Oxygen methyltransferase mutant with improved enzyme activity, preparation method and application

Abstract

The present invention discloses an oxygen methyltransferase mutant with improved enzyme activity and the construction of a biosynthetic chassis of a corresponding product, and belongs to the field of enzyme engineering and synthetic biology. The present invention mutates the hydrophilic amino acid glutamic acid inside the oxygen methyltransferase molecule into a highly hydrophobic leucine by means of site-directed mutagenesis. Leucine and valine change the hydrophobicity and substrate interaction inside the molecule, reshape the substrate pocket and the substrate access channel, and significantly improve the enzyme activity of the strain expressing oxygen methyltransferase. The transformed plasmid is used to construct a biosynthetic cell chassis of the corresponding metabolite in Escherichia coli, providing a basis for the biosynthesis of the relevant metabolites. The enzyme production capacity of the transformed strain is significantly improved, which is more suitable for industrial applications, can reduce production costs, and improve production efficiency.

Description

Technical Field

[0001] The invention relates to an oxygen methyl transfer mutant with improved enzyme activity, and utilizes the mutant to construct a biosynthetic chassis of corresponding metabolites, belonging to the fields of enzyme engineering and synthetic biology. Background Art

[0002] Coclaurine 7-O-methyltransferase (7-OMT) is an oxygen methyltransferase that catalyzes the methylation of the C7 hydroxyl group of coclaurine. It is a key enzyme in the synthesis of benzylisoquinoline alkaloids. The catalytic product, nor-opapaverine, is the direct precursor compound of opapaverine and plays an important role in the biosynthesis of related benzylisoquinoline alkaloids.

[0003] A 7OMT gene from Stephania tetrandra was used to construct a recombinant Escherichia coli strain containing the St7OMT gene using genetic engineering technology, and the expression of the St7OMT protein was achieved. In vitro enzyme catalytic activity verification confirmed that the St7OMT has the function of catalyzing the hydroxymethylation of C7, providing a 7OMT enzymatic element for the biosynthesis of related metabolites. However, in vitro catalytic verification found that its catalytic activity was low and the product conversion rate was low, which seriously limited the catalytic synthesis of related metabolites.

[0004] Papaverine is a Nelumbo nucifera The active compounds not only have anti-inflammatory effects on human peripheral blood mononuclear cells, but also have immunosuppressive effects on T lymphocytes and lupus nephritis mice, and also have the ability to inhibit TNF-α-induced MAPK and NF-κB signaling cascades.

[0005] The present invention is based on the St7OMT recombinant plasmid constructed in the early stage, changes the cavity channel of the protein by point mutation, and induces expression through Escherichia coli, thereby achieving the improvement of St7OMT enzyme activity and the change of substrate specificity, and utilizes the above-mentioned modified 7OMT plasmid to construct the metoprine biosynthesis chassis. Summary of the invention

[0006] The purpose of the present invention is to utilize the 7OMT gene (named St7OMT) identified from the medicinal plant Stephania tetrandra, to express the gene by heterologous recombination using Escherichia coli, and to obtain an oxygen methyltransferase mutant with improved enzyme activity using site-directed mutagenesis technology, and to disclose its application in product synthesis.

[0007] In order to achieve the above object, the present invention is implemented by the following scheme:

[0008] The present invention utilizes the wild-type St7OMT gene (amino acid sequence as shown in SEQ ID NO.1) derived from the medicinal plant Stephania tetrandra, and realizes the verification of the catalytic function of C7 hydroxymethylation through the heterologous expression system of Escherichia coli. In addition, the present invention also provides a method for improving the activity of oxygen methyltransferase, which is to mutate the alanine at position 118 inside the oxygen methyltransferase protein molecule as shown in SEQ ID NO.1; mutate the glutamine at position 121 to leucine or valine respectively; and mutate the arginine at position 151 to leucine or isoleucine. The mutant is selected from any one of the following:

[0009] A118V: Directed mutation of alanine at position 118 to valine;

[0010] Q121L: Directed mutation of glutamine at position 121 to leucine;

[0011] Q121V: Directed mutation of glutamine at position 121 to valine;

[0012] R151L: directed mutation of arginine at position 151 to leucine;

[0013] R151I: directed mutation of arginine at position 151 to isoleucine;

[0014] A118V / R151L: Directed mutation of alanine at position 118 to valine and arginine at position 151 to leucine;

[0015] A118V / R151I: Directed mutation of alanine at position 118 to valine and arginine at position 151 to isoleucine;

[0016] Q121V / R151L: Directed mutation of glutamine at position 121 to valine and arginine at position 151 to leucine;

[0017] Q121V / R151I: Directed mutation of glutamine at position 121 to valine and arginine at position 151 to isoleucine;

[0018] Q121L / R151L: Directed mutation of glutamine at position 121 to leucine and arginine at position 151 to leucine;

[0019] Q121L / R151I: Directed mutation of glutamine at position 121 to leucine and arginine at position 151 to isoleucine;

[0020] A118V / Q121L: Directed mutation of alanine at position 118 to valine and glutamine at position 121 to leucine;

[0021] A118V / Q121V: Directed mutation of alanine at position 118 to valine and glutamine at position 121 to valine;

[0022] A118V / Q121L / R151L: Simultaneously mutate alanine at position 118 to valine, glutamine at position 121 to leucine, and arginine at position 151 to leucine;

[0023] A118V / Q121L / R151I: Simultaneously mutate alanine at position 118 to valine, glutamine at position 121 to leucine, and arginine at position 151 to isoleucine;

[0024] A118V / Q121V / R151L: Simultaneously mutate the alanine at position 118 to valine, the glutamine at position 121 to valine, and the arginine at position 151 to leucine.

[0025] In one embodiment of the present invention, the gene encoding oxygen methyltransferase is connected to the vector pGEX-4T-1 and transformed into Escherichia coli. E. coli BL21(DE3). Select transformants and inoculate them into LB liquid medium with Amp resistance, culture at 37℃ for 12 h, and transfer them into new resistance medium with an inoculation volume of 1%. When the bacteria grow to OD 600 When the pH value was 0.6-0.8, IPTG was added for induction, and the culture temperature was lowered to 16°C for 24 h. The supernatant was collected and purified to obtain the oxygen methyltransferase mutant.

[0026] The present invention mutates the hydrophilic amino acid glutamine inside the oxygen methyltransferase molecule into highly hydrophobic leucine by site-directed mutagenesis. Leucine and valine change the hydrophobicity inside the molecule and the substrate interaction, and reshape the substrate pocket and the substrate entry and exit channel, thereby significantly improving the enzyme activity of the oxygen methyltransferase expressed by the strain, and increasing the oxygen methyltransferase activity by about 2-6 times. At the same time, the catalytic nitrogen-methylcoclaurine C7 hydroxyl catalysis is generated, and the construction of the metoprine biosynthesis chassis is completed by introducing the Ps6OMT and PsCNMT genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the result diagram of St7OMT function verification;

[0028] Figure 2It is a bar graph of the relative catalytic activities of wild-type St7OMT and its mutants;

[0029] Figure 3 The diagram shows the catalytic results of mutant A118V / Q121L / R151L on different substrates;

[0030] Figure 4 Designing a roadmap for engineering the E. coli chassis for metoprine biosynthesis;

[0031] Figure 5 Results of synthesizing papaverine for the E. coli chassis;

[0032] Figure 6 This is the mass spectrum of product A of the synthesis of papaverine in the E. coli chassis;

[0033] Figure 7 This is the mass spectrum of product B of the synthesis of papaverine in the Escherichia coli chassis. DETAILED DESCRIPTION

[0034] LB medium: tryptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, pH 7.0;

[0035] Phosphate buffer: 10 mM K2HPO4, 10 mM KH2PO4, pH 7.0;

[0036] The activity of O-methyltransferase was determined by high performance liquid chromatography detection, with the activity of wild enzyme as 100%: the catalytic activity of recombinant St7OMTs and mutants was determined in 50 mM potassium phosphate buffer (pH 7.5), the buffer included 100 μM substrate (S)-Coclurine, 100 μM SAM and 20 μg purified recombinant protein, and the reaction volume was 100 μL. Incubate at 30℃ for 4 h, quench with 100 μL methanol, and centrifuge at 12000 rpm for 2 min to precipitate the protein. An equal amount of heat-inactivated recombinant protein was taken as a negative control. The sample was filtered through a 0.22 μm filter and analyzed by HPLC-UV system. Mobile phase A was 0.03% trifluoroacetic acid and mobile phase B was acetonitrile. The product peak area measured by HPLC-UV at 280 nm was used to make a bar graph using a drawing software, and the enzyme activity was characterized by the product peak area.

[0037] Example 1 Construction of 7-oxomethyltransferase strain

[0038] The plasmid pGEX-4T-1 (vector) was double-digested with Xho I and EcoR I as restriction endonuclease sites, and the plasmid after digestion was purified and recovered using a gel recovery kit, and the concentration of the recovered product was tested by electrophoresis. The recovered target gene (St7OMT) was connected to the vector plasmid pGEX-4T-1. The connection system was: target gene (St7OMT) 4 μL, vector (pGEX-4T-1) 1 μL, 5 × CE II Buffer 4 μL, Exnase II 2 μL, ddH2O 9 μL, 37 ℃ connection for 30 minutes. The connected recombinant plasmid pGEX-4T-1-St7OMT was transformed into the competent cell. E.coil JM109, transform ampicillin LB plate, invert and culture at 37℃ overnight, pick colonies, positive identification, screen positive transformants for sequencing, sequencing was performed by Hangzhou Shangya. Compare the sequencing results, extract plasmids from the correct samples, and obtain pGEX-4T-1-St7OMT recombinant plasmid. The amino acid sequence encoded by the St7OMT gene is shown in SEQ ID NO.1.

[0039] Example 2 Verification of 7-Oxomethyltransferase enzyme activity

[0040] The plasmid sequenced correctly in Example 1 was transformed into Escherichia coli E. coli BL21(DE3). Select transformants and inoculate them into LB liquid medium, culture at 37℃ for 12 h, and then transfer them into LB medium with an inoculation volume of 3%. Grow the cells to OD 600 When the pH value was 1.5, IPTG was added for induction, and the culture temperature was lowered to 30°C for 24 h. The cells were collected, phosphate buffer was added, ultrasonically disrupted, centrifuged at 4°C, and the supernatant was taken. The protein was purified using a GST recombinant protein purification kit, the concentration of the purified protein was determined, substrate catalysis was performed, and the product was detected by HPLC-UV. The relative activity of the enzyme was expressed by the product peak area. The results are shown in Figure 1 shown.

[0041] Example 3 Obtaining St7OMT mutant

[0042] Five pairs of primers were designed (as shown in Table 1), and PCR amplification was performed using the constructed pGEX-4T-1-St7OMT as a template. Alanine at position 118 of St7OMT was mutated to valine, glutamine at position 121 was mutated to leucine or valine, and arginine at position 151 was mutated to leucine or isoleucine, obtaining mutants A118V, Q121L, Q121V, R151I, and R151L. PCR conditions were: 98°C for 5 min, 35 cycles (98°C for 5 min, 60°C for 30 s, 72°C for 6 min), and 72°C for 10 min. PCR amplification system: 1 μL template, 2 μL upstream and downstream primers each, 25 μL primeSTAR, and 20 μL sterilized double distilled water. The PCR products were purified and recovered using a gel recovery kit, and the concentration was determined. After transformation and sequencing, the transformants with successful mutations were named: pGEX-4T-1-St7OMT-A118V, pGEX-4T-1-St7OMT-Q121L, pGEX-4T-1-St7OMT-Q121V, pGEX-4T-1-St7OMT-R151I and pGEX-4T-1-St7OMT-R151L. Using the plasmids of the correctly sequenced strains pGEX-4T-1-St7OMT-R151L and pGEX-4T-1-St7OMT-R151I as templates, compound mutations were performed on alanine at position 118 and glutamine at position 121 to obtain mutant transformants, and the plasmids were named: pGEX-4T-1-St7OMT-A118V / R151L, pGEX-4T-1-St7OMT-A118V / R151I, pGEX-4T-1-St7OMT-Q121L / R151L, pGEX-4T-1-St7OMT-Q121L / R151I, respectively. GEX-4T-1-St7OMT-Q121V / R151I, pGEX-4T-1-St7OMT-Q121V / R151L, pGEX-4T-1-St7OMT-Q121V / A118V, pGEX-4T-1-St7OMT-Q121L / A 118V, pGEX-4T-1-St7OMT-A118V / Q121L / R151I, pGEX-4T-1-St7OMT-A118V / Q121L / R151L and pGEX-4T-1-St7OMT-A118V / Q121V / R151L.

[0043] The amino acid sequences of mutants A118V, Q121L, Q121V, R151L, R151I, A118V / R151L, A118V / R151I, Q121L / R151L, Q121L / R151I, Q121V / R151I, St7OMT-Q121V / R151L, St7OMT-Q121V / A118V, Q121L / A118V, A118V / Q121L / R151I, A118V / Q121L / R151L and A118V / Q121V / R151L are SEQ ID NOs. 2-17, respectively.

[0044] Table 1 Primer sequences

[0045]

[0046] Example 4 Enzyme activity verification of highly active 7-oxomethyltransferase mutant strains

[0047] The plasmid with the correct sequence was transformed into E. coli BL21(DE3), select transformants and inoculate them into LB liquid medium, culture at 37℃ for 12h, then transfer to TB medium with an inoculation volume of 3%. Grow the cells to OD 600 When the concentration of 1.5 was reached, IPTG was added for induction, and the culture temperature was lowered to 30°C for 24 h. The cells were collected and broken to collect the recombinant protein. The recombinant protein was purified by GST purification column and its catalytic activity was tested. The results are as follows: Figure 2 As shown. The experimental results show that compared with the wild-type enzyme, the mutants have improved activities in catalyzing the synthesis of norpapavine from erucine, that is, the mutants have stronger enzyme activity. Compared with WT (wild type), the enzyme activities of R151I, A118V, Q121L, Q121V, R151L, A118V / R151L, Q121V / R151L and A118V / Q121L / R151L increased by about 1.6, 2.0, 2.0, 4.0, 1.7, 4.0, 6.0 and 6.0 times, respectively.

[0048] Example 5 Substrate promiscuity analysis of mutant A118V / Q121L / R151L

[0049] The substrate promiscuity analysis was performed with substrates such as lagenaria, N-methyl lagenaria, and taurine. The results showed that compared with the wild-type enzyme, the mutant A118V / Q121L / R151L had better catalytic activity for a variety of substrates such as nor-methyl lagenaria, lagenaria, N-methyl lagenaria, and taurine. Figure 3 As shown, the mutant has catalytic effect on various benzylisoquinoline alkaloids containing C7 hydroxyl group.

[0050] Example 6 Construction of the papaverine cell chassis and product fermentation

[0051] Refer to Figure 4 Flow chart of artificially constructing the corresponding metabolite chassis cells. The detailed operation is as follows:

[0052] The published Ps6OMT and PsCNMT were constructed into the pRSFDuet vector and transformed into E. coli In BL21(DE3), after shaking, induction and feeding with substrate norcoclaurine and cofactor SAM, the production of claurine and nitrogen-methylclaurine was finally detected in the fermentation broth. The corresponding vector strain was then prepared into the corresponding competent cells by the CaCl2 method, and the pGEX-4T-1-St7OMT-A118V / Q121L / R151L plasmid was transformed into the competent cells to obtain a recombinant E. coli chassis with two free plasmids, named E. coli BL21(DE3)-6OMT-CNMT-7OMT was shaken, induced and fed with substrate higenamine and cofactor SAM, and finally the synthesis of papaverine was detected in the fermentation broth. Figure 5 As shown, the chassis cells achieved the synthesis of papaverine in the Escherichia coli chassis cells.

[0053] Figure 5 In the figure, Sample is the catalytic result of the engineered strain, Control is the catalytic result of the inactivated engineered strain, and STD-Norarmepavine is the chromatogram of the Norarmepavine standard. Figure 5 In the Sample catalysis results, product A and product B are included.

[0054] Figure 6 is the mass spectrum of product A, product A is papaverine, Figure 7 It is the mass spectrum of product B, and product B is nor-opapaverine.

[0055] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0056] Sequence Listing Used in the Present Invention

[0057] SEQ ID NO.1 (St7OMT wild type):

[0058] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLQYYEMDAWHHLSAAVEGAATPWEKCHGVDYREYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0059] SEQ ID NO.2 (St7OMT mutant A118V):

[0060] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFVLLQYYEMDAWHHLSAAVEGAATPWEKCHGVDYREYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0061] SEQ ID NO.3 (St7OMT mutant Q121L):

[0062] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLLYYEMDAWHHLSAAVEGAATPWEKCHGVDYREYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0063] SEQ ID NO.4 (St7OMT mutant Q121V):

[0064] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLVYYEMDAWHHLSAAVEGAATPWEKCHGVDYREYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0065] SEQ ID NO.5 (St7OMT mutant R151L):

[0066] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLQYYEMDAWHHLSAAVEGAATPWEKCHGVDYLEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0067] SEQ ID NO.6 (St7OMT mutant R151I):

[0068] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLQYYEMDAWHHLSAAVEGAATPWEKCHGVDYIEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0069] SEQ ID NO.7 (St7OMT mutant A118V / R151L):

[0070] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFVLLQYYEMDAWHHLSAAVEGAATPWEKCHGVDYLEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0071] SEQ ID NO.8 (St7OMT mutant A118V / R151I):

[0072] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFVLLQYYEMDAWHHLSAAVEGAATPWEKCHGVDYIEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0073] SEQ ID NO.9 (St7OMT mutant Q121V / R151L):

[0074] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLVYYEMDAWHHLSAAVEGAATPWEKCHGVDYLEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0075] SEQ ID NO.10 (St7OMT mutant Q121V / R151I):

[0076] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLVYYEMDAWHHLSAAVEGAATPWEKCHGVDYIEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0077] SEQ ID NO.11 (St7OMT mutant Q121L / R151L):

[0078] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLLYYEMDAWHHLSAAVEGAATPWEKCHGVDYLEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0079] SEQ ID NO.12 (St7OMT mutant Q121L / R151I):

[0080] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFALLLYYEMDAWHHLSAAVEGAATPWEKCHGVDYIEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0081] SEQ ID NO.13 (St7OMT mutant Q121L / A118V):

[0082] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFVLLLYYEMDAWHHLSAAVEGAATPWEKCHGVDYREYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0083] SEQ ID NO.14 (St7OMT mutant Q121V / A118V):

[0084] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFVLLVYYEMDAWHHLSAAVEGAATPWEKCHGVDYREYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0085] SEQ ID NO.15 (St7OMT mutant A118V / Q121L / R151L):

[0086] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFVLLLYYEMDAWHHLSAAVEGAATPWEKCHGVDYLEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0087] SEQ ID NO.16 (St7OMT mutant A118V / Q121L / R151I):

[0088] MTGGQQMGRGSMEEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFVLLLYYEMDAWHHLSAAVEGAATPWEKCHGVDYIEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDIVLDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

[0089] SEQ ID NO.17 (St7OMT mutant A118V / Q121V / R151L):

[0090] MTGGQQMGRGSMEEMKAQAQVWRYVYGFAESLTLKCAIQLNIPDILHHHNRPMTLSELATHLPLPSVNQDRLYRVMRYLVHMGLFDLLDSPDSPRYSLNSASKLLVKGQDKSLASFVLLVYYEMDAWHHLSAAVEGAATPWEKCHGVDYLEYFAKDAVANQLLSDAMTSHTSMVTEALVEGCKKGGVLHGVRSFVDVGGSTGVAARAVAKAFPGIKCSVFDLPHVIATAPECPEVTRIQGDMFVSIPETDVVFMKSVLHDWGDEDCVKILKKCKEAIAGKEGGKVVILDAESSYEFKGARLGMEMDMLVTVGGKERSEAEWQKLFKDAGYSRYKITPIVAIESVIEVFP

Claims

1. An oxygen methyltransferase mutant with improved enzyme activity, characterized in that: The method comprises mutating the glutamine at position 121 of the oxygen methyltransferase as shown in the amino acid sequence of SEQ ID NO. 1 to leucine, mutating the alanine at position 118 to valine, and mutating the arginine at position 151 to leucine.

2. The oxygen methyltransferase mutant with improved enzyme activity according to claim 1, characterized in that The amino acid sequences of the oxygen methyltransferase mutants are shown in SEQ ID NO.

15.

3. An application of the oxygen methyltransferase mutant according to claim 1 in the synthesis of papaverine, wherein the oxygen methyltransferase mutant is expressed in Escherichia coli, and a synthetic chassis for papaverine is constructed using Ps6OMT and PsCNMT, and the synthesis of papaverine is finally detected in the fermentation broth by adding the substrate higenamine and the cofactor SAM.

4. The method according to claim 3, wherein the oxygen methyltransferase mutant catalyzes the methylation of the C7 hydroxyl group in the synthesis of papaverine.

5. A method for increasing the activity of oxygen methyltransferase, characterized in that: The method comprises the following steps: mutating the 121st glutamine in the oxygen methyltransferase protein molecule of the amino acid sequence as shown in SEQ ID NO. 1 to leucine; mutating the 118th alanine to valine; and mutating the 151st arginine to leucine.

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