Recombinant escherichia coli with high yield of ergothioneine and method for preparing ergothioneine by using recombinant escherichia coli

Abstract

The invention provides recombinant escherichia coli with high yield of ergothioneine and a method for preparing ergothioneine, the recombinant escherichia coli co-expresses ergothioneine anabolic enzymes Egt1 and Egt2, the methyltransferase structural domain of the Egt1 is derived from trichoderma reesei, mycobacterium smegmatis EgtD or rhizopus nigricans, and the methyltransferase structural domain of the Egt2 is derived from the trichoderma reesei, the mycobacterium smegmatis EgtD or the rhizopus nigricans. The histidine sulfoxide synthase structural domain is derived from trichoderma reesei, schizosaccharomyces pombe or rhizopus nigricans, and the Egt2 is derived from claviceps purpurea, neurospora crassa or aspergillus niger. After the recombinant escherichia coli obtained by screening is cultured for 68 hours in a 5L fermentation tank, the yield of ergothioneine can reach 5g / L or above; wherein after recombinant escherichia coli co-expressing Egt1 enzyme (the structural domain of methyltransferase is derived from mycobacterium smegmatis EgtD, and the structural domain of histidine sulfoxide synthase is derived from trichoderma reesei) and Egt2 enzyme (derived from neurospora crassa) are cultured in a 5L fermentation tank for 68 hours, the yield of ergothioneine can reach 8.7 g / L, and the production intensity reaches 127.9 mg / (L.h).

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a recombinant Escherichia coli with high yield of ergothioneine and a method for preparing ergothioneine therefrom. Background Art

[0002] Ergothioneine is a sulfur-containing antioxidant with a long half-life in vivo, and has obvious advantages over glutathione and vitamin C with relatively short half-lives currently on the market. At present, ergothioneine is mainly applied in high-end cosmetics. Due to the low production yield of ergothioneine in the current industry and the high price (100,000 - 200,000 yuan / kg), the scale of use of ergothioneine is greatly restricted.

[0003] In recent years in the industry, the production and expression of ergothioneine in chassis microorganisms such as Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum have been achieved through synthetic biology tools. The ergothioneine synthesis pathway is as follows:

[0004]

[0005] Currently, the heterologous expression and production of ergothioneine can be achieved by cloning the enzyme cluster related to ergothioneine synthesis metabolism into different chassis microorganisms. For example, Naoyuki Tanaka et al. from the University of Tsukuba in Japan used egtABCDE from Mycobacterium smegmatis as the synthetic gene cluster to express ergothioneine in Escherichia coli BW25113 strain, and the production yield in the fermenter was 1.3 g / L [Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli]. Zhihui Chen et al. from the Institute of Microbiology, Chinese Academy of Sciences used Egt1 and Egt2 from Trichoderma reesei as the synthetic gene cluster to express ergothioneine with Escherichia coli as the chassis microorganism, and the expression level of ergothioneine was 4.34 g / L when fermented for 143 h in a 2L fermenter [Toward more efficient ergothioneine production using the fungal ergothioneine biosynthetic pathway]. Patent application CN201910789954.8 discloses the recombinant expression of the ergothioneine synthesis gene cluster from the mushroom Grifola frondosa in Saccharomyces cerevisiae, and the production yield of ergothioneine at the shake flask level is 2.5 mg / L.

[0006] Some researchers have also tried to optimize and combine the enzyme clusters related to ergothioneine anabolism, and attempt to combine synthetic gene clusters from different sources in order to obtain a higher-yielding engineered strain. Wang Li et al. from Jiangnan University used egtABCDE from Mycobacterium smegmatis and Egt1 from Schizosaccharomyces pombe as enzyme clusters related to ergothioneine anabolism and expressed them in Escherichia coli. The highest yield in a 3L fermenter was 710.53 mg / L [Construction and Optimization of an Escherichia coli Engineering Strain Producing Ergothioneine]. Patent application CN113993989A screened and combined ergothioneine synthase clusters from various molds, and finally found that the engineered strain with the metabolic synthase combination of NcEgt1 (Neurospora crassa ergothioneine synthase 1) and CpEgt2 (Claviceps purpurea ergothioneine synthase 2) had the strongest ability to synthesize ergothioneine, and the ergothioneine yield was 1.1 g / L during simulated fed-batch operation.

[0007] There is still room for improvement in the ergothioneine yield disclosed in the existing literature. Providing an engineered strain or production process with high ergothioneine yield is of great significance for the promotion and application of ergothioneine. Summary of the Invention

[0008] Due to the simple metabolic pathway of the ergot thioneine synthetic and metabolic enzyme system (Egt1, Egt2) derived from molds, more and more researchers have chosen the ergot thioneine metabolic enzyme system derived from molds for the biosynthesis of ergot thioneine. The ergot thioneine synthetic and metabolic enzyme Egt1 is composed of two domains, a methyltransferase (domain 1) and a histidine sulfoxide synthase (domain 2). Domain 1 transfers the methyl group on S-adenosylmethionine (SAM) to the α-amino group of L-histidine to generate L-histidine betaine (HER). Domain 2 catalyzes the addition reaction of cysteine and L-histidine betaine to generate the intermediate Hercynylcysteine sulfoxide (histidine trimethylbetaine cysteine sulfoxide). Subsequently, Egt2 enzyme catalyzes Hercynylcysteine sulfoxide to generate ergot thioneine. The inventors of the present invention found during the experiment that the methyltransferase activity and histidine sulfoxide synthase catalytic efficiency of Egt1 enzymes from different molds are different, resulting in the formation of L-histidine betaine impurities (N,N-dimethyl-L-histidine) by some molds-derived Egt1 during the biosynthesis of ergot thioneine. The physicochemical properties of this impurity are similar to those of ergot thioneine, making it difficult to separate during subsequent purification processes and affecting the subsequent purification of ergot thioneine. The inventors optimized the combination of domains of Egt1 metabolic enzymes from different molds and finally obtained multiple Egt1 enzymes with optimal metabolic enzyme domain combinations. This optimized combination can significantly reduce L-histidine betaine impurities. At the same time, the inventors unexpectedly found that the optimized novel metabolic enzyme Egt1 and the synthetic and metabolic enzyme cluster of wild-type Egt2 can rapidly synthesize ergot thioneine in Escherichia coli engineering strains, with high fermentation expression production intensity, and the ergot thioneine yield obtained by fermenting the recombinant Escherichia coli strain is significantly higher than the currently reported yields.

[0009] The primary object of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide a recombinant Escherichia coli with high ergot thioneine production.

[0010] Another object of the present invention is to provide a method for constructing the above recombinant Escherichia coli.

[0011] Another object of the present invention is to provide a method for preparing ergot thioneine using the above recombinant Escherichia coli.

[0012] To achieve the above invention objects, the present invention provides the following technical solutions:

[0013] The present invention provides a recombinant Escherichia coli with high ergot thioneine production, which co-expresses:

[0014] a) An Egt1 enzyme capable of converting L-histidine into histidine trimethylbetaine cysteine sulfoxide;

[0015] b) The Egt2 enzyme capable of converting histidine trimethylsulfonium cysteine sulfoxide into ergothioneine;

[0016] The methyltransferase domain of the Egt1 enzyme is derived from Trichoderma reesei, Mycobacterium smegmatis EgtD or Rhizopus nigricans, and the histidine sulfoxide synthase domain is derived from Trichoderma reesei, Schizosaccharomyces pombe or Rhizopus nigricans;

[0017] The Egt2 enzyme is derived from Claviceps purpurea, Neurospora crassa or Aspergillus niger.

[0018] In the present invention, as one of the embodiments, the methyltransferase domain and the histidine sulfoxide synthase domain are linked by a methyltransferase domain linker or a histidine sulfoxide synthase domain linker, preferably the histidine sulfoxide synthase domain linker.

[0019] In the present invention, as one of the embodiments, the amino acid sequence of the Egt1 enzyme is as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10; the amino acid sequence of the Egt2 enzyme is as shown in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:15.

[0020] In the present invention, as one of the embodiments, the amino acid sequences of the Egt1 enzyme and the Egt2 enzyme are selected from any one of the following combinations: SEQ ID NO:1 and SEQ ID NO:13, SEQ ID NO:3 and SEQ ID NO:13, SEQ ID NO:10 and SEQ ID NO:13, SEQ ID NO:1 and SEQ ID NO:14, SEQ ID NO:3 and SEQ ID NO:14, SEQ ID NO:6 and SEQ ID NO:14, SEQ ID NO:7 and SEQ ID NO:14, SEQ ID NO:9 and SEQ ID NO:14, SEQ ID NO:3 and SEQ ID NO:15, SEQ ID NO:6 and SEQ ID NO:15, SEQ ID NO:7 and SEQ ID NO:15; preferably SEQ ID NO:1 and SEQ ID NO:13, SEQ ID NO:3 and SEQ ID NO:13, SEQ ID NO:10 and SEQ ID NO:13, SEQ ID NO:1 and SEQ ID NO:14, SEQ ID NO:3 and SEQ ID NO:14.

[0021] In the present invention, as one of the embodiments, the Escherichia coli includes Escherichia coli MG1655, Escherichia coli W3110, Escherichia coli DH5α, Escherichia coli JM109, Escherichia coli BW25113 or Escherichia coli BL21(DE3); preferably Escherichia coli BL21(DE3).

[0022] The present invention also provides a method for constructing the aforementioned recombinant Escherichia coli, and the method includes:

[0023] Using a vector plasmid containing an Escherichia coli autonomous replicon, a protein expression element, and a resistance gene as the vector plasmid, recombining the nucleotide sequences encoding Egt1 enzyme and Egt2 enzyme into the vector plasmid, and transforming the recombinant vector plasmid into Escherichia coli;

[0024] The vector plasmids include pET28a, pET31b, pET9a, pET3a, pACYCduet-1.

[0025] As one of the embodiments, the Escherichia coli autonomous replicon is selected from at least one of pMB1, pBR322, CoIE1, R6K, P15A or pSC101, the protein expression element includes a promoter and a terminator, and the resistance gene is at least one of an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, a bleomycin resistance gene, a tetracycline resistance gene.

[0026] As one of the embodiments, the promoter is selected from at least one of a T7 promoter, a Lac promoter, a Trp promoter, a T7lac promoter, an araBAD promoter, a pTac promoter, a pL promoter;

[0027] The terminator is selected from at least one of a T7 terminator, an rrnB terminator, a T0 terminator.

[0028] It should be particularly noted that according to the amino acid sequence provided by the present invention, those skilled in the art can easily obtain the corresponding nucleotide sequence and optimize the nucleotide sequence according to the codon preference of Escherichia coli.

[0029] The present invention also provides a method for preparing ergothioneine, and the method includes: inoculating the aforementioned recombinant Escherichia coli into a culture medium for fermentation culture to obtain ergothioneine.

[0030] In the method of the present invention, as one of the embodiments, the method further includes:

[0031] (1) Taking the recombinant Escherichia coli seed solution and inoculating it into an LB medium for culture to prepare a primary seed solution and a secondary seed solution;

[0032] (2) When the OD600 of the secondary seed culture reaches 0.8 - 3.0, take the secondary seed culture and inoculate it into a fermenter containing the basal medium, and culture it at 28 - 37 °C, pH 6.0 - 7.5, with the dissolved oxygen controlled at 20% - 40%. Start feeding when the dissolved oxygen jumps.

[0033] (3) When the culture reaches an OD600 of 60 - 100, add IPTG with a final concentration of 0.05 mM - 0.5 mM for induction, and feed the amino acid solution during the induction stage.

[0034] In the method of the present invention, as one of the embodiments, step (3) further includes: controlling the dissolved oxygen at 20% - 40% during induction.

[0035] In the method of the present invention, as one of the embodiments, the basal medium includes organic nitrogen sources, carbon sources, inorganic salts, and trace elements; the feed includes organic nitrogen sources, carbon sources, and trace elements; the amino acid solution includes histidine, methionine, and cysteine.

[0036] In the method of the present invention, as one of the embodiments,

[0037] The organic nitrogen source includes one or more of yeast extract, yeast powder, and tryptone;

[0038] The carbon source includes glycerol or glucose;

[0039] The inorganic salts include sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate, magnesium sulfate heptahydrate, and ammonium sulfate;

[0040] The trace elements include copper sulfate pentahydrate, manganese sulfate monohydrate, sodium molybdate dihydrate, boric acid, zinc sulfate heptahydrate, ferrous sulfate heptahydrate, and cobalt chloride hexahydrate.

[0041] In the method of the present invention, as one of the preferred embodiments, the final concentration of IPTG in step (3) is 0.3 mM, and the contents of histidine, methionine, and cysteine in the amino acid solution are 20 - 40 g / L respectively.

[0042] The technical effects achieved by the present invention are as follows:

[0043] 1. By combining and optimizing the domains of Egt1 metabolic enzymes from different sources and screening them in combination with wild-type Egt2 metabolic enzymes from different mold sources, a recombinant Escherichia coli engineering strain with high expression of ergothioneine metabolic enzymes was finally obtained. The ergothioneine yield of this engineering strain can reach more than 5 g / L after 68 h of cultivation at the fermentor level. Among them, the combination of Mycobacterium smegmatis EgtD and Trichoderma reesei domain 2 was used as the Egt1 enzyme, the Egt2 enzyme was derived from Neurospora crassa, and Escherichia coli BL21(DE3) was used as the chassis microorganism. The ergothioneine yield can reach 8.7 g / L after 68 h of cultivation at the 5 L fermentor level, and the production intensity reaches 127.9 mg / (L·h).

[0044] 2. The L-histidine betaine impurity (N,N-dimethyl-L-histidine) generated during the expression of the recombinant Escherichia coli with high-yield ergothioneine screened in the present invention accounts for a relatively low proportion, which is beneficial to the subsequent separation and purification of ergothioneine. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Plasmid map of recombinant plasmid pET28a-(Mycobacterium smegmatis EgtD + Trichoderma reesei)Egt1-Neurospora crassa Egt2;

[0046] Figure 2 : Liquid chromatogram of ergothioneine. DETAILED DESCRIPTION OF THE INVENTION

[0047] The following will describe the embodiments of the present invention in detail in conjunction with the examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0048] Example 1 Construction of Recombinant Plasmid

[0049] Select the Trichoderma reesei domain 1 from fungi, the domain 1 from Rhizopus nigricans, the domain 1 from Neurospora crassa, and the EgtD of Mycobacterium smegmatis from bacteria as the methyltransferase domain, select the Trichoderma reesei domain 2, the Rhizopus nigricans domain 2, the Neurospora crassa domain 2, and the Schizosaccharomyces pombe domain 2 as the histidine sulfoxide synthase domain, and use the histidine sulfoxide synthase linker to connect the methyltransferase domain and the histidine sulfoxide synthase domain as the Egt1 enzyme; select the Egt2 enzymes from Claviceps purpurea, Neurospora crassa, and Aspergillus niger as the C-S lyase. The combinations of Egt1 enzymes and Egt2 enzymes from different sources are shown in Table 1, where Rhizopus nigricans Egt1 + Rhizopus nigricans Egt2, Colletotrichum gloeosporioides Egt1 + Colletotrichum gloeosporioides Egt2, and Trichoderma reesei Egt1 + Trichoderma reesei Egt2 are used as controls. The nucleotide sequences are reverse-translated according to the amino acid sequences in Table 1, and the optimized nucleotide sequences are obtained according to codon degeneracy and the codon preference of Escherichia coli. The restriction enzyme site XhoI is added to the 5' end of the nucleotide sequence, and the restriction enzyme site SgrAI is added to the 3' end. The optimized nucleotide sequences are synthesized by GenScript and digested with XhoI and SgrAI and then constructed into the pET28a vector digested with the same enzymes. Using pET28a as the vector plasmid, the promoter Tac is used as the Egt1 enzyme promoter, the T7 terminator is used as the Egt1 enzyme terminator, the promoter araBAD is used as the Egt2 promoter, and the T7 terminator is used as the Egt2 enzyme terminator. Taking the Egt1 enzyme with the methyltransferase domain from Mycobacterium smegmatis EgtD and the histidine sulfoxide synthase domain from Trichoderma reesei and the Egt2 enzyme from Neurospora crassa as an example, the plasmid map is as Figure 1 shown.

[0050] Table 1 Combinations of Egt1 enzymes and Egt2 enzymes from different sources

[0051]

[0052]

[0053]

[0054] Example 2 Preparation of Escherichia coli recombinant expression strains

[0055] Use the CaCl2 chemical transformation method to transform the constructed recombinant plasmid into Escherichia coli BL21(DE3) strain, dilute and spread it on the LB plate containing Kan, and culture it overnight at 37°C. Randomly pick monoclonal colonies from the plate, inoculate them into the LB+Kan (50 μg / mL) liquid medium, and culture them at 37°C and 220 rpm for 3 - 6 hours and then preserve the strains.

[0056] Example 3 Flask screening of recombinant expression strains

[0057] The ergothioneine seeds stored in an ultra-low temperature refrigerator were thawed on ice surface. After that, the seed liquid was transferred into LB liquid medium and cultured at 37 °C and 220 rpm. Induction started when OD=0.6-2.0. The final concentration of IPTG was 0.5 mM, the final concentration of histidine was 4 g / L, the final concentration of methionine was 2 g / L, the final concentration of cysteine was 3 g / L, and the final concentration of FeCl2 was 2 mg / L. After 24 hours of induction, 4 mL of the culture broth was centrifuged at 10000 rpm for 10 min, and the fermentation broth was collected for ergothioneine content detection (the detection method is shown in Example 5). The liquid chromatogram of ergothioneine is shown in Figure 2 , there was an impurity peak in front of the main peak of ergothioneine in the liquid chromatogram of the shake flask sample. This impurity was N,N-dimethyl-L-histidine. The shake flask yield of ergothioneine and the proportion of impurities in the shake flask sample are shown in Table 2.

[0058] Table 2 Shake flask expression results of recombinant expression strains

[0059]

[0060]

[0061] Example 4 Fermentation expression of recombinant expression strain in 5 L fermentor

[0062] Engineering strains with higher ergothioneine yield at the shake flask level were selected for small-scale fermentation test.

[0063] The stored glycerol cryopreserved bacteria were thawed on ice surface. After that, the seed liquid was transferred to 50 ml of LB medium and cultured at 37 °C and 220 rpm for 2-3 hours.

[0064] 1 ml of the primary seed liquid was transferred to 100 ml of LB medium and cultured at 37 °C and 220 rpm for 2-3 h. When OD600 reached 0.8-3.0, inoculation into the fermentor could be prepared, and the inoculation amount of the fermentor was 5%.

[0065] Take 95 ml of the secondary seed liquid and inoculate it into a 5 L fermenter containing 2.2 L of the basal medium. Cultivate it at 37 °C, pH 7.0, and dissolved oxygen of 40%. Start feeding when the dissolved oxygen jumps to 60%, and automatically control the feeding rate based on the dissolved oxygen feedback. When the culture reaches an OD600 of about 80, lower the fermentation temperature to 30 °C, and add IPTG with a final concentration of 0.3 mM for induction. During the induction stage, control the dissolved oxygen at 20% - 40%, and automatically control the feeding rate based on the dissolved oxygen feedback. Start a constant-rate feeding of the amino acid solution at a feeding rate of 68 ml / h during the induction stage. End the cultivation after 68 h of fermentation in the fermenter. Take 1 ml of the fermentation broth and centrifuge it at 10000 rpm for 10 min, collect the fermentation broth for ergothioneine content detection, and the detection method is shown in Example 5. The composition of the medium components is shown in Table 3.

[0066] Table 3 Composition of the medium components

[0067]

[0068] The yields of the recombinant expression strains in a 5 L fermenter are shown in Table 4, where the ergothioneine metabolic enzyme combinations of Rhizopus nigricans Egt1 and Rhizopus nigricans Egt2, Colletotrichum gloeosporioides Egt1 and Colletotrichum gloeosporioides Egt2, Trichoderma reesei Egt1 + Trichoderma reesei Egt2 are used as the control group. As can be seen from Table 4, the recombinant Escherichia coli expressing Egt1 enzymes and Egt2 enzymes from different sources screened in the present invention can produce high yields of ergothioneine. The ergothioneine yield in a 5 L fermenter can reach more than 5 g / L. Among them, using Mycobacterium smegmatis EgtD and the domain 2 of Trichoderma reesei as the Egt1 enzyme, the Egt2 enzyme is derived from Neurospora crassa, and using Escherichia coli BL21(DE3) as the chassis microorganism, the ergothioneine yield after 68 h of cultivation at the 5 L fermenter level can reach 8.7 g / L, and the production intensity reaches 127.9 mg / (L·h).

[0069] Table 4 Yields of the recombinant expression strains in a 5 L fermenter

[0070]

[0071]

[0072] Example 5 Method for detecting ergothioneine content

[0073] Detection by HPLC method: Use an InfimityⅡ1260 type high-performance liquid detection system, C18 column (Welch AQ-C18, 4.6×250 mm, 5 μm). Mobile phase: Phase A: phosphoric acid / water = 1 / 999, Phase B: acetonitrile / water = 95 / 5, column temperature 35 °C, flow rate 1 ml / min, ultraviolet absorption 254 nm / 210 nm, injection volume: 5 - 10 μl, detection duration 28 min.

[0074] Ergothioneine concentration (mg / ml) = ergothioneine RF (mean) × ergothioneine peak area of ​​the test sample × dilution factor

[0075] Ergothioneine RF value = ergothioneine standard concentration per unit volume / ergothioneine peak area

[0076] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the principles and essence of the present invention, and these modifications and improvements are also considered as protections of the present invention.

Claims

1. A recombinant Escherichia coli with high yield of ergothioneine, characterized in that: The recombinant Escherichia coli co-expresses: a) Egt1 enzyme capable of converting L-histidine into histidine trimethylsulfonium cysteine sulfoxide; b) Egt2 enzyme capable of converting histidine trimethylsulfonium cysteine sulfoxide into ergothioneine; The methyltransferase domain of the Egt1 enzyme is derived from Trichoderma reesei, Mycobacterium smegmatis EgtD or Rhizopus nigricans, and the histidine sulfoxide synthase domain is derived from Trichoderma reesei, Schizosaccharomyces pombe or Rhizopus nigricans; The Egt2 enzyme is derived from Claviceps purpurea, Neurospora crassa or Aspergillus niger.

2. The recombinant Escherichia coli according to claim 1, characterized in that: The methyltransferase domain and the histidine sulfoxide synthase domain are linked by a methyltransferase domain linker or a histidine sulfoxide synthase domain linker, preferably the histidine sulfoxide synthase domain linker.

3. The recombinant Escherichia coli according to claim 2, characterized in that: The amino acid sequence of the Egt1 enzyme is as shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:10; The amino acid sequence of the Egt2 enzyme is as shown in SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO:

15.

4. The recombinant Escherichia coli according to claim 3, characterized in that: The amino acid sequences of the Egt1 enzyme and the Egt2 enzyme are selected from any one of the following combinations: SEQ ID NO:1 and SEQ ID NO:13, SEQ ID NO:3 and SEQ ID NO:13, SEQ ID NO:10 and SEQ ID NO:13, SEQ ID NO:1 and SEQ ID NO:14, SEQ ID NO:3 and SEQ ID NO:14, SEQ ID NO:6 and SEQ ID NO:14, SEQ ID NO:7 and SEQ ID NO:14, SEQ ID NO:9 and SEQ ID NO:14, SEQ ID NO:3 and SEQ ID NO:15, SEQ ID NO:6 and SEQ ID NO:15, SEQ ID NO:7 and SEQ ID NO:

15.

5. The recombinant Escherichia coli according to claim 1, characterized in that: The Escherichia coli includes Escherichia coli MG1655, Escherichia coli DH5α, Escherichia coli W3110, Escherichia coli JM109, Escherichia coli BW25113 or Escherichia coli BL21(DE3); preferably Escherichia coli BL21(DE3).

6. A method for constructing the recombinant Escherichia coli according to any one of claims 1-5, characterized in that: Using a plasmid containing an Escherichia coli autonomous replicon, a protein expression element and a resistance gene as a vector plasmid, recombining the nucleotide sequences encoding the Egt1 enzyme and the Egt2 enzyme into the vector plasmid, and transforming the recombinant vector plasmid into Escherichia coli; The vector plasmid is selected from pET28a, pET31b, pET9a, pET3a or pACYCduet-1.

7. A method for preparing ergothioneine, characterized in that: Inoculating the recombinant Escherichia coli according to any one of claims 1-5 into a medium for fermentation culture to obtain ergothioneine.

8. The method according to claim 7, characterized in that, The method includes: (1) Taking the recombinant Escherichia coli seed liquid and inoculating it into an LB medium for culture to prepare a primary seed liquid and a secondary seed liquid; (2) When the OD600 of the secondary seed culture reaches 0.8 - 3.0, take the secondary seed culture and inoculate it into a fermenter containing the basal medium, and culture it at 28 - 37 °C, pH 6.0 - 7.5, with the dissolved oxygen controlled at 20% - 40%. Start feeding when the dissolved oxygen jumps. (3) When the culture reaches an OD600 of 60 - 100, add IPTG with a final concentration of 0.05 mM - 0.5 mM for induction, and feed the amino acid solution during the induction stage.

9. The method according to claim 8, characterized in that: The basal medium includes organic nitrogen sources, carbon sources, inorganic salts, and trace elements; The feed includes organic nitrogen sources, carbon sources, and trace elements; The amino acid solution includes histidine, methionine, and cysteine.

10. The method according to claim 9, characterized in that: The organic nitrogen source includes one or more of yeast extract, yeast powder, and tryptone; The carbon source includes glycerol or glucose; The inorganic salts include sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate, magnesium sulfate heptahydrate, and ammonium sulfate; The trace elements include copper sulfate pentahydrate, manganese sulfate monohydrate, sodium molybdate dihydrate, boric acid, zinc sulfate heptahydrate, ferrous sulfate heptahydrate, and cobalt chloride hexahydrate.

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