A methyltransferase and its use in ergothioneine biosynthesis

Abstract

This invention discloses a methyltransferase and its application in ergothioneine biosynthesis. The methyltransferase comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO:1, and differs from the amino acid sequence shown in SEQ ID NO:1 by one or more amino acid residues at positions F47, Q64, T89, R120, and C285. The methyltransferase provided by this invention can efficiently catalyze the synthesis of HER from histidine, thereby improving the yield of ergothioneine synthesis and providing a new technical solution for the green biomanufacturing of ergothioneine, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of enzyme catalysis and relates to a methyltransferase and its application in the biosynthesis of ergothioneine. Specifically, the enzyme can efficiently catalyze the production of histidine from histidine, and histidine betaine can serve as a key intermediate in the synthesis of ergothioneine. Background Technology

[0002] Ergothioneine (EGT) is a rare natural amino acid derivative with unique antioxidant, anti-inflammatory, and cell-protective activities, showing broad application prospects in food additives, cosmetic active ingredients, and pharmaceutical development. EGT is a powerful antioxidant with many physiological functions, such as protecting skin cells from UV radiation, maintaining redox homeostasis, and promoting neuronal stem cell differentiation. Therefore, EGT has wide applications in the pharmaceutical and cosmetic fields. With the continued growth in market demand for high-purity ergothioneine, developing efficient and economical biomanufacturing methods has become a current research hotspot.

[0003] EGT was initially discovered in ergot fungi, but it was later found to be primarily synthesized by fungi and bacteria. However, humans cannot produce EGT endogenously and must obtain it through dietary intake.

[0004] The biosynthetic pathway of EGT has been elucidated and understood in microorganisms such as *Mycobacterium smegmatis* (Alamgir et al., 2015), *Neurospora crassa* (Han et al., 2021), and *Chlorobium limicola* (Burn et al., 2017). The aerobic EGT synthesis pathway includes Egt A (γ-glutamylcysteine ​​synthase), Egt B (mononuclear non-heme ironase), Egt C (amidoletransferase), Egt D (S-adenosylmethionine (SAM)-dependent histidine methyltransferase), and Egt E (pyridoxal phosphate-mediated C-S lyase) (Seebeck, 2010). This gene cluster is also widely found in actinomycetes (egt D and egt B), cyanobacteria, acidobacteria, Firmicutes, Proteobacteria, and other prokaryotes (Jones et al., 2014). Furthermore, in some *Methylobacteria* species, L-cysteine ​​and histidine betaine (HER) can be directly catalyzed by Egt B to form Cys-HER (Hirasawa et al, 2023). In *Neurospora crassa*, another aerobic pathway for EGT synthesis involves Egt1 and Egt2. Egt1 catalyzes the conversion of L-histidine to HER, which in turn catalyzes the synthesis of Cys-HER from HER and L-cysteine. Egt2 promotes the binding of PLP to Cys-HER, forming ergothioneine sulfinic acid or disulfide bond intermediates. These intermediates are reduced to release EGT via DTT or a thiol system (thioredoxin).

[0005] In current technologies, ergothioneine production mainly relies on direct extraction from edible fungi or microbial fermentation. Natural extraction methods are limited by the availability of fungal resources, low content, and complex extraction processes, making them unsuitable for industrial-scale production. While microbial fermentation has the potential for sustainable production, it generally faces bottlenecks such as low yield and high cost. Specifically, the methyl donor S-adenosylmethionine is expensive, and its direct addition would significantly increase production costs. Furthermore, some studies have attempted to prepare histidine betaine using chemical synthesis, but the chemical methylation process suffers from poor selectivity, numerous byproducts, and does not meet the requirements of green production. With the rapid development of synthetic biology, directly adding amino acids to microbial cells to synthesize ergothioneine through the regulation of enzyme elements and metabolic networks is a promising method. However, intracellular synthesis by microorganisms faces challenges such as limited metabolic flux and low methyl cycle efficiency.

[0006] In summary, the existing technology has the following defects: (1) the biological extraction method is limited by the limited fungal resources, low content and complex extraction process, which makes it difficult to meet the needs of industrial production; (2) as the first step in the microbial synthesis route, the efficiency of methyltransferase affects the entire metabolic pathway. The low methylation efficiency limits the throughput of the entire microbial synthesis route.

[0007] Therefore, developing a novel methyltransferase that can efficiently catalyze the conversion of histidine to histidine betaine and constructing a low-cost, high-yield ergothioneine biosynthesis system has significant industrial application value. Summary of the Invention

[0008] To address the aforementioned shortcomings of existing technologies, this invention provides a methyltransferase and its application in ergothioneine biosynthesis. Specifically, it provides a methyltransferase that efficiently generates histidine betaine from histidine and S-adenosylmethionine as substrates, the product of which can be further converted into ergothioneine, providing a new technical solution for the green biomanufacturing of ergothioneine.

[0009] The present invention specifically provides the following technical solutions to solve the above-mentioned technical problems: A first aspect of the present invention provides a methyltransferase comprising an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 1, and comprising one or more differences in amino acid residues at positions F47, Q64, T89, R120, and C285 compared to the amino acid sequence shown in SEQ ID NO: 1, wherein positions 47, 89, and 120 refer to the amino acid residue numbers in SEQ ID NO: 1; and the amino acid residue difference at position C285 is C285N.

[0010] In some embodiments, the amino acid residue difference at position F47 is F47V.

[0011] In some implementations, the amino acid residue difference at position Q64 is Q64E.

[0012] In some implementations, the amino acid residue difference at position T89 is T89A.

[0013] In some embodiments, the amino acid residue difference at position R120 is R120E.

[0014] In some embodiments, the methyltransferase contains one or more differences in amino acid residues at positions F47, T89, and R120 compared to the amino acid sequence shown in SEQ ID NO: 1.

[0015] In some specific embodiments, the methyltransferase differs from the amino acid sequence shown in SEQ ID NO: 1 by any of the following amino acid residues: F47V; T89A; R120E; F47V and T89A; F47V and R120E; T89A and R120E; F47V, T89A, and R120E.

[0016] A second aspect of the invention provides an isolated nucleic acid that encodes a methyltransferase as described in the first aspect of the invention.

[0017] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described in the second aspect of the present invention.

[0018] A fourth aspect of the present invention provides a transformant comprising a nucleic acid as described in the second aspect of the present invention, or a recombinant expression vector as described in the third aspect of the present invention.

[0019] A fifth aspect of the present invention provides a method for preparing a methyltransferase, the method comprising culturing a transformant as provided in a fourth aspect of the present invention to obtain a culture product containing the methyltransferase.

[0020] In some embodiments, the method further includes the step of obtaining the methyltransferase from the culture product.

[0021] A sixth aspect of the present invention provides a reaction system for preparing histidine betaine, the reaction system comprising: The substrate and the methyltransferase as described in the first aspect of the invention, wherein the substrate is histidine and S-adenosylmethionine.

[0022] In some embodiments, the reaction system further includes ascorbic acid and / or TCEP.

[0023] In some embodiments, the concentration of histidine is 0.5-10 mM.

[0024] In some embodiments, the concentration of histidine is 5-10 mM.

[0025] In some embodiments, the concentration of histidine is 5 mM.

[0026] In some embodiments, the concentration of the S-adenosylmethionine is 1-30 mM.

[0027] In some embodiments, the concentration of the S-adenosylmethionine is 10-30 mM.

[0028] In some embodiments, the concentration of the S-adenosylmethionine is 15 mM.

[0029] In some embodiments, the concentration of ascorbic acid is 0.5-10 mM.

[0030] In some embodiments, the concentration of ascorbic acid is 0.5-5 mM.

[0031] In some implementations, the concentration of ascorbic acid is 2 mM.

[0032] In some implementations, the concentration of TCEP is 0.5-10 mM.

[0033] In some implementations, the concentration of TCEP is 0.5-5 mM.

[0034] In some implementations, the concentration of TCEP is 2 mM.

[0035] A seventh aspect of the present invention provides a method for preparing histidine betaine, the method comprising: Histidine betaine is prepared by contacting and reacting a methyltransferase as described in the first aspect of the invention with a substrate; the substrate being histidine and S-adenosylmethionine.

[0036] In some embodiments, the method includes using a reaction system as described in the sixth aspect of the invention.

[0037] The eighth aspect of the present invention provides a genetically engineered bacterium that, compared with the starting strain, has a gene encoding a methyltransferase as described in the first aspect of the present invention.

[0038] In some implementations, the starting strain is Escherichia coli.

[0039] In some embodiments, the Escherichia coli is Escherichia coli BL21(DE3).

[0040] In some embodiments, the genetically engineered bacteria further comprises bacteria derived from methylbacteria ( Methylobacterium egtB and / or egtE.

[0041] In some embodiments, the egtB comprises an amino acid sequence as shown in SEQ ID NO: 13.

[0042] In some embodiments, egtE comprises an amino acid sequence as shown in SEQ ID NO: 14.

[0043] In some specific embodiments, the genetically engineered bacteria contains genes encoding egtB and / or egtE.

[0044] In some specific embodiments, the genetically engineered bacteria contains the pACYC-EgtB-EgtE plasmid, which contains the nucleotide sequence shown in SEQ ID NO: 15.

[0045] In some specific embodiments, the gene encoding the methyltransferase as described in the first aspect of the invention is inserted between the NCO I and Xho I sites in the pACYC-EgtB-EgtE plasmid.

[0046] A ninth aspect of the present invention provides a method for preparing ergothioneine, the method comprising: Cultivate genetically engineered bacteria as described in the eighth aspect of the present invention.

[0047] In some embodiments, the culture medium used for the culture is LB or TB medium.

[0048] In some embodiments, the culture medium used for the culture is TB medium.

[0049] In some embodiments, the culture medium used for the culture contains L-cysteine, L-histidine, and L-methionine.

[0050] In some embodiments, the concentrations of L-cysteine, L-histidine, and L-methionine are 0.05%-1%, respectively.

[0051] In some embodiments, the concentrations of L-cysteine, L-histidine, and L-methionine are 0.05%-0.5%, respectively.

[0052] In some embodiments, the concentrations of L-cysteine, L-histidine, and L-methionine are each 0.1%.

[0053] The tenth aspect of the present invention provides the use of methyltransferases as described in the first aspect of the present invention, nucleic acids as described in the second aspect of the present invention, recombinant expression vectors as described in the third aspect of the present invention, transformants as described in the fourth aspect of the present invention, or reaction systems as described in the sixth aspect of the present invention, or genetically engineered bacteria as described in the eighth aspect of the present invention, in the preparation of histidine betaine and / or ergothioneine.

[0054] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0055] The reagents and raw materials used in this invention are all commercially available.

[0056] The positive and progressive effects of this invention are as follows: The Mycobacterium smegmatis constructed in this invention ( Mycolicibacterium smegmatis The mutant of egtD protein, derived from , can efficiently catalyze the synthesis of HER from histidine, thereby increasing the yield of ergothioneine synthesis and providing a new technical solution for the green biomanufacturing of ergothioneine, with good application prospects. Attached Figure Description

[0057] Figure 1 For mutant expression identification (bacterial lysis supernatant).

[0058] Figure 2 This was to verify the activity of methyltransferase.

[0059] Figure 3 To verify the superposition mutant.

[0060] Figure 4 This was to validate the fermentation process of methyltransferase. Detailed Implementation

[0061] The purpose of this invention is to design an egtD protein mutant to increase the synthesis rate of histidine betaine, thereby enhancing the level of ergothioneine synthesis in microorganisms. Specifically, multi-scale computational biology methods, including homology modeling, molecular docking, conservation analysis, molecular dynamics simulation, and model prediction, are integrated to pinpoint key amino acid residues within a 6 Å substrate binding pocket. By considering their dynamic trajectory and evolutionary conservation, potential beneficial mutation sites are screened and combined. The mutant is expressed using an *E. coli* expression system, and its activity is verified by catalyzing a reaction with S-adenosylmethionine (SAM) and histidine as substrates. A methyltransferase is then integrated into *E. coli* to verify the strain's ability to synthesize ergothioneine. The specific reaction route is shown in the figure below.

[0062] The technical solution is explained in detail in four points: 1. Discovery and screening of methyltransferase genes Based on the requirements of enzyme function, in Mycobacterium smegmatis ( Mycolicibacterium smegmatis Based on the egtD protein from which the protein originates, amino acid mutations were performed to obtain F47V, Q64E, T89A, R120E, and C285N, respectively. These mutants were then stacked to obtain mutants.

[0063] 2. Construction and expression of methyltransferase expression vectors

[0064] The amino acid sequence of the egtD enzyme was codon optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and cloned into the commercial plasmid peTduet-1. The synthesized plasmids were integrated into BL21(DE3), and the corresponding engineered bacteria were obtained by antibiotic screening. The engineered bacteria were expressed separately, the bacterial cells were collected, and the supernatant was collected after cell disruption.

[0065] 3. Screening by methyltransferase reaction

[0066] The supernatant from the lysis of engineered bacteria with different protein mutants was mixed with the substrates S-adenosylmethionine (SAM) and histidine on ice, and a catalytic reaction was carried out under the same conditions. Samples were taken during the reaction, and the reaction was terminated by high-temperature treatment and centrifugation. The conversion rate, histidine betaine (HER) yield, and enzyme selectivity for the substrate histidine were analyzed by HPLC.

[0067] 4. Application of methyltransferases

[0068] The methyltransferase egtD mutant, and the methyltransferase from Methylobacterium ( Methylobacterium The egtB and egtE genes were integrated into the BL21(DE3) strain and fermented to verify whether the screened methyltransferase could synthesize histidine betaine in vivo and promote ergothionein synthesis.

[0069] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0070] Example 1: Screening of methyltransferase genes and verification of protein expression

[0071] 1.1 Construction of methyltransferase mutants

[0072] To obtain highly efficient methyltransferase mutants using S-adenosylmethionine and histidine as substrates, we started with the previously reported Mycobacterium smegma egtD protein (PDB ID: 4PIM). We integrated multi-scale computational biology methods, including homology modeling, molecular docking, conservation analysis, molecular dynamics simulation, and model prediction, to pinpoint key amino acid residues within a 6 Å substrate binding pocket. Considering its dynamic trajectory and evolutionary conservation, we screened and combined potential beneficial mutation sites. Five mutation sites were designed: F47V, Q64E, T89A, R120E, and C285N.

[0073] The amino acid sequence of the egtD enzyme was codon optimized and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and cloned into the commercial plasmid pETduet-1. The DNA sequence encoding the egtD mutation was amplified by PCR. After gel extraction and purification, the PCR product was fused and ligated using a seamless cloning kit (Shanghai Beyotime Biotechnology Co., Ltd., Cat No. D7010M) to construct the mutant expression plasmid.

[0074] The egtD sequences are shown in Table 1: Table 1. Amino acid and nucleotide sequences of egtD

[0075] The primer sequences used are shown in Table 2: Table 2 Primer sequences for constructing the EgtD mutant vector

[0076] The mutants were named as follows: M1 (F47V), M2 (Q64E), M3 (T89A), M4 (R120E), and M5 (C285N).

[0077] Example 2: Transformation and Expression Verification of Methyltransferase

[0078] The mutant from Example 1 was transformed into the BL21(DE3) host, and the corresponding engineered strain was obtained through resistance screening. The specific operation steps are as follows: (1) Preparation of chemically competent cells 5 μL of laboratory-preserved *Escherichia coli* BL21(DE3) glycerol bacteria was streaked onto an LB agar plate (components shown in Table 3) for activation. Single activated colonies were picked and transferred to LB agar and incubated at 37°C with a shaker until OD500 reached. 600 Competent cells were prepared at a concentration of 0.6. The preparation method is as follows: ice bath for 30 min; centrifugation to collect bacterial cells; resuspending bacterial cells in 15% glycerol; centrifugation to collect bacteria; collecting bacterial cells and resuspending them in 0.1M CaCl2 and 15% glycerol, then aliquoting and storing in an ultra-low temperature freezer at -80℃. All the above operations were performed in a low temperature and sterile environment.

[0079] (2) Plasmid transformation

[0080] The competent cells were thawed on ice, and 2 μL of plasmid synthesized by Sangon Biotech was added. The mixture was then incubated in an ice-water bath for 30 min, heat-shocked at 42 °C for 90 s, and 800 μL of LB was added. The mixture was then cultured in a shaker at 37 °C for 1 h. After the culture was completed, the mixture was centrifuged at 4000 rpm for 2 min, and the precipitate was resuspended and spread onto LB agar plates containing ampicillin. The plates were then cultured at 37 °C for 18 h.

[0081] Table 3 LB medium formulation

[0082] (3) Induced expression

[0083] Pick a single colony from the transformation plate and transfer it to an LB tube. Add the appropriate antibiotic and incubate overnight at 37°C and 220 rpm on a shaker. Transfer the culture solution at a ratio of 1% to an Erlenmeyer flask containing 10% volumetric medium and incubate at 37°C and 220 rpm on a shaker until OD (outlet volume) is reached. 600 When the bacterial count reaches between 0.6 and 0.8, add 0.35 mM IPTG and induce at 28°C and 220 rpm for a total of 20 h. After induction, collect the bacterial cells by centrifugation.

[0084] (4) Protein expression detection

[0085] The collected bacterial cells were resuspended in double-distilled water, and the resuspended bacterial solution was then disrupted using an ultrasonic homogenizer. The homogenization program was as follows: amplitude bar 6, engineering number 1, on for 2 seconds and off for 3 seconds, 40% power, running for 20 minutes. After disruption, the cells were centrifuged at 10000 g for 20 minutes, and SDS-PAGE was performed to detect expression levels (see [link to SDS-PAGE analysis]). Figure 1 All mutants expressed well.

[0086] Example 3: Verification of methyltransferase gene activity

[0087] The reaction systems shown in Table 4 were used to catalyze the selected enzymes under the conditions of 5 mM His (histidine) and 15 mM SAM substrate. Table 4 Screening protein catalytic reaction systems

[0088] The collected bacterial cells were resuspended in water to OD. 600 =40, the bacterial cells were disrupted according to the method described in Example 2 for protein detection, and the supernatant from centrifugation was the crude enzyme solution. The reaction mixture was added according to the proportions in Table 4. All reaction components were mixed on ice, and the resulting reaction system was carried out in a shaker at 30°C. Samples were taken at 0, 30, and 60 minutes of the reaction. The samples were then subjected to high-temperature treatment in a 95°C metal bath for 10 minutes, followed by centrifugation at 12000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter membrane, and the yield was analyzed by liquid chromatography. The results are shown in Table 4. Figure 2 .

[0089] Among the screened mutants, M1, M3, and M4 showed increased egtD activity by 24%, 40%, and 52% respectively compared to the wild-type mutant. These mutants were then stacked to obtain mutants M6 (F47V, T89A), M7 (F47V, R120E), M8 (T89A, R120E), and M9 (F47V, T89A, R120E). These mutants were screened again. The cell culture and disruption methods are described in Example 2. The reaction components were added according to the proportions in Table 4. The reaction components were mixed on ice, and the resulting reaction system was carried out in a shaker at 30°C. Samples were taken at 0, 30, and 60 minutes of the reaction. The samples were then subjected to high-temperature treatment in a 95°C metal bath for 10 minutes, followed by centrifugation at 12000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter and the yield was analyzed by liquid chromatography. The results are shown in [Figure 1]. Figure 3 .

[0090] All the screened methyltransferases can catalyze the production of histidine betaine using His and SAM as substrates. Among them, the M7 enzyme exhibited extremely high activity, producing 0.169 g / L of histidine betaine within 1 hour, which is about 70% higher than the activity of egtD.

[0091] Example 4: Application of methyltransferase mutants in ergothioneine synthesis

[0092] Construction of ergothionein-synthesizing strains: Wild-type EgtD and the screened methyltransferase M7 were respectively constructed into the NCO I and Xho I sites of the laboratory-preserved pACYC-EgtB-EgtE vector (SEQ ID NO: 15) to obtain the pACYC-EgtB-EgtE-egtD and pACYC-EgtB-EgtE-M7 vectors, respectively. These vectors were then transformed into BL21(DE3) to obtain recombinant strains S1 and S2 capable of synthesizing ergothioneine.

[0093] The amino acid sequence of egtB is as follows: MIARETLADELALARERTLRLVEFDDAELHRQYNPLMSPLVWDLAHIGQQEELWLLRDGNPDRPGMLAPEVDRLYDAFEHSRASRVNLPLLPPSDARAYCATVRAKALDTLDTLPEDDPGFRFALVISHENQHDETMLQALNLREGPPLLDTGIPLPAGRPGVAGTSVLVPGGPFVLGVDALTEPHSLDNERPAHVVDIPSFRIGRVPVTNAEWREFIDDGGYDQPRWWSPRGWAHRQEAGLVAPQFWNPDGTRTRFGHIEEIPGDEPVQHVTFFEAEAYAAWAGARLPTEIEWEKACAWDPVAGARRRFPWGSAQPSAALANLGGDARRPAPVGAYPAGASAYGAEQMLGDVWEWTSSPLRPWPGFTPMIYEQYSTPFFEGTTSGDYRVLRGGSWAVAPGILRPSFRNWDHPIRRQIFSGVRLAWDV (SEQ ID NO: 13) The amino acid sequence of egtE is as follows: MMLAQQWRDARPKVAGLHLDSGACSRQSFAVIDATTAHARHEAEVGGYVAAEAATPALDAGRAAVASLIGFAASDVVYTSGSNHAIDLLLSSWPGKRTLACLPGEYGPNLSAMAANGFQVRALPVDDDGRVLVDEASHELSAHPVALVHLTALASHRGIAQPAAELVEACHNAGIPVVIDAAQALGHLDCNVGADAVYSSSRKWLAGPRGVGVLAVRPELAERLQPRIPPSDWPIPMSVLEKLELGEHNAAARVGFSVAVGEHLAAGPTAVRERLAEVGRLSRQVLAEVDGWRVVEPVDQPTAITTLESTDGADPASVRSWLIAERGIVTTACELARAPFEMRTPVLRISPHVDVTVDELEQFAAALREAP (SEQ ID NO: 14) Flask fermentation of strains S1 and S2: The strain was activated by streaking on LB agar plates, and single colonies were picked and incubated at 37°C for 16 h. The cultured bacterial solution was then transferred at a ratio of 1% to shake flasks containing fermentation medium. After incubation at 37°C and 220 rpm for 3 h, 0.5 mM IPTG was added, and the culture was induced at 28°C and 220 rpm. The composition of the culture medium is shown in Table 5. Table 5 Components of shake flask fermentation medium

[0094] Samples were taken after 72 hours of incubation. The samples were then subjected to high-temperature treatment in a 95℃ metal bath for 10 minutes, followed by centrifugation at 12000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm filter membrane, and the yield was analyzed by liquid chromatography. The results are shown in [Figure number missing]. Figure 4 Fermentation culture for 72 hours produced a total of 0.72 g / L ergothioneine, while 0.44 g / L histidine betaine remained in the fermentation broth.

[0095] In summary, this patent, based on the requirements of enzyme function and building upon the previously reported egtD enzyme, integrates multi-scale computational biology methods such as homology modeling, molecular docking, conservation analysis, and molecular dynamics simulation to obtain a highly active egtD mutant M7. This mutant exhibits 70% higher activity than the wild-type egtB. After 72 hours of shake-flask fermentation, it produces 0.72 g / L ergothioneine and 0.44 g / L histidine betaine, providing a new technical solution for the green biomanufacturing of ergothioneine and possessing significant industrial application value.

[0096] pACYC-EgtB-EgtE vector sequence (SEQ ID NO: 15): While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A methyltransferase, characterized in that, The methyltransferase is an enzyme whose amino acid sequence is as shown in SEQ ID NO: 1, by performing any one of the following mutations: The R at position 120 mutates to E; The R at position 120 mutates to E and the F at position 47 mutates to V; The R at position 120 mutates to E and the T at position 89 mutates to A; or, The R at position 120 is mutated to E, the F at position 47 is mutated to V, and the T at position 89 is mutated to A.

2. An isolated nucleic acid, characterized in that, The nucleic acid encodes the methyltransferase as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 2.

4. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 2, or the recombinant expression vector as described in claim 3.

5. A method for preparing methyltransferase, characterized in that, The method includes culturing the transformant as described in claim 4 to obtain a culture product containing the methyltransferase; the method further includes the step of obtaining the methyltransferase from the culture product.

6. A reaction system for preparing histidine betaine, characterized in that, The reaction system includes: The substrate and the methyltransferase as described in claim 1, wherein the substrate is histidine and S-adenosylmethionine.

7. The reaction system as described in claim 6, characterized in that, The reaction system also includes ascorbic acid and / or TCEP.

8. A method for preparing histidine betaine, characterized in that, The method includes: Histidine betaine is prepared by contacting and reacting the methyltransferase as described in claim 1 with a substrate; wherein the substrate is histidine and S-adenosylmethionine; the method includes using the reaction system as described in claim 6 or 7.

9. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria, compared to the starting strain, have a gene encoding the methyltransferase as described in claim 1.

10. The genetically engineered bacteria as described in claim 9, characterized in that, The starting strain is *Escherichia coli*; and / or, the genetically engineered bacteria further comprises *Methylobacterium* (…). Methylobacterium egtB and / or egtE; The amino acid sequence of egtB is shown in SEQ ID NO: 13, and the amino acid sequence of egtE is shown in SEQ ID NO:

14.

11. The genetically engineered bacteria as described in claim 10, characterized in that, The Escherichia coli mentioned is Escherichia coli BL21(DE3).

12. A method for preparing ergothioneine, characterized in that, The method includes: Cultivate the genetically engineered bacteria as described in any one of claims 9-11; wherein the genetically engineered bacteria comprises bacteria derived from methylbacterium ( Methylobacterium egtB and egtE; The amino acid sequence of egtB is shown in SEQ ID NO: 13, and the amino acid sequence of egtE is shown in SEQ ID NO:

14.

13. The method as described in claim 12, characterized in that, The culture medium used for the culture is LB or TB medium; and / or, the culture medium used for the culture contains L-cysteine, L-histidine and L-methionine.

14. The use of the methyltransferase as described in claim 1, the nucleic acid as described in claim 2, the recombinant expression vector as described in claim 3, the transformant as described in claim 4, or the reaction system as described in claim 6 or 7, or the genetically engineered bacteria as described in any one of claims 9-11, in the preparation of histidine betaine.

15. The use of the genetically engineered bacteria as described in any one of claims 9-11 in the preparation of ergothionein; wherein, The genetically engineered bacteria include those derived from Methylobacterium ( Methylobacterium egtB and egtE; The amino acid sequence of egtB is shown in SEQ ID NO: 13, and the amino acid sequence of egtE is shown in SEQ ID NO: 14.

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