Application of methyltransferase in converting emodin into physcion

Abstract

The invention belongs to the field of biological information analysis and enzyme engineering, and particularly relates to application of methyltransferase in converting emodin into emodin monomethyl ether. The invention discloses an application of methyltransferase in converting emodin into physcion. The amino acid sequence of the methyltransferase comprises an amino acid sequence as shown in SEQ ID NO: 1-5 (particularly the methyltransferase as shown in SEQ ID NO: 1). The invention also provides a recombinant microorganism expressing the methyltransferase.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioinformatics analysis and enzyme engineering, and particularly relates to application of a methyltransferase in converting emodin into emodin methyl ether. BACKGROUND

[0002] Rhubarb is a traditional herbal medicine that has been widely used in East Asia for about two thousand years. Anthraquinones, including emodin and its derivatives, are the key bioactive components of rhubarb. Emodin methyl ether, a derivative of emodin with methylation at the 3-hydroxyl group, is a characteristic component of traditional Chinese medicine rhubarb, and has various pharmacological activities, such as anti-inflammatory and antioxidant effects, anticancer effects, etc.

[0003] Currently in China, emodin methyl ether has been developed as a commercially available plant-derived fungicide for treating powdery mildew, downy mildew and gray mold on plants, etc.

[0004] Emodin methyl ether is extremely low in plants, and the expensive purification process and geographically restricted planting seriously hinder its application. Compared with the plant extraction method, the biological manufacturing method such as microbial fermentation or whole cell transformation has the characteristics of high efficiency, green and sustainability. There are a large number of literatures on the biosynthesis of emodin, and its biosynthetic pathway has been completely resolved. The key to realizing the efficient biosynthesis of emodin methyl ether is to find a methyltransferase with high catalytic ability of emodin to emodin methyl ether. SUMMARY

[0005] The present application adopts the following technical solutions: In a first aspect, the present application provides application of a methyltransferase in converting emodin into emodin methyl ether, wherein the amino acid sequence of the methyltransferase comprises the amino acid sequence as shown in SEQ ID NO: 1-5.

[0006] Preferably, the amino acid sequence of the methyltransferase is as shown in SEQ ID NO: 1.

[0007] Preferably, the nucleotide sequence encoding the methyltransferase comprises the nucleotide sequence as shown in SEQ ID NO: 6-10.

[0008] Further preferably, the nucleotide sequence encoding the methyltransferase is as shown in SEQ ID NO: 6.

[0009] In a second aspect, the present application provides a recombinant microorganism, wherein the recombinant microorganism expresses a methyltransferase, and the amino acid sequence of the methyltransferase comprises the amino acid sequence as shown in SEQ ID NO: 1-5.

[0010] Preferably, the amino acid sequence of the methyltransferase is as shown in SEQ ID NO: 1.

[0011] Preferably, the nucleotide sequence encoding the methyltransferase comprises the nucleotide sequence shown in SEQ ID NO: 6-10.

[0012] Further preferably, the nucleotide sequence encoding the methyltransferase is shown in SEQ ID NO: 6.

[0013] Preferably, the recombinant microorganism is Escherichia coli.

[0014] Preferably, the methyltransferase is expressed in a plasmid or on a chromosome.

[0015] A third aspect of the present invention provides a method for preparing recombinant microorganisms, the method comprising introducing a nucleotide sequence encoding a methyltransferase into the recombinant microorganisms, wherein the amino acid sequence of the methyltransferase comprises the amino acid sequences shown in SEQ ID NO: 1-5.

[0016] Preferably, the amino acid sequence of the methyltransferase is shown in SEQ ID NO: 1.

[0017] Preferably, the nucleotide sequence encoding the methyltransferase comprises the nucleotide sequence shown in SEQ ID NO: 6-10.

[0018] A further preferred embodiment is the nucleotide sequence encoding the methyltransferase, as shown in SEQ ID NO: 6.

[0019] Preferably, the recombinant microorganism is Escherichia coli.

[0020] In a fourth aspect, the present invention provides a method for producing emodin methyl ether, the method comprising using the recombinant microorganisms described above or the recombinant microorganisms obtained by the preparation method described above.

[0021] Preferably, the method includes mixing the recombinant microorganisms described above or the recombinant microorganisms obtained by the preparation method described above with emodin.

[0022] In a fifth aspect, the present invention provides the application of the above-described recombinant microorganism or the recombinant microorganism obtained by the above-described preparation method in the preparation of a bactericide.

[0023] Preferably, the fungicide is a plant-derived fungicide.

[0024] In a sixth aspect, the present invention provides a bactericide comprising the recombinant microorganism described above or the recombinant microorganism obtained by the preparation method described above.

[0025] In a seventh aspect, the present invention provides the use of the above-described recombinant microorganism or the recombinant microorganism obtained by the above-described preparation method in the conversion of emodin into emodin methyl ether.

[0026] In an eighth aspect, the present invention provides the application of the above-described recombinant microorganism or the recombinant microorganism obtained by the above-described preparation method in the preparation of emodin methyl ether. The term "and / or" as used in this invention includes all combinations of items connected by the term, and should be regarded as each combination having been individually listed in this application. For example, "A and / or B" includes "A", "B", and "A and B"; and "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0027] The terms "comprising" or "including" in this invention are open-ended descriptions, containing the specified ingredients or steps described, as well as other specified ingredients or steps that do not materially affect them. Attached Figure Description

[0028] To more clearly illustrate the specific implementation schemes of this invention, the accompanying drawings used in the scheme description are now explained in detail.

[0029] Figure 1 The structural formulas are those of emodin and its methylated product physcion (emodin methyl ether).

[0030] Figure 2 This is a schematic diagram of homologous clusters retrieved from the NCBI database using simulated emodin methyl ether BGCs.

[0031] Figure 3 This is a schematic diagram of the phylogenetic tree analysis of PtaI and its homologous proteins.

[0032] Figure 4 This is a protein similarity network diagram labeled with homologous gene clusters, phylogenetic trees, and protein subfamily analysis results (different colors in the outer circle represent different protein subfamilies; the presence or absence of color in the inner circle indicates whether it belongs to a homologous cluster of GedC+PtaI or RugA+PtaI. Specifically, a colored inner circle indicates that the protein belongs to a homologous cluster of GedC+PtaI or RugA+PtaI, while an uncolored inner circle indicates that it does not belong to the aforementioned homologous clusters).

[0033] Figure 5 This is a schematic diagram showing the comparison between the BGCs containing the selected candidate methyltransferases.

[0034] Figure 6 This is a multiple sequence alignment diagram of the five candidate methyltransferases selected in the final screening within their catalytic domains.

[0035] Figure 7 The images show PCR electrophoresis diagrams of the transformant colonies after each plasmid was transformed into E. coli BL21.

[0036] Figure 8 HPLC chromatogram (250 nm) of each transformant catalyzing emodin to emodin methyl ether.

[0037] Figure 9 HPLC detection (250 nm) of emodin to emodin methyl ether catalyzed by transformants carrying Tp3OMT and PtaI.

[0038] Figure 10 A bar chart showing the catalytic conversion of emodin to emodin methyl ether by transformants carrying Tp3OMT and PtaI. Detailed Implementation

[0039] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out according to the conditions provided by the manufacturer.

[0040] All gene expression in this invention was performed on E. coli BL21, and gene expression was carried out in the plasmid vector pCold II. The methyltransferases involved in this invention were all obtained from the NCBI database.

[0041] Example 1. Utilizing bioinformatics analysis to identify emodin methyltransferases 1) Artificial assembly of emodin methyl ether BGCs Using SnapGene software, combined with Figure 1 The emodin methyl ether generation pathway shown involves splicing the core emodin synthesis genes rugA and gedC downloaded from NCBI with the methyltransferase encoding gene ptaI. Here, the non-coding region about 500bp upstream of ptaI is used as the linker sequence. The entire base sequence after linking is a small artificially assembled BGC.

[0042] 2) Fishing for homologous BGCs Using the cblaster tool on the Cagecat website (https: / / cagecat.bioinformatics.nl / tools / search), the input file was the FASTA format file of the artificially assembled emodin methyl ether BGCs constructed above. The Min identity and Min query coverage parameters were set to 30% and 50% respectively, and the Min unique query hits and Min hits in clusters parameters were both set to 2. The remaining parameters could be left at their default settings. Homologous BGCs retrieved from the NCBI database are as follows: Figure 2 As shown.

[0043] 3) Construction of PtaI protein similarity network Using the ENZYMESIMILARITY tool on the EFI website (https: / / efi.igb.illinois.edu / efi-est / ), input the protein sequence of PtaI. Set the UniProt BLAST query e-value to 50, the Maximum number of sequence retrieved to 500, and UniProt as the Sequence database. Leave the other parameters at their default settings. After completing the initial calculations, continue using the website to generate a protein similarity network. Set the sequence length range to 150-500 bytes and the e-value to 75. Open the generated XGMML file with Cytoscape software for visualization.

[0044] 4) Construction of the phylogenetic tree of PtaI and its homologous proteins The protein sequences of each node in the previously generated sequence similarity network were downloaded and aggregated, and a phylogenetic tree was constructed using MEGA11 software. The neighbor-joining algorithm was chosen for phylogenetic tree construction, with a step value of 1500. The completed phylogenetic tree is shown below. Figure 3 As shown.

[0045] 5) Subfamily analysis of PtaI and its homologous proteins The protein subfamily classifications corresponding to PtaI and its homologous proteins were retrieved from the UniProt database using sequence similarity networks. This information, along with the information from the aforementioned homology cluster analysis and phylogenetic tree analysis, was then annotated into the protein similarity network to enrich and improve it.

[0046] 6) Improvement of sequence similarity networks The information from the homology cluster analysis, phylogenetic tree analysis, and protein subfamily analysis described above is annotated into the protein similarity network to enrich and improve the protein sequence similarity network. The improved protein sequence similarity network is as follows: Figure 4 As shown, the protein sequence similarity network enriched with this information is used to mine candidate methyltransferases.

[0047] 7) Screening and degeneracy of homologous clusters The protein sequence similarity network, enriched with the aforementioned information, guided the discovery of 13 candidate methyltransferase sequences. To further screen these sequences, the Clinker tool from the Cagecat website was used to align the BGCs containing these 13 methyltransferases to their respective clusters. The alignment results are as follows: Figure 5 As shown, the synthesis of emodin methyl ether requires not only non-reducing polyketide synthase and methyltransferase, but also β-lactamase. Therefore, BGCs that do not contain this enzyme were excluded. Furthermore, the gene distribution and structure within some methyltransferase-corresponding BGCs showed high consistency, which can be considered as the distribution of the same gene cluster in different strains. Only one methyltransferase from the same gene cluster was selected. Following the method described above, the number of 13 candidate methyltransferases was reduced to 5. Multiple sequence alignment was performed on these candidate methyltransferases, and the final alignment results are shown below. Figure 6 As shown.

[0048] Example 2. Verification of the catalytic activity of emodin methyltransferase 1) Synthesis of gene fragments The methyltransferase gene sequence involved in this invention is derived from the NCBI public database, and the codons were optimized and synthesized by Qingke Biotechnology Co., Ltd.

[0049] 2) Construction of recombinant plasmids To express various fungal methyltransferases in *E. coli* BL21, the methyltransferase genes were loaded into the pCold II plasmid (Takara). Induction of expression at low temperature improved protein expression levels and solubility. The vectors were double-digested with NdeI and KpnI, and exogenous fragments were amplified using primers containing homologous arms (primer sequences are shown in Table 1). The amplified fragments were purified by electrophoresis using an Omega D2500 gel extraction kit. The purified target bands were ligated to the linearized plasmids using the YEASEN Hieff Clone Universal One Step Cloning Kit. All constructed plasmids were verified by sequencing. The nucleotide sequences of each methyltransferase in the plasmids are shown in SEQ ID NO: 6-10, and the amino acid sequences of the expressed methyltransferases are shown in SEQ ID NO: 1-5.

[0050] Table 1 Primers used in this invention

[0051] 3) Preparation and transformation of competent Escherichia coli cells E. coli BL21 culture, preserved at a low temperature, was streaked onto SOB solid medium plates and incubated overnight at 37 °C. Single colonies were picked and added to 5 mL of liquid SOB medium, and incubated overnight at 37 °C with shaking. 1 mL of the bacterial culture was then added to 100 mL of liquid SOB medium and incubated until OD reached... 600 The concentration was 0.6. After incubating on ice for 10 min, all bacterial culture was transferred in batches to 50 mL centrifuge tubes and centrifuged at 4000 rpm and 4 °C for 10 min, discarding the supernatant. 40 mL of pre-chilled 0.1 M CaCl2 solution was added, and the mixture was incubated on ice for 50 min. After centrifugation at 4000 rpm and 4 °C for 10 min, the supernatant was discarded. 6 mL of 0.1 M CaCl2 solution containing 15% glycerol was added, and the bacterial pellet was gently resuspended with a pipette tip and aliquoted into 100 μL portions. The prepared recombinant plasmid was transformed into E. coli BL21 competent cells using the heat shock method, and the transformation was verified by colony PCR. The verification results are as follows: Figure 7 As shown.

[0052] 4) Detection of exogenous protein expression levels in transformants Transformants induced at low temperature for 24 h were centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in PBS buffer. They were then sonicated on ice at 30% power for a total of 30 min. After sonication, the supernatant and precipitate from each transformant were subjected to SDS-PAGE electrophoresis. The results showed that, except for Ms3OMT, which was expressed as inclusion bodies, all other proteins were normally expressed in E. coli BL21.

[0053] 5) In vitro conversion of emodin Different transformants were inoculated into 5 mL LB medium containing carboxybenzyl in a -80 ℃ preservation solution and cultured overnight at 37 ℃ and 220 rpm. The seed culture was then inoculated at a rate of 2% into 250 mL Erlenmeyer flasks containing 50 mL LB medium and cultured for 2.5 h. OD 600 The effective value ranged from 0.4 to 0.6. The bacterial culture was cooled to 15 °C in ice water, and then IPTG was added to a final concentration of 0.5 mM. Induction was carried out at 15 °C and 170 rpm for 24 h. After induction, the culture was centrifuged and resuspended in 50 mM NaH2PO4 solution, so that the final 100 μL of reaction system contained 10 mg of wet bacterial cells. The substrate concentration of emodin was 200 μM, and the concentration of SAM (S-adenosylmethionine) was 2 mM. The reaction was carried out at 30 °C and 200 rpm for 8 h or 24 h. After the reaction, four volumes of ethyl acetate were added, and the culture was extracted by sonication for 30 min. The culture was then evaporated to dryness using a rotary evaporator, reconstituted with methanol, and then analyzed by HPLC.

[0054] 6) Detection of emodin and emodin methyl ether ① HPLC analysis conditions: Agilent TC-C18 reversed-phase analytical column (4.6 mm × 250 mm, 5 μM). Mobile phase: 0–1 min: 50% acetonitrile, 1–20 min: 50% acetonitrile to 100% acetonitrile, 20–25 min: 100% acetonitrile, with 1% trifluoroacetic acid added to the aqueous phase.

[0055] ② HPLC analysis conditions optimization: HPLC results showed that emodin and emodin methyl ether were well separated and had early peak times. To save sample analysis time, the detection conditions were optimized as follows: 0-1 min: 60% acetonitrile, 1-10 min: 60% acetonitrile to 98% acetonitrile, 10-10.1 min: 98% acetonitrile to 60% acetonitrile, 10.1-12 min: 60% acetonitrile, with 1% trifluoroacetic acid added to the aqueous phase.

[0056] In vitro transformation experiments showed that, except for Ms3OMT, which was expressed in inclusion body form and could not be expressed normally and had no transformation effect, all five methyltransferases mined by the enzyme mining method developed in this invention exhibited activity in catalyzing the conversion of emodin to physcion (see [link to original text]). Figure 8 However, the activities of different enzymes vary greatly. For example, Tm3OMT has a poor catalytic effect, inferior to the control enzyme PtaI, but Tp3OMT has a significantly higher catalytic activity than the control enzyme PtaI (see [link to relevant documentation]). Figures 8-9 The HPLC conditions for the two are different. Figure 9 (After optimization) In addition, SAM, as a cofactor of methyltransferases, is expensive and unsuitable for use in actual production. When producing emodin methyl ether using whole-cell transformation, the catalytic efficiency of the group without added SAM did not change significantly (participating in...). Figure 9 and Figure 10 This indicates that the SAM present in the bacteria can meet the requirements of the catalytic process, providing a more economical production method for the synthesis of emodin methyl ether.

[0057] In summary, this invention verifies that the methyltransferase Tp3OMT can efficiently convert emodin into emodin methyl ether, laying the foundation for subsequent production of emodin methyl ether using whole-cell conversion.

[0058] The above description is merely an example of the embodiments of the present invention. It should be noted that any improvements and modifications made based on the description and drawings of the present invention, whether directly or indirectly applied to other related technical fields, should also be considered within the protection scope of the present invention.

[0059] sequence list Tp3OMT (SEQ ID No.1):MAPSKTQDLETPFTTGAIGTDVPFWRSYIAGRPSPTEDFFELINDYHTKHGDPNTGVAHDVGTGPGNIAERLAKYFDHVVGSDVNDAALAAAPAIVSKEHLQRMTFVKSPAEGLAAAPIPEDVGKGKTDLVVVSECMPLLDAPKALEAFHTLLRPGGTVAIYFY ARPIFTGDDADKLNAIYDKIATRICTFLLPFKGTPGFPFHARGAEAMGSWLDNIEFPASHWSNVQRHKWNNDIPLLFNSKEGYDFEIERVDRTGPEDVKKEVIDRGFWAEEWDAERVAGYLASVYPNYRKKAGERYSEIEQMVEELREAMGGSGATRTVSFPVVLILATSK Ms3OMT (SEQ ID No.2): MALSSTQDLETPFTTAVLGSDVDFWRSYIAGRPSPDESFFHLINEYHSSHGQSKTAIAHDVGTGPDNIAQRLLAYYDHVVGSDLNDQALAAAPALIPSELYKRTTFIKSPAEALASGVVPNGVGLGKTDLITVSECIPLLDAPKALDAFHELLRPEGTLAIYFYGRAIFAEGDIHTLNAIYDAIATRICTFLLPFKGTPGFPFHVRGAEALVSRLDNIAFPSESWKGVERYKWNYDYPLLFNSKEGYDFDFEPVDRRAEGEKTIEVIDKQFWAKEWGVEEVKTYLDSVYPNWQKKAGARSTEVDELLEELRNALGGGGSKKKVTFPVVLILATKK Ab3OMT (SEQ ID No.3): MSQTSTRLETPFTTAALGSDVSFWKSYIASRPNPPETFFRLINEYHTNHSDGRMEVAHDVGTGPGNIAARLASYYDQVVGSDVNEQALAAAPKLISPDLLERMTFVQSPAEKLVTAGLPTELQQTDLVVVSECMPLLDAPRALEAFHALLRPGGTLAIYFYGRPIFPDPVCDEIYDRMATRICQFLLPFKGTPGEPFHRRGAEGLVSFLDNIALPGGDWQNVERYKWNCDYPLLFNSPEGFDFEFHPVDRRAPGEVTHDIVDREFWVHEWDADDVKAYLDSVYPHYRHQAGESYAEIEAMREELRAAMGGKQKVTFSVVLVLGSKK Dc3OMT (SEQ ID No.4): MSTTATTTKQKVETPFTSAALGTDVGFWRAYVAGRPTPTEDFFRLINEYHKSHGNPQFRVAHDVGTGPGNIAERLAPYYEHVVGSDVNESALAAAPSLVPFQYLNRMTFVKSPAEGLASGVVPSEVGVGKTDLITVSECIPLLDTQKALEAFHALLKPGGTLAIYFYGRPVFTDGDAKMCDEIYDKIATRICQFNLPMKDTPGFPFHLRGVEALESWLDNIPIPAEQWTGVQRHKWNYHHPLLFNSHDGFDFEFSRVDRRGEGEKTIEMIDNSFWAEEWDVGQARTYLESVYPNIRMKAGEKWTEVEEMLKELNQAMGGERRKVTFTVSLILATKM Tm3OMT (SEQ ID No.5): MSLTTTNTPATVETPFTTGALSSDADFWAKYIASRPTPSEEFFRVIQDYHSSHGDSTTALAHDVGTGPGNIAERLLRYYDHVVGSDVNKRALVAAPSLIPQDKVQRMTFVHSPAENLLAGGVVPADVGAGKTDLVVVSECMPLLDAPKALDAFHGLLRVGGTLAIYFYGRPIFTSGQDPAHLDSLYDRIATKICSFLLPFKGTPGFPFHKRGAEALVSCLDNIPLPSGAWADVVRYKWNTNVTLLFNSKAGFDFDFEPVDRREDGEVTKEIRDRTWWQDEWDADRVKSYLDSVYPSYIEKAGDRYVEIEQGIEELREAMGGGKVKVSFPVALILATKK Tp3OMT (SEQ ID No.6): Ms3OMT (SEQ ID No.7): Ab3OMT (SEQ ID No.8): ATGTCTCAGACCTCTACTCGTCTGGAAACTCCGTTCACCACTGCAGCTCTGGGTTCTGACGTTAGCTTCTGGAAATCTTACATCGCGAGCCGTCCAAATCCACCGGAAACCTTCTTCCGTCTGATCAACGAATACCACACCAACCACTCTGACGGTCGTATGGAAGTTGCGCACGATGTTGGTACTGGTCCGGGTAACATCGCAGCGCGTTTGGCGTCTTACTACGACCAGGTTGTTGGTTCCGACGTTAACGAACAGGCACTTGCTGCTGCACCGAAACTGATCTCTCCAGACCTGCTGGAACGTATGACCTTCGTTCAGAGTCCGGCAGAGAAACTGGTTACCGCAGGTCTGCCAACCGAACTGCAGCAGACCGACCTGGTTGTTGTTTCCGAATGTATGCCGCTGCTGGATGCACCACGTGCACTGGAAGCATTCCACGCTCTGCTGCGTCCAGGTGGTACTCTGGCGATCTACTTCTACGGTCGTCCGATCTTTCCAGATCCGGTTTGCGACGAAATCTACGACCGTATGGCGACTCGTATCTGCCAGTTTCTGCTGCCGTTCAAAGGTACTCCGGGTGAACCGTTCCATCGTCGTGGTGCAGAAGGTCTGGTTTCTTTTCTGGACAACATCGCTCTGCCAGGTGGTGATTGGCAGAACGTTGAACGTTACAAGTGGAACTGCGACTATCCGCTGCTGTTCAACTCTCCGGAAGGTTTCGACTTCGAGTTCCATCCGGTAGATCGTCGTGCGCCAGGTGAAGTTACTCACGACATCGTTGATCGTGAATTTTGGGTTCACGAATGGGATGCTGATGACGTTAAAGCGTACCTGGACTCTGTTTATCCGCACTACCGTCATCAGGCTGGTGAAAGCTACGCGGAAATCGAAGCAATGCGTGAAGAACTGCGTGCTGCAATGGGTGGTAAACAGAAAGTGACCTTCTCTGTTGTGCTGGTTCTGGGTTCCAAGAAGTAA Dc3OMT (SEQ ID No.9): Tm3OMT (SEQ ID No.10):

Claims

1. The application of methyltransferase in the conversion of emodin to emodin methyl ether, characterized by: The amino acid sequence of the methyltransferase comprises the amino acid sequence shown in SEQ ID NO: 1-5, preferably as shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that the nucleotide sequence encoding the methyltransferase comprises the nucleotide sequences shown in SEQ ID NO: 6-10, preferably as shown in SEQ ID NO:

6.

3. A recombinant microorganism, characterized by: The recombinant microorganism expresses a methyltransferase, wherein the amino acid sequence of the methyltransferase comprises the amino acid sequence shown in SEQ ID NO: 1-5, preferably as shown in SEQ ID NO:

1.

4. The recombinant microorganism according to claim 3, characterized in that... The recombinant microorganism is Escherichia coli.

5. The recombinant microorganism according to claim 3 or 4, characterized in that the nucleotide sequence encoding the methyltransferase comprises the nucleotide sequence shown in SEQ ID NO: 6-10, preferably as shown in SEQ ID NO:

6.

6. The recombinant microorganism according to any one of claims 3-5, characterized in that... The methyltransferase is expressed in a plasmid or on a chromosome.

7. A method for preparing recombinant microorganisms, characterized in that: The preparation method includes introducing a nucleotide sequence encoding a methyltransferase into a recombinant microorganism, wherein the amino acid sequence of the methyltransferase comprises the amino acid sequence shown in SEQ ID NO: 1-5, preferably as shown in SEQ ID NO: 1; Preferably, the recombinant microorganism is Escherichia coli.

8. The preparation method according to claim 7, characterized in that: The nucleotide sequence encoding the methyltransferase comprises the nucleotide sequence shown in SEQ ID NO: 6-10, preferably as shown in SEQ ID NO:

6.

9. A method for producing emodin methyl ether, characterized in that: The method includes recombinant microorganisms obtained using the recombinant microorganisms of claims 3-6 or any of the preparation methods of claims 7-8; Preferably, the method comprises mixing the recombinant microorganisms of claims 3-6 or the recombinant microorganisms obtained by any of the preparation methods of claims 7-8 with emodin.

10. The application of the recombinant microorganisms according to claims 3-6 or the recombinant microorganisms obtained by any of the preparation methods according to claims 7-9 in the preparation of bactericides; preferably, the bactericide is a plant-derived bactericide.

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