of methylated flavonoids at this site increased by about 45%. The same fermentation method was used for two rounds of screening. The final experimental results showed that the mutant synthesized the highest yield of 52.80 mg / L of chrysanthemum yellow, which was 74% higher than that of the wild type, about 1.7 times;

[0111] Adding methionine to the culture medium is an effective method to increase the amount of SAM synthesis. The constructed mutant plasmid was co-expressed with the SAM pathway gene, and the amount of scutellarin synthesis after overexpression of the SAM pathway gene was verified by adding or not adding methionine. The results are shown in Figure 2. Figure 3As shown (where the horizontal axis represents: no optimization item None, exogenous addition of methionine Met, overexpression of SAM synthetase metK, exogenous addition of methionine and overexpression of SAM synthetase metK-Met, Native represents the wild-type strain, and the vertical axis represents the yield of ginseng flavonoids), it is confirmed that the synthesis amount of ginseng flavonoids is higher under the condition of overexpression of SAM synthetase metK, and the maximum yield of the target product can reach 104.26 mg / L.

[0112] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A method for preparing a 3′-O-methyltransferase mutant, characterized in that: The method comprises: The 284th amino acid of the amino acid sequence of the wild-type plant O-methyltransferase is mutated from tryptophan W to alanine A. The amino acid sequence of the wild-type plant O-methyltransferase is shown in SEQ ID NO: 1, and the amino acid sequence of the 3′-O-methyltransferase mutant is shown in SEQ ID NO:

2.

2. The method according to claim 1, wherein The method uses the caffeic acid-O-methyltransferase protein with the amino acid sequence shown in SEQ ID NO: 5 as a template to obtain a protein skeleton model. Mutation sites were selected within a certain range, and the principle of screening mutants was to enhance the binding ability of the enzyme to the substrate and increase the synthesis of the methylated product. The optimal mutation target was determined to be the mutation of amino acid 284 of SEQ ID NO: 1 from tryptophan W to alanine A. SEQ ID NO: 1 has 78% homology with SEQ ID NO:

5.

3. The method according to claim 1 or 2, wherein: The wild-type plant oxygen methyltransferase is derived from rice.

4. The method according to claim 1 or 2, wherein: The nucleotide sequence of the gene encoding the 3′-O-methyltransferase mutant is shown in SEQ ID NO:

3.

5. A method for preparing a recombinant plasmid, characterized in that: The method comprises: The recombinant plasmid contains the gene encoding the 3'-O-methyltransferase mutant according to claim 4.

6. The method according to claim 5, wherein: The method further includes making the recombinant plasmid further contain a gene encoding overexpression of S-adenosyl-L-methionine synthetase.

7. A method for preparing a mutant strain, characterized in that: The method comprises: The mutant strain contains the gene encoding the 3'-O-methyltransferase mutant according to claim 4 and a gene encoding overexpressing S-adenosyl-L-methionine synthetase.

8. A method for preparing a mutant strain, characterized in that: The method comprises: Transforming the mutant strain into the recombinant plasmid according to claim 5, or The mutant strain is co-transformed with a recombinant plasmid overexpressing S-adenosyl-L-methionine synthetase and the recombinant plasmid according to claim 5.

9. The method according to claim 8, wherein The recombinant plasmid for overexpressing S-adenosyl-L-methionine synthetase is a plasmid pACYCDuet-1 comprising a gene encoding the overexpression S-adenosyl-L-methionine synthetase.

10. The method according to any one of claims 7 to 9, wherein The mutant strain is derived from Corynebacterium glutamicum, Escherichia coli or Bacillus subtilis.

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