A mutant of bovine mango alkaloid 7-O-methyltransferase with improved enzyme activity and its application

Abstract

The present invention provides a mutant of reticuline 7-O-methyltransferase with improved enzyme activity. By means of site-directed mutagenesis, the hydrophobic amino acid methionine in the reticuline 7-O-methyltransferase molecule is mutated into isoleucine with stronger hydrophobicity, and the amino acids valine and leucine with strong hydrophobicity are respectively mutated into the weakly hydrophobic amino acids methionine and phenylalanine, changing the overall internal hydrophobicity of the molecule and the interaction with the substrate, and remodeling the substrate pocket cavity, significantly improving the enzyme activity of the strain expressing reticuline 7-O-methyltransferase, and the reticuline 7-O-methyltransferase activity is maximally increased by about 18 times.

Description

Technical Field

[0001] The present invention relates to a mutant of reticuline 7-O-methyltransferase with improved enzyme activity, belonging to the fields of enzyme engineering and synthetic biology. Background Art

[0002] Reticuline 7-O-methyltransferase is an O-methyltransferase that catalyzes the methylation of the C7 hydroxyl group of reticuline. It is a key enzyme in the biosynthesis of benzylisoquinoline alkaloids. The catalytic product, laudanidine, is a precursor compound of papaverine and morphine, and plays an important role in the biosynthesis of related benzylisoquinoline alkaloids.

[0003] A reticuline 7-O-methyltransferase gene derived from Stephania tetrandra was used to construct an Escherichia coli recombinant strain containing the reticuline 7-O-methyltransferase gene by genetic engineering technology. The expression of the reticuline 7-O-methyltransferase protein and the verification of its in vitro enzyme catalytic activity were achieved. It was confirmed that the reticuline 7-O-methyltransferase has the function of catalyzing the C7 hydroxyl group methylation of reticuline, providing a reticuline 7-O-methyltransferase element for the biosynthesis of related metabolites. However, through in vitro catalytic verification, it was found that it also catalyzes the C7 hydroxyl group of corytuberine and N-methylcorytuberine (mainly catalyzing reticuline). The catalytic activity of reticuline 7-O-methyltransferase is relatively low, especially the conversion rate of R-reticuline is low, which severely limits the catalytic synthesis of related metabolites.

[0004] Based on the previously constructed recombinant plasmid of reticuline 7-O-methyltransferase, the present invention changed the cavity channel and internal hydrophobicity of the protein through site-directed mutagenesis, and induced expression in Escherichia coli, thereby achieving the improvement of the enzyme activity of reticuline 7-O-methyltransferase and the change of stereoselectivity. Summary of the Invention

[0005] The purpose of the present invention is to use the reticuline 7-O-methyltransferase gene identified from the medicinal plant Stephania tetrandra, heterologously recombinantly express this gene using Escherichia coli, and obtain a mutant of reticuline 7-O-methyltransferase with improved enzyme activity by using site-directed mutagenesis technology.

[0006] To achieve the above purpose, the present invention is realized through the following scheme:

[0007] The present invention utilizes the wild-type stepharine 7-O-methyltransferase gene derived from the medicinal plant Stephania tetrandra (the amino acid sequence is shown in SEQ ID NO.1), and through the heterologous expression system of Escherichia coli, the catalytic function verification of C7 hydroxymethylation is realized. In addition, the present invention also provides a method for improving the activity of stepharine 7-O-methyltransferase, which is to mutate methionine at position 251 inside the stepharine 7-O-methyltransferase protein molecule with the amino acid sequence shown in SEQ ID NO.1 into isoleucine; and / or mutate valine at position 236 into methionine; and / or mutate leucine at position 237 into phenylalanine respectively. The mutant is selected from any one of the following:

[0008] M251I: Directly mutate methionine at position 251 into isoleucine;

[0009] V236M: Directly mutate valine at position 236 into methionine respectively;

[0010] L237F: Directly mutate leucine at position 237 into phenylalanine respectively;

[0011] In one embodiment of the present invention, the gene encoding stepharine 7-O-methyltransferase is ligated with the vector pET-28a(+), and then transformed into Escherichia coli E. coli BL21(DE3). Select the transformant and inoculate it into the LB liquid medium with kanamycin resistance, culture at 37 °C for 12 h, then transfer it to a new resistant medium, and the inoculation amount is 1%. When the cell density reaches OD 600 of 0.6 - 0.8, add IPTG for induction, and reduce the culture temperature to 16 °C, and culture for 24 h. Collect the supernatant, and purify to obtain the stepharine 7-O-methyltransferase mutant (R7OMT).

[0012] In the present invention, through site-directed mutagenesis, the hydrophobic amino acid methionine inside the stepharine 7-O-methyltransferase molecule is mutated into isoleucine with stronger hydrophobicity, and the amino acid valine with strong hydrophobicity is mutated into the amino acid methionine with weak hydrophobicity, and the hydrophobic amino acid leucine is mutated into phenylalanine with stronger hydrophobicity. This is because the phenyl group has a stronger electron affinity and π-electron conjugation effect, which can better attract water molecules, thus showing stronger hydrophobicity. By changing the overall hydrophobicity inside the molecule and the interaction with the substrate, and remodeling the substrate pocket cavity, the enzyme activity of the stepharine 7-O-methyltransferase expressed by the strain is significantly improved, and the activity of the stepharine 7-O-methyltransferase is maximally increased by about 18 times. Description of the Drawings

[0013] Figure 1 It is a result diagram for the function verification of stepharine 7-O-methyltransferase;

[0014] Figure 2 Bar graph of the relative catalytic activities of wild-type StR7OMT and its mutants;

[0015] Figure 3 Catalytic result graph of mutant M251I-V236M-L237F for substrates with different configurations;

[0016] Figure 4 10% SDS-PAGE purified protein gel diagram of wild enzyme and its mutants. Detailed implementation method

[0017] LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, NaCl 10 g / L, pH 7.0;

[0018] Phosphate buffer: 10 mM K2HPO4, 10 mM KH2PO4, pH 7.0;

[0019] The enzyme activity of anonaine 7-O-methyltransferase was determined by high-performance liquid chromatography detection method, with the enzyme activity of wild enzyme as 100%. The catalytic activities of recombinant StR7OMT and mutants were determined in 50 mM potassium phosphate buffer (pH 7.5). The buffer included 100 μM substrates (S)-coclaurine, norcoclaurine and (S)-anonaine, 100 μM S-adenosylmethionine (SAM) and 20 μg purified recombinant protein, and the reaction volume was 100 μL. Incubate at 30 °C for 4 h, quench with 100 μL methanol, centrifuge at 12000 rpm for 2 min to precipitate proteins. Take an equal amount of heat-inactivated recombinant protein as a negative control. After the samples were filtered through a 0.22 μm filter, they were analyzed using a UPLC-qTOF-MS system. Mobile phase A was 0.1% formic acid, and mobile phase B was acetonitrile. The peak area of the product was measured, and a bar graph was made through drawing software, and the enzyme activity was characterized according to the peak area of the product.

[0020] Example 1 Construction of anonaine 7-O-methyltransferase strain

[0021] Using Bam I 、 Hind ⅢIt is a restriction endonuclease cleavage site. The plasmid pET-28a(+) vector was digested with double enzymes, and the digested plasmid was purified and recovered using a gel extraction kit, and the concentration of the recovered product was examined by electrophoresis. The recovered target gene, annonaine 7-O-methyltransferase, was ligated with the vector plasmid pET-28a(+). The ligation system: 4 μL of the target gene pET-28a(+), 1 μL of the vector (pET-28a(+)), 4 μL of 5 × CE II buffer, 2 μL of homologous recombinase (Exnase II), 9 μL of ddH2O, and ligated at 37°C for 30 min. The ligated recombinant plasmid was transformed into competent E.coil JM109, transformed onto a kanamycin LB plate, and cultured overnight at 37°C in an inverted position. Colonies were picked, positively identified, and positive transformants were screened for sequencing, which was performed by Hangzhou Shangya. Comparing the sequencing results, plasmids were extracted from the samples with correct sequencing to obtain the pET-28a-StR7OMT recombinant plasmid. The amino acid sequence encoded by the StR7OMT gene is shown in SEQ ID NO.1.

[0022] Example 2 Verification of the enzyme activity of annonaine 7-O-methyltransferase

[0023] The plasmid with correct sequencing in Example 1 was transformed into Escherichia coli E. coli BL21(DE3). The transformants were selected and inoculated into LB liquid medium, cultured at 37°C for 12 h, then transferred to LB medium with an inoculation amount of 1%. When the cell density reached OD 600 of 0.6 - 0.8, IPTG was added for induction, and the culture temperature was reduced to 30°C and cultured for 24 h. The cells were collected, phosphate buffer was added, sonicated, centrifuged at 4°C, and the supernatant was taken. The protein was purified using a His-tag recombinant protein purification kit, the concentration of the purified protein was measured, the substrate was catalyzed, and the product was detected by HPLC-UV. The relative enzyme activity was represented by the product peak area. The results are as Figure 1 shown. Laudanosine produced characteristic fragment ions with mass numbers of approximately 206 and 137, respectively, upon cleavage in the mass spectrum.

[0024] Example 3 Obtaining of StR7OMT mutants

[0025] Three pairs of primers (shown in Table 1) were designed and used to perform PCR amplification with the constructed pET-28a-StR7OMT as the template. The methionine at position 251 inside StR7OMT was directionally mutated to isoleucine, the valine at position 236 was directionally mutated to methionine, and the leucine at position 237 was directionally mutated to phenylalanine to obtain the M251, V236M, and L237F mutants. The PCR conditions were: 98°C for 5 min, 35 cycles (98°C for 5 min, 60°C for 30 s, 72°C for 7 min), and 72°C for 10 min. The PCR amplification system was: 1 μL of template, 2 μL each of upstream and downstream primers, 25 μL of high-fidelity enzyme (Prime-STAR), and 20 μL of sterilized double-distilled water. The PCR products were purified and recovered using a gel extraction kit, the concentration was measured, and after transformation and sequencing, the successfully mutated transformants were named pET-28a-StR7OMT-M251I, pET-28a-StR7OMT-V236M, and pET-28a-StR7OMT-L237F respectively. Using the plasmids of the correctly sequenced strains pET-28a-StR7OMT-M251I, pET-28a-StR7OMT-V236M, and pET-28a-StR7OMT-L237F for compound mutations, mutant transformants were obtained respectively, and the plasmids were named pET-28a-StR7OMT-M251I-V236M-L237F.

[0026] The amino acid sequences of the mutants M251I, V236M, L237F, and M251I-V236M-L237F are SEQ ID NO.2 - 5 respectively.

[0027] Table 1 Primer sequences

[0028]

[0029] Example 4 Enzyme activity verification of the mutant strain of high-activity bullock's heart alkaloid 7-O-methyltransferase

[0030] The correctly sequenced plasmid was transformed E. coli into BL21(DE3), and the transformants were selected and inoculated into LB liquid medium. Incubate at 37°C for 12 h, then transfer to LB medium with an inoculation amount of 1%. When the cell density reached OD 600 of 0.6 - 0.8, IPTG was added for induction, and the culture temperature was reduced to 16°C and cultured for 24 h. The cells were collected and broken to collect the recombinant protein, which was purified by a His purification column to obtain the recombinant protein, and its catalytic activity was detected. The results are as Figure 2As shown. The experimental results show that compared with the wild enzyme, the activities of the mutants in catalyzing the synthesis of laudanosine from reticuline are all improved, that is, the mutants have stronger enzyme activities. Compared with WT (wild type), the enzyme activities of M251I, V236M and L237F are increased by about 1.37, 2.4 and 2.3 times respectively.

[0031] Example 5 Analysis of Different Chiral Substrates of Mutant M251I-V236M-L237F

[0032] Substrate chiral analysis was carried out using substrates such as R-coclaurine, S-coclaurine, R-reticuline and S-reticuline. Taking the conversion rate of the most active S-reticuline as 100%, the results show that compared with the wild enzyme, the mutant M251I-V236M-L237F has good catalytic activities for both R-coclaurine and R-reticuline substrates. The results are as Figure 3 shown. The catalytic efficiency of M251I-V236M-L237F for R-reticuline reaches 18 times. It can be seen that the catalytic effect of the mutant on the R configuration is improved. The purification results of the His-tagged wild recombinant protein and its mutant are as Figure 4 . Among them, M: Marker, EV: empty vector control, lanes 1-2: WT, lane 3: M251I, lane 4: V236M, lane 5: L237F, lanes 6-7: M251I-V236M-L237F.

[0033] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

[0034] Sequence Listing

[0035] SEQ ID NO.1 (Wild Type of Reticuline-7-O-Methyltransferase):

[0036] MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDVLSSIPNGDVLFFKSMFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP

[0037] SEQ ID NO.2 (Annonaine-7-O-methyltransferase mutant M251I):

[0038] MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDVLSSIPNGDVLFFKSIFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP

[0039] SEQ ID NO.3 (Annonaine-7-O-methyltransferase mutant V236M):

[0040] MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDMLSSIPNGDVLFFKSMFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP

[0041] SEQ ID NO.4 (Annonaine-7-O-methyltransferase mutant L237F):

[0042] MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDVFSSIPNGDVLFFKSMFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP

[0043] SEQ ID NO.5 (Annonaine-7-O-methyltransferase mutant M251-V236M-L237F):

[0044] MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDMFSSIPNGDVLFFKSIFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP

[0045] SEQ ID NO.6:

[0046] gatgtgctattctttaagtcgatattccatggatttgaggatg

[0047] SEQ ID NO.7:

[0048] catcctcaaatccatggaatatcgacttaaagaatagcacatc

[0049] SEQ ID NO.8:

[0050] cttcgtggccggggacatgttaagttccattccca

[0051] SEQ ID NO.9:

[0052] tgggaatggaacttaacatgtccccggccacgaag

[0053] SEQ ID NO.10:

[0054] ggccggggacgtcttcagttccattccc

[0055] SEQ ID NO.11:

[0056] gggaatggaactgaagacgtccccggcc。

Claims

1. A mutant of annonaine 7-O-methyltransferase with improved enzyme activity, characterized in that: It is to mutate the methionine at position 251 of the anonasine 7-O-methyltransferase with the amino acid sequence as shown in SEQ ID NO.1 into isoleucine, mutate the valine at position 236 into methionine, and mutate the leucine at position 237 into phenylalanine.

2. A recombinant genetic engineering bacterium, characterized in that, Comprising the annonaine 7-O-methyltransferase mutant with enhanced enzyme activity described in claim 1, and the engineered bacterium is Escherichia coli E. coli BL21.

3. Use of the mutant of reticuline 7-O-methyltransferase with enhanced enzyme activity according to claim 1 in the synthesis of benzylisoquinoline alkaloids, characterized in that, The anonasine 7-O-methyltransferase mutant catalyzes the C7 hydroxymethylation of anonasine.

4. Use of the recombinant genetically engineered bacterium according to claim 2 in the synthesis of benzylisoquinoline alkaloids, characterized in that, The anonasine 7-O-methyltransferase mutant catalyzes the C7 hydroxymethylation of anonasine.

5. A method for enhancing the activity of oxygen methyltransferase, characterized in that, It is to mutate the methionine at position 251 of the anonasine 7-O-methyltransferase with the amino acid sequence as shown in SEQ ID NO.1 into isoleucine, mutate the valine at position 236 into methionine, and mutate the leucine at position 237 into phenylalanine.

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