Caulis sinomenii methyltransferase and application thereof in synthesis of sinomenine

Abstract

The invention discloses a caulis sinomenii methyltransferase and an application of the caulis sinomenii methyltransferase in synthesis of sinomenine. The amino acid sequence of the caulis sinomenii methyltransferase is as shown in SEQ ID NO: 2. According to the invention, a new methyltransferase SaOMT3 is identified from a medicinal plant caulis sinomenii for the first time, and it is found that the methyltransferase participates in biosynthesis of sinomenine. Functional identification on SaOMT3 can greatly promote the research on the biosynthesis path of sinomenine, provide sufficient raw material guarantee for the creation of new sinomenine drugs, and also provide reference for the research of other medicinal plants. Meanwhile, the caulis sinomenii methyltransferase SaOMT3 disclosed by the invention is good in substrate specificity, high in catalytic efficiency and very high in application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a Sinomenium acutum methyltransferase and application thereof in synthesis of sinomenine. BACKGROUND

[0002] Sinomenium acutum (Thunb.) Rehd. et Wils.) is a dry stem of Sinomenium acutum (Thunb.) Rehd. et Wils.) in the family of Ranunculaceae, and is a traditional Chinese medicine for treating rheumatism and arthralgia. Modern pharmacological studies have confirmed that the main active ingredient of Sinomenium acutum is alkaloid, and sinomenine is the core component for playing the roles of anti-inflammatory, immunosuppression and treating rheumatoid arthritis. Sinomenine has a unique chemical structure, and its pharmacological effect is similar to that of morphine analogs but without addiction. In clinical application, sinomenine shows good efficacy and safety, and has become a raw material of modern Chinese medicine preparations (such as sinomenine hydrochloride tablets) for treating rheumatoid arthritis.

[0003] Studies have shown that sinomenine belongs to benzylisoquinoline alkaloids (BIAs). In the biosynthetic pathway thereof, (S)-coclaurine is a core active intermediate. Coclaurine and its derivatives have been confirmed to have various biological activities, and are key nodes connecting primary metabolism and numerous BIAs (such as sinomenine, morphine and berberine) with significant pharmacological activities. The biosynthesis of sinomenine needs to go through complex enzymatic modifications such as multiple hydroxylation, methylation and oxidative cyclization. Among them, the methylation reaction, especially the reaction catalyzed by O-methyltransferase (OMT), is a key step for constructing the characteristic methoxy structure of sinomenine. Therefore, identifying and developing enzymes with methylation function have important value for the biosynthesis of sinomenine anti-inflammatory compounds. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides a Sinomenium acutum methyltransferase SaOMT3.

[0005] The present application also provides a nucleotide sequence encoding the Sinomenium acutum methyltransferase SaOMT3. The present application also provides biological materials related to the nucleotide sequence encoding the Sinomenium acutum methyltransferase SaOMT3.

[0006] The present application also provides applications of the above-mentioned Sinomenium acutum methyltransferase SaOMT3, nucleotide sequence and biological materials.

[0007] The present application also provides a preparation method of coclaurine.

[0008] The present invention also proposes the application of the above-mentioned method for preparing linderine in the synthesis of sinomenine.

[0009] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a Sinomenium acutum methyltransferase SaOMT3, wherein the Sinomenium acutum methyltransferase SaOMT3 is one of the following A1) to A4): A1) A protein with the amino acid sequence shown in SEQ ID NO:2; A2) A protein having the same function as the protein described in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:2. A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence of A1) or A2); A4) An amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:2 and has the same function as the protein shown in SEQ ID NO:2.

[0010] In some embodiments of this application, the amino acid sequence has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the sequence shown in SEQ ID NO:2.

[0011] In a second aspect, the present invention provides a nucleotide sequence encoding the Sinomenium acutum methyltransferase SaOMT3, said nucleotide sequence comprising any one of the following nucleotide sequences (B1) to (B3): B1) The nucleotide sequence shown in SEQ ID NO.1; B2) A nucleotide sequence with the same function formed by substituting, deleting or adding at least one nucleotide from the nucleotide sequence shown in B1); The nucleotide sequences shown in B3) and B1) have more than 90% sequence identity and the same function.

[0012] In some embodiments of this application, the nucleotide sequence of B3) has 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the nucleotide sequence shown in B1).

[0013] According to a third aspect of the invention, a biomaterial is provided, the biomaterial comprising any of the following: C1) An expression cassette containing the aforementioned nucleotide sequence; C2) A recombinant vector containing the aforementioned nucleotide sequence or expression cassette; C3) Recombinant cells containing the aforementioned nucleotide sequence, expression cassette, or recombinant vector.

[0014] An expression cassette is a construct containing regulatory elements necessary for the expression of a target nucleotide sequence in recombinant cells. Specific regulatory elements include promoters, polyadenylation sites, etc.

[0015] A recombinant vector is a sequence containing all elements necessary for the replication, integration, amplification, and / or expression of a target nucleotide sequence in recombinant cells. These elements include, but are not limited to, promoters, polyadenylation sites, terminators, replication initiation sites, transcription initiation sequences, enhancers, selection elements, and reporter genes. Optional types of recombinant vectors include plasmids, bacteriophages, lentiviruses, adenoviruses, and adeno-associated viruses.

[0016] Recombinant cells are cells used to replicate, integrate, amplify, and / or express target nucleotide sequences. Recombinant cells include, but are not limited to, bacterial, fungal, insect, plant, and animal cells. Specifically, they can be competent cells used to mediate nucleotide sequence transformation, or target plant cells transformed with the aforementioned nucleotide sequences. Specifically, competent cells include *Escherichia coli* BL21(DE3) competent cells.

[0017] According to a fourth aspect of the present invention, the use of the above-mentioned Sinomenium acutum methyltransferase SaOMT3, nucleotide sequence or biological material in any of the following is proposed: (1) Synthesis of linderine; (2) Synthesize sinomenine.

[0018] In some embodiments of the present invention, the lindera alkaloid is synthesized by an enzymatic reaction, wherein the methyl donor of the enzymatic reaction includes S-adenosylmethionine (SAM).

[0019] In some embodiments of the present invention, the substrate for the enzymatic reaction includes norcodonine.

[0020] According to a fifth aspect of the present invention, a method for preparing linderine is provided, the method comprising the following steps: preparing linderine by an enzymatic reaction using S-adenosylmethionine as a methyl donor, wherein the catalytic enzyme of the enzymatic reaction is Sinomenium acutum methyltransferase SaOMT3, and the sequence of Sinomenium acutum methyltransferase SaOMT3 is as described in the first aspect of the present invention.

[0021] In some embodiments of the present invention, the substrate for the enzymatic reaction includes norcodonine.

[0022] In some embodiments of the present invention, the concentration of S-adenosylmethionine is 8-12 mmol / L. Specifically, it can be 8, 9, 10, 11, or 12 mmol / L.

[0023] In some embodiments of the present invention, the temperature of the enzymatic reaction is 32-40°C and the pH is 7-8.5.

[0024] In some embodiments of the present invention, the optimal temperature for the enzymatic reaction is 40°C and the optimal pH is 8.5.

[0025] According to a sixth aspect of the present invention, the application of the above-described method for preparing linderine in the synthesis of sinomenine is proposed.

[0026] The beneficial effects of this invention are as follows: This invention is the first to identify a novel methyltransferase SaOMT3 from the medicinal plant *Sinomenium acutum*, and discovers that this methyltransferase participates in the biosynthesis of sinomenine. Functional identification of SaOMT3 will greatly advance the research on the biosynthetic pathway of sinomenine, provide sufficient raw material guarantees for the creation of new sinomenine drugs, and also provide a reference for the research of other medicinal plants. Furthermore, the *Sinomenium acutum* methyltransferase SaOMT3 of this invention exhibits good substrate specificity and high catalytic efficiency, and has high application value. Attached Figure Description

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is an image showing the agarose gel electrophoresis detection results in an embodiment of the present invention, where M is a 2000bp DNA marker, and lane 1 contains... SaOMT3 Results of bacterial culture PCR validation of the gene; Figure 2 This is an SDS-PAGE gel electrophoresis image of the pET-28a-SaOMT3 fusion protein in the embodiments of the present invention, where M is the protein marker (15-180kDa), and 1, 2, 3, and 4 are SaOMT3 purified enzymes (1, 2, 3, and 4 are the target protein, miscellaneous proteins, supernatant protein, and total protein, respectively). Figure 3 The diagram shows the reaction results of SaOMT3 with the substrate and the catalytic activity in the embodiments of the present invention. A is a liquid phase diagram of the reaction between SaOMT3 and norcodonine; B is an MS diagram of the catalytic product; and C is a structural diagram of the substrate and the product. Figure 4 The figures show the detection results of the enzymatic kinetic parameters for the conversion of norcodonopsis alkaloid to causaline catalyzed by SaOMT3 in the embodiments of the present invention. Among them, A is the detection result of the effect of temperature on catalytic activity, B is the detection result of the effect of pH on catalytic activity, and C is the detection result of the effect of substrate concentration on catalytic activity. Detailed Implementation

[0028] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0029] The reagents, methods, and equipment used in the following examples are all conventional reagents, methods, and equipment in this technical field. Test methods in the following examples that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions.

[0030] Example 1: Sinomenium acutum methyltransferase SaOMT3 Genes and the proteins they encode This embodiment provides a *Sinomenium acutum* methyltransferase. SaOMT3 Genes and the proteins they encode.

[0031] Sinomenium methyltransferase SaOMT3 The nucleotide sequence of the gene is as follows:

[0032] The amino acid sequence of Sinomenium acutum methyltransferase SaOMT3 is as follows: MGSTQNQLAAMTLSAEDEEEAACMYAMQLASASVLPMVLKAAIELDVLEIIAKAGPGAYVAPSQIVAQLPNCINPQAAVMLDRTLRLLASFRILTCKLDEGGERVERTYGLGPVCKFLVKNEDGVSMAPLVLMNQDKVLMESWYHLKDAILDGGIPFNKAYGMTAFEYHGTDPRFNKVFNRGMSD HSTITMKKLLETYKGFDGLKSLVDVGGGIGATLNMIISKHPTIKGINFDLPHVIEDAPSYPGVEHVGGDMFVSVPKGDGIFMKWILHDWSDGHCAKFLKNCYEALPDDGKVIIVESILPEFPETNLAANGSFQLDNIMLAHNPGGKERTAKDFEALAKGAGFVGFNVVCGAYNSWVMEFCKKL( SEQ ID NO:2).

[0033] Example 2: Sinomenium acutum methyltransferase SaOMT3 Application of genes and their encoded proteins in the synthesis of sinomenine This embodiment provides *Sinomenium acutum* methyltransferase. SaOMT3 The application of the gene and its encoded protein in the synthesis of sinomenine is specifically verified as follows: 1. SaOMT3 Gene amplification Total RNA was extracted from *Sinomenium acutum* using the OminiPlant RNA Kit. High-quality cDNA was obtained by following the instructions of the HiScript III 1st Strand cDNA Synthesis Kit, and this cDNA was used as a template for polymerase chain reaction (PCR) amplification. SaOMT3 The full-length gene fragment was used, and the amplification primers were designed based on the transcriptome. The sequences are shown in Table 1.

[0034] Table 1 Primer sequences

[0035] The target fragment was amplified using primers SaOMT3-F and SaOMT3-R, and amplified using Taq DNA Polymerase PCR polymerase. A 50µL PCR reaction system was prepared according to Table 2.

[0036] Table 2. PCR reaction system for amplifying the target gene (50µL)

[0037] Place the above PCR reaction system in a PCR instrument and amplify the target gene according to the PCR reaction procedure in Table 3.

[0038] Table 3 PCR reaction procedure

[0039] After the reaction was complete, 2.5 µL of the product was taken and the size of the PCR product was detected by 1% agarose gel electrophoresis.

[0040] 2. Recovery, transformation, and identification of positive clones of the target fragment. The target fragment was recovered using the FastPure Gel DNA Extraction Mini Kit. The target fragment was then homologously recombinated with the PET28a vector (provided by the laboratory), transformed with E. coli DH5α, and single colonies were selected for culture on LB medium. The bacterial culture was then verified by PCR. The reaction system was prepared according to Table 4.

[0041] Table 4. PCR reaction system of 10µL bacterial culture

[0042] After the reaction, 2.5 µL of the PCR product was analyzed by 1% agarose gel electrophoresis. The electrophoresis result is shown in the figure below. Figure 1 As shown in the image. Sequencing was performed on bacterial culture samples with correct PCR results.

[0043] The sequencing results were correct. SaOMT3 The nucleotide sequence of the gene is shown in SEQ ID NO:1. The gene has 1104 nucleotides and encodes 367 amino acids, the amino acid sequence of which is shown in SEQ ID NO:2.

[0044] 3. Expression and detection of SaOMT3 protein The target bacterial culture with correct sequencing was expanded, and the recombinant plasmid pET-28a-SaOMT3 was extracted and transformed into E. coli BL21(DE3) competent cells. Single colonies were picked and cultured in LB medium at 37°C and 220 rpm for 12 h to obtain the stock solution. The stock solution was then inoculated at a dilution of 1:100 into LB medium with a KAN concentration of 50 µg / mL and cultured at 37°C and 220 rpm until OD500 was reached. 600 =0.4~0.6, add 0.5mM / mL IPTG at a ratio of 1:1000, and induce at 16℃ and 220rpm for 12-16h.

[0045] Collect the induced bacterial culture and centrifuge at 4000 rpm for 10 min at 4°C. Resuspend in pre-chilled (4°C) pH 9.0 imidazole (10 mmol / L) buffer, centrifuge again to collect the cells, and repeat once more. Add 15 mL of the above imidazole buffer to each gram of wet bacteria. Then, perform low-temperature disruption on an ultrasonic cell disruptor at 350 W for 5 seconds followed by a 40-second pause. Stop disruption when the bacterial culture becomes clear. Finally, centrifuge the disrupted culture at 4°C, 4000 rpm for 5 min, collect the supernatant as crude enzyme solution, and store it at -20°C for later use.

[0046] Based on the Tris tag on the fusion protein, the target protein was purified using a nickel affinity chromatography column. The target protein was eluted with imidazole solutions at concentrations of 10 mmol / L, 20 mmol / L, and 300 mmol / L. The purity and molecular weight of the target protein were determined by 10% SDS-PAGE. (See attached image). Figure 2 The target protein was found at 42.3 kDa, and the target protein was purified for subsequent experiments.

[0047] 4. In vitro enzyme activity detection The steps for in vitro enzyme activity detection are as follows: (1) Enzyme activation reaction system: 10 µg purified enzyme protein, 100 μM norcodonine, 10 mM SAM methyl donor, and 50 mM Hepes buffer (pH=8.0) were added to a final volume of 100 µL. The reaction solution was reacted at 37 °C for 1 h, then 100 μL of methanol was added, the mixture was vortexed and vortexed for 2 min at 4 °C and 12000 rpm. The supernatant obtained by filtration was used for detection by high performance liquid chromatography.

[0048] (2) Detection conditions of high performance liquid chromatography (HPLC) The HPLC conditions are shown in Table 5 below.

[0049] Table 5 HPLC gradient elution conditions

[0050] Liquid chromatography-mass spectrometry (LC-MS) conditions: detection mode: positive ion mode; capillary voltage: 3500V; capillary temperature: 320℃; sheath gas pressure: 30arb; auxiliary gas pressure: 10arb; resolution: 35000; collision energy: 50V; flow rate: 0.3mL / min.

[0051] Test results as follows Figure 3 As shown in the figure, when SaOMT3 uses norcodine as a substrate, one product peak can be observed. By comparing the retention time and mass-to-charge ratio (m / z) with the standard, it was found that peak a is the substrate norcodine and peak b is the product codine.

[0052] 5. Detection of enzyme kinetic parameters Using norcorydaline as a substrate and SAM as a methyl donor, the effects of temperature, pH, and different substrate concentrations on catalytic activity were determined.

[0053] (1) Temperature: Using norcodonine as the substrate and SAM as the methyl donor, the changes in enzyme activity at different reaction temperatures (30℃, 32℃, 35℃, 37℃, 40℃, 42℃, and 45℃) were determined. The reaction system was the same as the in vitro enzyme activity detection reaction system described above, containing 10 μg of protein. The reaction was carried out at each temperature for 30 min, and 100 μL of methanol was added to terminate the reaction. The mixture was centrifuged at 4℃ and 12000 rpm for 2 min, and the supernatant was filtered and analyzed by HPLC. Each reaction temperature was repeated three times. The conversion rate of the substrate was estimated based on the peak area ratio in the chromatogram.

[0054] (2) pH: The effect of 50 mM Hepes buffer at different pH values ​​(5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10) on enzyme activity was tested. The reaction system was the same as the in vitro enzyme activity detection reaction system described above. The reaction system was reacted at 37℃ for 30 min, and 100 μL of methanol was added to terminate the reaction. The system was centrifuged at 4℃ and 12000 rpm for 2 min. The supernatant was collected, filtered, and then analyzed by HPLC. Each reaction temperature was set to be repeated 3 times. The conversion rate of the substrate was estimated based on the peak area ratio in the chromatogram.

[0055] (3) Different substrate concentrations: The reaction system was the same as the in vitro enzyme activity detection reaction system described above. Different concentrations of norcodine were added as substrates at 37℃: 20, 10, 5, 2, 1, 0.5, 0.2, and 0.1 μM. The reaction was carried out at 37℃ for 30 min, then 100 μL of methanol was added to terminate the reaction. The mixture was centrifuged at 4℃ and 12000 rpm for 2 min. The supernatant was filtered and analyzed by HPLC. Each reaction temperature was repeated three times. The conversion rate of the substrate was estimated based on the peak area ratio in the chromatogram. The Michaelis constant was calculated by fitting the Michaelis equation.

[0056] Test results as follows Figure 4 As shown in the figure, enzyme kinetic studies indicate that SaOMT3 exhibits the highest enzyme activity at 40℃, with good conversion rates within the 32–40℃ range. The optimal pH of the reaction solution is 8.5, suitable for reactions within the pH range of 7–8.5. Using SAM as the methyl donor at a constant concentration of 10 mM, enzyme kinetics were conducted with different substrate concentrations of norcodonine. The product, codonine, was measured, and calculations were performed using the Michaelis-Menten equation. The results showed that SaOMT3 achieved a Km of 3.24 nM at pH 8.5 and a reaction temperature of 40℃.

[0057] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A type of methyltransferase SaOMT3 from *Sinomenium acutum*, characterized in that, The *Sinomenium acutum* methyltransferase SaOMT3 is any one of the following A1) to A4): A1) A protein with the amino acid sequence shown in SEQ ID NO:2; A2) A protein having the same function as the protein described in A1) obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:

2. A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the amino acid sequence of A1) or A2); A4) An amino acid sequence that has at least 90% identity with the amino acid sequence shown in SEQ ID NO:2 and has the same function as the protein shown in SEQ ID NO:

2.

2. A nucleotide sequence encoding the Sinomenium acutum methyltransferase SaOMT3, wherein the nucleotide sequence comprises any one of the following nucleotide sequences (B1) to (B3): B1) The nucleotide sequence shown in SEQ ID NO.1; B2) A nucleotide sequence with the same function formed by substituting, deleting or adding at least one nucleotide from the nucleotide sequence shown in B1); The nucleotide sequences shown in B3) and B1) have more than 90% sequence identity and the same function.

3. A biomaterial, characterized in that, The biomaterial comprises any one of the following: C1) An expression cassette comprising the nucleotide sequence as described in claim 2; C2) A recombinant vector comprising the nucleotide sequence as described in claim 2 or the expression cassette as described in C1); C3) A recombinant cell comprising the nucleotide sequence of claim 2, the expression cassette of C1), or the recombinant vector of C2).

4. The use of the *Sinomenium acutum* methyltransferase SaOMT3 according to claim 1, the nucleotide sequence according to claim 2, or the biological material according to claim 3 in any of the following: (1) Synthesis of linderine; (2) Synthesize sinomenine.

5. The application according to claim 4, characterized in that, The lindera alkaloid is synthesized by an enzymatic reaction, wherein the methyl donor of the enzymatic reaction includes S-adenosylmethionine.

6. The application according to claim 4, characterized in that, The substrate for the enzymatic reaction includes norcodonine.

7. A method for preparing linderine, characterized in that, The method includes the following steps: preparing the product by using S-adenosylmethionine as a methyl donor via an enzymatic reaction; The catalytic enzyme for the enzymatic reaction is Sinomenium acutum methyltransferase SaOMT3, and the sequence of Sinomenium acutum methyltransferase SaOMT3 is as described in claim 1.

8. The method according to claim 7, characterized in that, The substrates for the enzymatic reaction include norcodonine; And / or, the concentration of the S-adenosylmethionine is 8-12 mmol / L.

9. The method according to claim 7, characterized in that, The enzymatic reaction is carried out at a temperature of 32-40℃ and a pH of 7-8.

5.

10. The use of the method according to any one of claims 7-9 in the synthesis of sinomenine.

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