Bovine heart alkali 7-O-methyltransferase mutant with improved enzyme activity and application of bovine heart alkali 7-O-methyltransferase mutant

Abstract

The invention provides a bovine heart alkali 7-O-methyltransferase mutant with improved enzyme activity and application of the bovine heart alkali 7-O-methyltransferase mutant. Hydrophobic amino acid methionine amine in a bovine heart alkali 7-O-methyltransferase molecule is mutated into isoleucine with strong hydrophobicity in a site-directed mutagenesis mode; and amino acids valine and leucine with strong hydrophobicity are respectively mutated into amino acids methionine and phenylalanine with weak hydrophobicity, so that the hydrophobicity and substrate interaction in the whole molecule are changed, the pocket cavity of the substrate is remodeled, the enzymatic activity of the bovine heart alkali 7-O-methyltransferase expressed by the strain is obviously improved, and the yield of the bovine heart alkali 7-O-methyltransferase is improved. And the activity of the bovine heart alkali 7-O-methyltransferase is improved by about 18 times to the maximum extent.

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 a recombinant Escherichia coli 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 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), and 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 object 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 using site-directed mutagenesis technology.

[0006] To achieve the above object, the present invention is realized through the following solutions: 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 achieved. In addition, the present invention also provides a method for improving the activity of stepharine 7-O-methyltransferase, which is to mutate the methionine at the 251st position inside the stepharine 7-O-methyltransferase protein molecule with the amino acid sequence shown in SEQ ID NO.1 into isoleucine; and / or mutate the valine at the 236th position into methionine; and / or mutate the leucine at the 237th position into phenylalanine respectively. The mutants are selected from any one of the following: M251I: Directly mutate the methionine at the 251st position into isoleucine; V236M: Directly mutate the valine at the 236th position into methionine respectively; L237F: Directly mutate the leucine at the 237th position into phenylalanine respectively; In an embodiment of the present invention, the gene encoding stepharine 7-O-methyltransferase is ligated with the vector pET-28a(+), and Escherichia coli E. coli BL21(DE3) is transformed. The transformants are selected and inoculated into the LB liquid medium with kanamycin resistance, cultured at 37 °C for 12 h, transferred to a new resistant medium, and the inoculation amount is 1%. When the cell density reaches OD 600 of 0.6 - 0.8, IPTG is added for induction, and the culture temperature is reduced to 16 °C, and cultured for 24 h. The supernatant is collected, and the stepharine 7-O-methyltransferase mutant (R7OMT) is purified.

[0007] 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. The hydrophobicity and substrate interaction inside the whole molecule are changed, and the substrate pocket cavity is reshaped, significantly improving the enzyme activity of the strain expressing stepharine 7-O-methyltransferase, and the activity of stepharine 7-O-methyltransferase is maximally increased by about 18 times. Description of the Drawings

[0008] Figure 1 It is a diagram showing the results of the function verification of stepharine 7-O-methyltransferase; Figure 2 It is a bar chart of the relative catalytic activities of wild-type StR7OMT and its mutants; Figure 3 Catalytic results of mutant M251I-V236M-L237F on substrates with different configurations; Figure 4 10% SDS-PAGE purified protein gel of wild enzyme and its mutants. Detailed implementation method

[0009] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0; Phosphate buffer: 10 mM K 2 HPO 4 、10 mM KH 2 PO 4 , pH 7.0; The enzyme activity of annonaine 7-O-methyltransferase was determined by high-performance liquid chromatography. Taking the enzyme activity of the wild enzyme as 100%, the catalytic activities of recombinant StR7OMT and mutants were determined in 50 mM potassium phosphate buffer (pH 7.5), and the buffer contained 100 μM substrates (S)-coclaurine, norcoclaurine and (S)-annonaine, 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 chart was made using drawing software, and the enzyme activity was characterized according to the peak area of the product.

[0010] Example 1 Construction of annonaine 7-O-methyltransferase strain Using Bam I 、 Hind Ⅲ as the restriction enzyme digestion sites, the plasmid pET-28a(+) vector was digested with double enzymes, and the digested plasmid was purified and recovered using a gel recovery 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(+). Ligation system: 4 μL of target gene pET-28a(+), 1 μL of vector (pET-28a(+)), 4 μL of 5 × CE II buffer, 2 μL of homologous recombinase (Exnase II), 9 μL of ddH 2 O, ligate at 37 °C for 30 min. The ligated recombinant plasmid was transformed into competent E.coilJM109 was transformed on a kanamycin LB plate and cultured overnight at 37°C in an inverted position. Colonies were picked for positive identification, and positive transformants were selected for sequencing, which was performed by Shangya in Hangzhou. After comparing the sequencing results, plasmids were extracted from the samples with correct sequencing to obtain the recombinant plasmid pET-28a-StR7OMT. The amino acid sequence encoded by the StR7OMT gene is shown in SEQ ID NO.1.

[0011] Example 2 Verification of the Enzyme Activity of Annonaine 7-O-Methyltransferase The plasmid with correct sequencing in Example 1 was transformed into Escherichia coli E. coli BL21(DE3). Transformed colonies were picked and inoculated into LB liquid medium and 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, and the cells were sonicated and centrifuged at 4°C. The supernatant was taken and the protein was purified using a His-tag recombinant protein purification kit. The concentration of the purified protein was measured, and substrate catalysis was carried out, 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.

[0012] Example 3 Obtaining of StR7OMT Mutants 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, to directedly mutate methionine at position 251 inside StR7OMT into isoleucine, valine at position 236 into methionine, and leucine at position 237 into phenylalanine, obtaining 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 of each upstream and downstream primer, 25 μL of high-fidelity enzyme (Prime-STAR), and 20 μL of sterilized double-distilled water. A gel extraction kit was used to purify and recover the PCR products, 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. Using the plasmids of the strains pET-28a-StR7OMT-M251I, pET-28a-StR7OMT-V236M, and pET-28a-StR7OMT-L237F with correct sequencing to perform compound mutations, mutant transformants were obtained respectively, and the plasmids were named: pET-28a-StR7OMT-M251I-V236M-L237F.

[0013] The amino acid sequences of mutants M251I, V236M, L237F, and M251I-V236M-L237F are SEQ ID NO.2 - 5 respectively.

[0014] Table 1 Primer sequences

[0015] Example 4 Enzyme activity verification of the mutant strain of high-activity reticuline 7-O-methyltransferase The plasmid with correct sequencing was transformed E. coli into BL21(DE3), the transformants were selected and inoculated into LB liquid medium, and 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 16°C and cultured for 24 h. The cells were collected and broken to collect the recombinant protein, and the recombinant protein was obtained after purification by His purification column, and its catalytic activity was detected. The results are as Figure 2 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.

[0016] Example 5 Analysis of Different Chiral Substrates of Mutant M251I-V236M-L237F Substrate chiral analysis was performed using substrates such as R-coclaurine, S-coclaurine, R-annonaine, and S-annonaine. Taking the conversion rate of the most active S-annonaine as 100%, the results showed that compared with the wild enzyme, the mutant M251I-V236M-L237F had good catalytic activity towards both R-coclaurine and R-annonaine substrates. The results were as follows Figure 3 shown. The catalytic efficiency of M251I-V236M-L237F towards R-annonaine reached 18-fold. It can be seen that the catalytic effect of the mutant on the R configuration was improved. The purification results of the His-tagged wild recombinant protein and its mutant were as follows 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.

[0017] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and alterations 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.

[0018] Sequence Listing SEQ ID NO.1 (Wild-Type Annonaine-7-O-Methyltransferase): MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDVLSSIPNGDVLFFKSMFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP SEQ ID NO.2 (Annonaine-7-O-Methyltransferase Mutant M251I): MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDVLSSIPNGDVLFFKSIFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP SEQ ID NO.3 (Annonaine-7-O-methyltransferase mutant V236M): MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDMLSSIPNGDVLFFKSMFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP SEQ ID NO.4 (Annonaine-7-O-methyltransferase mutant L237F): MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDVFSSIPNGDVLFFKSMFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP SEQ ID NO.5 (Annona reticulata alkaloid - 7 - O - methyltransferase mutant M251 - V236M - L237F): MEEELKARVQLSKHMFAFAETISLRCAVQLGLPDKIYEHGPLTLSELASKLPIKSLNMDRFEQTMRYMVHMNLFEATTDGNEETKYGLTPMSRLLLNNSHNRKSLATFVMAQTDPEELFISGRLVESLGGTKSCWELQYGVPVFEKMENDEKWSKVSDGMNGYTMSMIDAVVDGIKRENIIDESVTTLVDVGGNTGIAAKAILNAFPHLKCTVMDLVDVVENVPKDPQLNFVAGDMFSSIPNGDVLFFKSIFHGFEDDLSVKILNNCKKAMHPTKGRLIVVEMVLDIETMPEFSHARLGLAMQMMFLGGQERTKKGWERLIHKAGFTRYKIVPIAAAESIIVIYP SEQ ID NO.6: gatgtgctattctttaagtcgatattccatggatttgaggatg SEQ ID NO.7: catcctcaaatccatggaatatcgacttaaagaatagcacatc SEQ ID NO.8: cttcgtggccggggacatgttaagttccattccca SEQ ID NO.9: tgggaatggaacttaacatgtccccggccacgaag SEQ ID NO.10: ggccggggacgtcttcagttccattccc SEQ ID NO.11: gggaatggaactgaagacgtccccggcc。

Claims

1. A mutant of taurine 7-O-methyltransferase with improved enzyme activity, characterized in that: The method comprises the following steps: the methionine at position 251 of the oxygen methyltransferase of the amino acid sequence as shown in SEQ ID NO. 1 is mutated to isoleucine, the valine at position 236 is mutated to methionine, and the leucine at position 237 is mutated to phenylalanine.

2. The taurine 7-O-methyltransferase mutant with improved enzyme activity according to claim 1, characterized in that: The amino acid sequence of the tadalafil 7-O-methyltransferase mutant is shown in SEQ ID NO.

5.

3. A recombinant genetically engineered bacterium, characterized in that: The invention comprises the taurine 7-O-methyltransferase mutant with improved enzyme activity as claimed in claim 1, wherein the engineering bacteria is Escherichia coli E. coli BL21.

4. Use of the taurine 7-O-methyltransferase mutant with improved enzyme activity according to claim 1 in the synthesis of benzylisoquinoline alkaloids.

5. Use of the recombinant genetically engineered bacteria according to claim 3 in the synthesis of benzylisoquinoline alkaloids.

6. The use according to claim 4 or 5, wherein the hydroxyl group of taurine is methylated by the taurine 7-O-methyltransferase mutant in the synthesis of benzylisoquinoline alkaloids.

7. A method for increasing the activity of oxygen methyltransferase, characterized in that: The method comprises mutating the 251st methionine in the choline 7-O-methyltransferase protein molecule with the amino acid sequence as shown in SEQ ID NO.1 to isoleucine, mutating the 236th valine to methionine, and mutating the 237th leucine to phenylalanine.

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