A method for synthesizing ergothioneine by biological enzyme

Abstract

The application discloses a method for synthesizing ergothioneine by using a biological enzyme, and belongs to the field of synthetic biology. The method comprises the following steps: overexpressing ergothioneine synthase Ccegt1 and ergothioneine synthase Ncegt2 in Escherichia coli, preparing ergothioneine synthase, and using a crude enzyme solution of the ergothioneine synthase to synthesize ergothioneine by using a biological enzyme. The ergothioneine is synthesized by using HER and cysteine as substrates, and under the catalysis of ergothioneine synthase Egt1 and ergothioneine synthase Egt2, and the yield of the ergothioneine can reach more than 10 g / L after 8 hours of reaction, and the method has the advantages of high yield, simple composition and easy purification.

Description

Technical Field

[0001] This invention relates to a method for the bioenzymatic synthesis of ergothioneine, belonging to the field of synthetic biology. Background Technology

[0002] Ergothioneine (EGT) is a naturally occurring amino acid, first isolated from ergot fungi in 1909. Its structure was determined in 1911, revealing it to be a histidine betaine derivative. Ergothioneine is a precious natural sulfur-containing amino acid, highly soluble in water, methanol, and ethanol. It possesses various beneficial effects, including antioxidant, anti-aging, cardiovascular disease inhibition, maintenance of DNA biosynthesis, free radical scavenging, and nerve cell generation. In its dissolved state, it exists as a tautomer in both thiols and thioketones, primarily in the thioketone form under physiological pH conditions. Ergothioneine is a potent natural amino acid antioxidant, exhibiting higher thermal stability and better pH stability compared to other thiols such as glutathione, and is less prone to spontaneous oxidation. EGT is synthesized only by microorganisms such as edible fungi, cyanobacteria, mycobacteria, and non-yeast fungi; other species cannot synthesize EGT. Plants absorb EGT through symbiotic nitrogen-fixing systems and soil fungi, while humans and other mammals can only ingest and accumulate EGT in their bodies through diet. EGT is widely found in various foods, but most foods contain very little of it; the food with the highest content is edible fungi.

[0003] Currently, ergothioneine can be prepared through chemical synthesis and bio-fermentation. Chemical synthesis requires sophisticated processes for synthesis and purification. Compared to chemical synthesis and natural biological extraction, bio-fermentation offers advantages such as lower cost and readily available raw materials, but its fermentation cycle is longer. Microbial fermentation places high demands on the metabolic pathway technology of the strains, requiring the knockout of genes affecting metabolites, some of which are difficult to knock out. Furthermore, it necessitates overexpression of key genes or overexpression of transcribed proteins in the metabolic pathway. Constructing the fermentation strain requires a significant initial investment of time. The biosynthetic pathway of ergothioneine was elucidated relatively late. In 2014, the ergothioneine synthesis pathway in the fungus Neurospora crassa was elucidated. Egt1 is a bifunctional enzyme that catalyzes two consecutive reactions: L-histidine (L-His) reacts with S-adenosylmethionine (SAM) to form hesciline (HER), and then HER reacts with L-cysteine ​​(L-Cys) and O2 to synthesize hesciline cysteine ​​sulfoxide (Cys-HER) again under the action of Egt1. Egt2 is a CS lyase that depends on pyridoxal phosphate to catalyze the conversion of Cys-HER to EGT, while also producing pyruvate and ammonia byproducts. Current literature reports on the preparation of ergothioneine by biofermentation, mostly using ergothioneine biosynthesis genes (Tregt 1 / 2 and Ncegt 1 / 2) from Trichoderma reesei or Neurospora crassa. Through fed-batch fermentation, after culturing in a 2L fermenter for 143 hours, the extracellular EGT yield reached 4.34 g / L. Summary of the Invention

[0004] This invention provides a recombinant Escherichia coli that co-expresses ergothionein synthase Ccegt1 (SEQ ID NO.1) and ergothionein synthase Ncegt2 (SEQ ID NO.2).

[0005] In one embodiment, the recombinant Escherichia coli uses the pBAD plasmid as an expression vector.

[0006] In one embodiment, the gene sequence encoding ergothioneine synthase Ccegt1 is shown in SEQ ID NO.1; and the gene sequence encoding ergothioneine synthase Ncegt2 is shown in SEQ ID NO.2.

[0007] The present invention also provides a biocatalyst that can be used to catalyze the production of ergothioneine, said biocatalyst being (a) or (b):

[0008] (a) A cell catalyst containing the recombinant Escherichia coli;

[0009] (b) Cell lysate of the recombinant Escherichia coli.

[0010] This invention also provides the application of ergothioneine synthase Ccegt1 and ergothioneine synthase Ncegt2 in the preparation of ergothioneine.

[0011] In one embodiment, the application uses the ergothioneine synthases Ccegt1 and Ncegt2 as catalysts, and herbicides and L-cysteine ​​as substrates, to convert and produce ergothioneine.

[0012] In one embodiment, the gene sequence encoding the ergothionein synthase Ccegt1 is shown in SEQ ID NO.1.

[0013] In one embodiment, the gene sequence encoding the ergothionein synthase Ncegt2 is shown in SEQ ID NO.2.

[0014] The present invention also provides a method for preparing ergothionein, wherein ergothionein is produced in a reaction system containing hexamethasone, L-cysteine, tris(2-carboxyethyl) phosphate hydrochloride (TCEP), pyridoxal phosphate (PLP), dithiothreitol (DTT) and ascorbic acid, under the catalysis of ergothionein synthase Ccegt1 and ergothionein synthase Ncegt2.

[0015] In one embodiment, the reaction is carried out at 30–35°C.

[0016] In one embodiment, the pH of the reaction system is 7.0 to 7.5.

[0017] In one embodiment, the reaction system is established in Tris-acetic acid buffer.

[0018] In one embodiment, the reaction system contains (by final concentration): 1–4 mM tris(2-carboxyethyl)phosphohydrochloride, 2–10 mM dithiothreitol, 1–3 mM ascorbic acid, 0.8–2 mM pyridoxal phosphate, 0.2 mM ferrous sulfate heptahydrate, 15 g / L hexamethasone, and 1.58–4.5 g / L cysteine.

[0019] In one embodiment, 1.58 g / L of cysteine ​​is added every half hour during the reaction.

[0020] In one implementation, the reaction lasts for at least 10 hours.

[0021] The present invention also provides the use of the recombinant Escherichia coli or the method thereon in the preparation of ergothioneine or ergothioneine-containing products.

[0022] Beneficial effects:

[0023] 1. This invention uses a bioenzymatic method to synthesize ergothioneine. Using HER and cysteine ​​as substrates, ergothioneine is synthesized efficiently and rapidly under the catalysis of ergothioneine synthase Egt1 and ergothioneine synthase Egt2. The yield of ergothioneine can reach more than 10 g / L after 8 hours of reaction. It has the advantages of high yield, simple composition and easy purification.

[0024] 2. The present invention also optimized the catalytic reaction system. Under the optimized reaction conditions (at 35°C, in Tris-acetic acid buffer at pH 7.5, 1 mM ascorbic acid, 10 mM DTT, 0.8 mM PLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate and 70 g / L HER, wherein 4.5 g / L L-cysteine ​​was added to the initial reaction system and 4.5 g / L L-cysteine ​​was added every half hour), the yield of ergothioneine can reach 41.8 g / L after 10 hours of reaction. Attached Figure Description

[0025] Figure 1 This is a roadmap for the enzymatic synthesis of ergothioneine.

[0026] Figure 2 A schematic diagram of the structure of plasmid PBAD-Ccegt1-Ncegt2 constructed in this application;

[0027] Figure 3 Determination of ergothionein content at different time points

[0028] Figure 4 The image shows the HPLC results of enzyme-catalyzed synthesis of ergothioneine in one embodiment of this application. A is 800 mg / L ergothioneine standard, and B is the sample after centrifugation of the crude enzyme solution and dilution of the supernatant by 70 times.

[0029] Figure 5 The yield of ergothioneine synthesized by different ergothioneine-producing strains. Detailed Implementation

[0030] Example 1: Construction of Escherichia coli expression vector

[0031] The database is used to screen ergothionein synthases from different species using intelligent computing software. These gene codons are then optimized for gene synthesis.

[0032] Using pBAD plasmid (purchased from Ubisoft) as a template, pBAD-F and pBAD-R primers were used to amplify the vector fragment. The synthesized Ccegt1 gene (nucleotide sequence shown in SEQ ID NO.1) was used as a template, and F2 and R2 primers were used to amplify the Ccegt1 fragment. The synthesized Ncegt2 gene (nucleotide sequence shown in SEQ ID NO.2) was used as a template, and F1 and R1 primers were used to amplify the Ncegt2 fragment. The plasmid pBAD-Ccegt1-Ncegt2 for ergothioneine production was constructed. The three fragments were ligated using the Novizan Recombinant Cloning Kit (Clone MultiS One Step Cloning Kit, (C113-02)). The ligation was performed and the fragments were transformed into competent Trans1-T1 (Phage Chemically Comptent Cell) cells using chemical transformation. Positive clones were selected, and the plasmid was extracted for sequencing.

[0033] Table 1 Primers and Sequences

[0034]

[0035] Example 2: Transformation and Induced Expression of Ergothionein Synthesis Plasmid

[0036] To prepare electrocompetent cells, 50 μL of BW25113 glycerol bacteria was inoculated into 5 mL of antibiotic-free LB medium and cultured overnight. The cells were then transferred to 100 mL of antibiotic-free LB medium at a 1% inoculation rate and cultured at 37°C until OD500 reached. 600 =0.6, pre-cool on ice for 10 min, then centrifuge the bacterial culture at 4000 rpm for 10 min, discard the supernatant, resuspend the cells in pre-cooled 10% glycerol, centrifuge at 4000 rpm for 10 min, and repeat the washing with 10% glycerol twice. Resuspend the cells in 1 mL of pre-cooled 10% glycerol and aliquot into sterile EP tubes (100 μL per tube). Take five tubes of competent cells and add 1 μL of the plasmid constructed in Example 1. Add the above system to a 2 mm electroporation cuvette, electroporate at 2.5 kV, quickly add 900 μL of antibiotic-free LB medium, resuspend, transfer to sterile EP tubes, incubate at 37°C, 200 rpm for 60 min, centrifuge at 4000 rpm for 5 min, discard part of the supernatant, resuspend, plate on LB agar containing 100 mg / L ampicillin, incubate upside down at 37°C overnight, pick single clones for PCR identification, and use the correct strains for subsequent ergothioneine synthesis. Single colonies were inoculated into 5 mL of LB broth containing Amp (100 mg / L) resistance and cultured overnight at 37°C and 220 rpm. The bacterial culture obtained in the previous step was then transferred at a 1% inoculum to 500 mL of LB broth containing Amp (100 mg / L) resistance and cultured at 37°C and 220 rpm on a shaker. 600When the OD value reaches 0.6-0.8, add arabinose to a final concentration of 2 g / L and induce at 30°C for 16 h to reach OD. 600 5. Centrifuge the fermentation broth at 4°C and 7000 rpm for 15 min, discard the supernatant, and resuspend the bacterial cells in 125 mL of 1×PBS buffer (pH 7.5) to OD500. 600 The concentration is 20. After resuspending the bacterial culture, it is sonicated and centrifuged to collect the supernatant, which is the crude enzyme solution.

[0037] Example 3: Optimization of conditions for crude enzyme synthesis of ergothionein

[0038] The crude enzyme solution obtained in Example 2 was used to catalyze the production of ergothioneine.

[0039] (1) Effects of different buffer solutions on enzyme catalysis

[0040] Bacterial cells were resuspended in four different buffers: 50 mM PBS, Tris-HCl, Tris-acetic acid, and HEPES, respectively, to adjust the OD of the bacterial suspension. 600 =20, the bacterial culture was sonicated and then centrifuged to obtain the supernatant, yielding a crude enzyme solution. Add (based on final concentration) the following to 5 mL of the crude enzyme solution: 1 mM TCEP, 2 mM DTT, 1 mM ascorbic acid, 0.8 mM PLP, 0.2 mM ferrous sulfate heptahydrate, 15 g / L HER, and cysteine ​​at an initial concentration of 1.58 g / L, to prepare a 5 mL reaction system. During the reaction, cysteine ​​was added every half hour to achieve a final concentration of 1.58 g / L. The reaction was carried out at 30°C for 150 minutes, and the formation of ergothioneine was determined.

[0041] Table 2. Effects of buffer solution on enzyme catalysis

[0042] EGT(g / L) 5.1 5.5 6 4.3

[0043] (2) Effect of different pH values ​​on enzyme catalysis

[0044] The crude enzyme solution was prepared according to the method in Example 2, except that Tris-acetic acid was used as the buffer. To 5 mL of the crude enzyme solution, the following (based on final concentrations) were added: 1 mM TCEP, 2 mM DTT, 1 mM ascorbic acid, 0.8 mM PLP, 0.2 mM ferrous sulfate heptahydrate, 15 g / L HER, and an initial concentration of 1.58 g / L cysteine, to prepare a 5 mL reaction system. During the reaction, cysteine ​​was added every half hour to achieve a final concentration of 1.58 g / L, and the pH of the Tris-acetic acid buffer was adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The reaction was carried out at 30°C for 150 minutes, and samples were taken for HPLC analysis to determine the formation of ergothioneine.

[0045] Table 3 Effect of pH on enzyme catalysis

[0046] EGT(g / L) 4.5 4.9 6 6.3 5.8

[0047] (3) Effects of different temperatures on enzyme catalysis

[0048] The crude enzyme solution was prepared according to the method in Example 2, except that Tris-acetic acid was used as the buffer. To 5 mL of the crude enzyme solution, the following were added (by final concentration): 1 mM TCEP, 2 mM DTT, 1 mM ascorbic acid, 0.8 mM PLP, 0.2 mM ferrous sulfate heptahydrate, 15 g / L HER, and an initial concentration of 1.58 g / L cysteine, to prepare a 5 mL reaction system. During the reaction, cysteine ​​was added every half hour to achieve a final concentration of 1.58 g / L, and the pH of the Tris-acetic acid buffer was adjusted to 7.5. The reaction was carried out at 25°C, 30°C, 35°C, and 40°C for 150 minutes, respectively.

[0049] Table 4 Effect of temperature on enzyme catalysis

[0050] EGT(g / L) 5 6.5 7 5.1

[0051] (4) Effects of different concentrations of PLP on enzyme catalysis

[0052] The crude enzyme solution was prepared according to the method in Example 2, except that Tris-acetic acid was used as the buffer. To 5 mL of the crude enzyme solution, the following were added (based on final concentration): 1 mM TCEP, 2 mM DTT, 1 mM ascorbic acid, 0.2 mM ferrous sulfate heptahydrate, 15 g / L HER, and an initial concentration of 1.58 g / L cysteine, to prepare a 5 mL reaction system. The concentration of PLP in the reaction system was controlled at 0.5, 1.0, 2.0, and 3.0 mM, respectively. During the reaction, cysteine ​​was added every half hour to achieve a final concentration of 1.58 g / L. The reaction was carried out at 35°C with the pH of the Tris-acetic acid buffer adjusted to 7.5 for 150 minutes.

[0053] Table 5 Effects of PLP on enzyme catalysis

[0054] EGT(g / L) 6.5 7.1 6.3 6.1

[0055] (5) Effects of different concentrations of ferrous sulfate heptahydrate on enzyme catalysis

[0056] The crude enzyme solution was prepared according to the method in Example 2, except that Tris-acetic acid was used as the buffer. To 5 mL of the crude enzyme solution, the following were added (based on final concentration): 1 mM TCEP, 2 mM DTT, 1 mM ascorbic acid, 1 mM PLP, 15 g / L HER, and an initial concentration of 1.58 g / L cysteine, to prepare a 5 mL reaction system. The concentrations of ferrous sulfate heptahydrate in the reaction system were controlled at 0.1, 0.2, 0.4, and 0.6 mM, respectively. Cysteine ​​was added every half hour during the reaction to achieve a final concentration of 1.58 g / L. The reaction was carried out at 35°C with the pH of the Tris-acetic acid buffer adjusted to 7.5 for 150 minutes.

[0057] Table 6. Effects of ferrous sulfate heptahydrate on enzyme catalysis

[0058] EGT(g / L) 7.1 7.2 6 5.5

[0059] (6) Effects of different concentrations of DTT on enzyme catalysis

[0060] The crude enzyme solution was prepared according to the method in Example 2, except that Tris-acetic acid was used as the buffer. To 5 mL of the crude enzyme solution, the following were added (based on final concentration): 1 mM TCEP, 1 mM PLP, 1 mM ascorbic acid, 0.2 mM ferrous sulfate heptahydrate, 15 g / L HER, and an initial concentration of 1.58 g / L cysteine, to prepare a 5 mL reaction system. The concentration of DTT in the reaction system was controlled at 2, 4, 6, and 8 mM, respectively. During the reaction, cysteine ​​was added every half hour to achieve a final concentration of 1.58 g / L. The reaction was carried out at 35°C with the pH of the Tris-acetic acid buffer adjusted to 7.5 for 150 minutes.

[0061] Table 7 Effects of DTT on enzyme catalysis

[0062] EGT(g / L) 7.1 7.8 7 6.7

[0063] (7) Effects of different concentrations of TCEP on enzyme catalysis

[0064] The crude enzyme solution was prepared according to the method in Example 2, except that Tris-acetic acid was used as the buffer. To 5 mL of the crude enzyme solution, the following were added (based on final concentration): 4 mM DTT, 1 mM PLP, 1 mM ascorbic acid, 0.2 mM ferrous sulfate heptahydrate, 15 g / L HER, and an initial concentration of 1.58 g / L cysteine, to prepare a 5 mL reaction system. The concentration of TCEP in the reaction system was controlled at 1, 2, 3, and 6 mM, respectively. During the reaction, cysteine ​​was added every half hour to achieve a final concentration of 1.58 g / L. The reaction was carried out at 35°C with the pH of the Tris-acetic acid buffer adjusted to 7.5 for 150 minutes.

[0065] Table 8 Effects of TCEP on enzyme catalysis

[0066] EGT(g / L) 7.9 7.5 7.4 7.3

[0067] (8) Effects of different concentrations of ascorbic acid on enzyme catalysis

[0068] The crude enzyme solution was prepared according to the method in Example 2, except that Tris-acetic acid was used as the buffer. To 5 mL of the crude enzyme solution, the following were added (by final concentration): 4 mM DTT, 1 mM PLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, 15 g / L HER, and an initial concentration of 1.58 g / L cysteine, to prepare a 5 mL reaction system. The concentration of ascorbic acid in the reaction system was controlled at 1, 2, 3, and 5 mM, respectively. Cysteine ​​was added every half hour during the reaction to achieve a final concentration of 1.58 g / L. The reaction was carried out at 35°C with the pH of the Tris-acetic acid buffer adjusted to 7.5 for 150 minutes.

[0069] Table 9. Effects of ascorbic acid on enzyme catalysis

[0070] EGT(g / L) 7.8 7.5 7.4 7.4

[0071] Example 5: Detection of co-expressed enzyme activity of ergothioneine synthase

[0072] The synthesis of ergothioneine was carried out in an optimized reaction system. The specific steps are as follows:

[0073] The bacterial cells were resuspended in 50 mM Tris acetate buffer to adjust the OD of the bacterial suspension. 600 =20, the bacterial culture was ultrasonically disrupted, then centrifuged to obtain the supernatant, yielding crude enzyme solution. Add (based on final concentration) 1 mM ascorbic acid, 4 mM DTT, 1 mM MPLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, 20 g / L HER, and an initial concentration of 1.58 g / L cysteine ​​to 5 mL of the crude enzyme solution to prepare a 5 mL reaction system. During the reaction, cysteine ​​was added every half hour to achieve a final concentration of 1.58 g / L. The reaction was carried out at 30℃, and the ergothioneine content at different time points was determined. Ergothioneine was synthesized efficiently and rapidly under the catalysis of ergothioneine synthase Egt1 and ergothioneine synthase Egt2, with a yield exceeding 10 g / L after 8 hours of reaction. Figure 3 ).

[0074] Example 6: Ergothioneine crude enzyme catalysis

[0075] The bacterial cells were resuspended in 50 mM Tris acetate buffer to adjust the OD of the bacterial suspension. 600=200, the bacterial culture was ultrasonically disrupted, then centrifuged to collect the supernatant, obtaining a crude enzyme solution. To 5 mL of the crude enzyme solution, the following were added (based on final concentration): 1 mM ascorbic acid, 10 mM DTT, 1 mM MPLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, 70 g / L HER, and an initial concentration of 4.5 g / L cysteine, to prepare a 5 mL reaction system. During the reaction, cysteine ​​was added every half hour to achieve a final concentration of 4.5 g / L. The reaction was carried out at 35°C and pH 7.5 for 10 hours. Samples were taken for HPLC analysis, and the results are as follows. Figure 4 As shown, 41.8 g / L of ergothioneine can be generated after 10 hours of reaction.

[0076] Comparative Example

[0077] Following the construction method of the ergothioneine production plasmid pBAD-Ccegt1-Ncegt2 in Example 1, plasmids pBAD-Ncegt1-Ncegt2 and pBAD-Tregt1-Ncegt2 were constructed using the two most commonly reported ergothioneine synthetic proteins from different sources: Ncegt1 (Genbank accession number: XP_956324), Ncegt2 (Genbank accession number: XP_001728131), and Tregt1 (Genbank accession number: XP_006968620). The crude enzyme catalytic activity was compared according to the method in Example 2. Plasmids pBAD-Ncegt1-Ncegt2 and pBAD-Tregt1-Ncegt2 were constructed as described in Example 1, wherein the nucleotide sequence of Ncegt1 is shown in SEQ ID NO.3; the nucleotide sequence of Ncegt2 is shown in SEQ ID NO.2; and the nucleotide sequence of Tregt1 is shown in SEQ ID NO.4.

[0078] The crude enzyme solution was prepared according to the method in Example 2, except that Tris acetate buffer was used as the buffer. 5 ml of the crude enzyme solution was added to the reaction system. The crude enzyme reaction system contained (by final concentration): 1 mM ascorbic acid, 10 mM DTT, 1 mM MPLP, 1 mM TCEP, 0.2 mM ferrous sulfate heptahydrate, 20 g / L HER, and initially added 1.58 g / L cysteine. 1.58 g / L cysteine ​​was added every half hour, and finally OD was added. 600 The crude enzyme solution obtained after cell disruption from a 200 μL bacterial suspension was reacted at 35°C. Ergothionein content was determined at different time points, and the results are shown below. Figure 5 It can be seen that the ergothioneine production plasmid pBAD-Ccegt1-Ncegt2 constructed in this application has a significantly better ability to catalyze the synthesis of ergothioneine than the other two production strains.

[0079] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant Escherichia coli, characterized in that, The ergothioneine synthase gene Ccegt1 (SEQ ID NO.1) and the ergothioneine synthase gene Ncegt2 (SEQ ID NO.2) were co-expressed.

2. The recombinant Escherichia coli according to claim 1, characterized in that, The recombinant Escherichia coli was expressed using the pBAD plasmid as an expression vector.

3. A method for preparing ergothionein synthase, characterized in that, The recombinant Escherichia coli according to claim 1 or 2 is cultured in a culture medium for a period of time, the bacterial cells are collected, and the bacterial cells are broken to obtain crude ergothionein synthase solution.

4. A biocatalyst that can be used to catalyze the production of ergothioneine, characterized in that, The biocatalyst is (a) or (b): (a) A cell catalyst containing the recombinant Escherichia coli of claim 1 or 2; (b) Cell lysate of recombinant Escherichia coli as described in claim 1 or 2, or crude ergothionein synthase solution prepared by the method described in claim 3.

5. A method for preparing ergothioneine, characterized in that, Ergothioneine was produced by conversion using ergothioneine synthases Ccegt1 and Ncegt2 as catalysts and histidine betaine and L-cysteine ​​as substrates. The gene sequence encoding ergothioneine synthase Ccegt1 is shown in SEQ ID NO.1; the gene sequence encoding ergothioneine synthase Ncegt2 is shown in SEQ ID NO.

2.

6. The method according to claim 5, characterized in that, Ergothionein is produced in a reaction system containing histidine betaine, L-cysteine, tris(2-carboxyethyl) phosphate hydrochloride, pyridoxal phosphate, dithiothreitol, and ascorbic acid, catalyzed by ergothionein synthases Ccegt1 and Ncegt2.

7. The method according to claim 5 or 6, characterized in that, The method involves a reaction at 30-35°C with a pH of 7.0-7.

5.

8. The method according to claim 5 or 6, characterized in that, L-cysteine ​​was added every half hour during the reaction.

9. The method according to claim 7, characterized in that, L-cysteine ​​was added every half hour during the reaction.

10. The use of the recombinant Escherichia coli of claim 1 or 2, or the biocatalyst of claim 4, or the method of any one of claims 5 to 9 in the preparation of ergothioneine or ergothioneine-containing products.

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