Microorganism for efficiently producing ergothioneine as well as preparation method and application thereof

Abstract

The invention belongs to the field of industrial application of microbial fermentation, and relates to a microorganism for efficiently producing ergothioneine as well as a preparation method and application of the microorganism. Specifically, the method comprises the following steps: firstly, artificially creating a promoter with a group response property by utilizing saturation mutation and screening of a fluorescence microscope; then, the promoter is used for mediating host bacterium heterologous histidine methyltransferase, sulfoxide synthase and PLP dependent C-S lyase to improve the yield of ergothioneine; on the basis, an endogenous promoter HisJ of host bacteria is replaced, and endogenous cysteine lyase yhaM is knocked out, so that the yield of the ergothioneine is increased again. According to combination of the above strategies, a heuristic and commercialized production thought is provided for efficient synthesis of ergothioneine, and a foundation is laid for reduction of the synthesis cost of ergothioneine.

Description

Technical Field

[0001] The invention belongs to the field of industrial application of microbial fermentation and relates to a microorganism for efficiently producing ergothioneine and a preparation method and application thereof. Background Art

[0002] Ergothioneine was first discovered in 1909 by German chemist David RML Schardinger in Claviceps purpurea. At the time, he identified this sulfur-containing compound and thought it was some kind of "ergotin". However, it was not until the 1980s that scientists realized that it had important physiological effects in organisms. In 2005, studies found that ergothioneine has antioxidant, anti-inflammatory effects, and plays a protective role in many cells and tissues, especially in the nervous system, eyes, and liver. The original source of ergothioneine was a natural product, usually obtained by extracting ergot or certain other fungi. However, with the growth of demand and the demand for more economical and sustainable production methods, commercial production methods have changed significantly. Early commercial production methods mainly relied on extracting ergothioneine from ergot or other natural sources. This process is not only costly, but also limited by the source of raw materials. With the advancement of biotechnology, more and more companies have begun to use fermentation to produce ergothioneine. By using specific microorganisms (such as certain bacteria or fungi) for fermentation, higher yields can be obtained in large-scale production. The fermentation method can not only reduce production costs, but also improve the controllability of production and avoid fluctuations in the supply of natural raw materials. With the rapid development of synthetic biology and genetic engineering technology, some companies have begun to modify microorganisms through genetic engineering to enable them to efficiently synthesize ergothioneine during the fermentation process. This technology can further improve production efficiency and make the production process more environmentally friendly.

[0003] Currently, the synthesis of ergothioneine by recombinant Escherichia coli has become the mainstream method for commercial production of ergothioneine. However, due to the high toxicity of cysteine ​​in the substrate to Escherichia coli and the inability of other amino acids to be better utilized by Escherichia coli, the yield of ergothioneine has been difficult to obtain good commercial production in large-scale fermentation tanks. Summary of the invention

[0004] In order to improve the efficiency of preparing ergothioneine by microbial fermentation and improve the output of industrial fermentation level, the present invention provides the following technical scheme:

[0005] The first aspect of the present invention is to provide a microorganism for efficiently producing ergothioneine, characterized in that the microorganism is a genetically engineered Escherichia coli, and the Escherichia coli expresses histidine methyltransferase EgtD, sulfoxide synthase Egt1 from Trichoderma reesei, and PLP-dependent C-S lyase EgtE from Mycobacterium;

[0006] Further, the Escherichia coli uses promoter Pfic to mediate the expression of EgtD, Egt1 and EgtE; preferably, the nucleotide sequence of Pfic is shown as SEQ ID NO:7;

[0007] Further, the amino acid sequence of EgtD is as shown in SEQ ID NO:1;

[0008] Further, the amino acid sequence of Egt1 is as shown in SEQ ID NO:2;

[0009] Further, the amino acid sequence of EgtE is as shown in SEQ ID NO:3;

[0010] Further, the coding sequences of EgtD, Egt1 and EgtE are codon-optimized to adapt to the transcription and translation of Escherichia coli; preferably, the optimized coding sequence of EgtD is as shown in SEQ ID NO:4; preferably, the optimized coding sequence of Egt1 is as shown in SEQ ID NO:5; preferably, the optimized coding sequence of EgtE is as shown in SEQ ID NO:6.

[0011] Further, the Escherichia coli is Escherichia coli K12 MG1655 strain;

[0012] Further, the endogenous promoter HisJ of the Escherichia coli is replaced by promoter Ptac; preferably, the nucleotide sequence of Ptac is shown as SEQ ID NO:12.

[0013] Further, the endogenous gene yhaM of the Escherichia coli is inactivated, preferably, the inactivation is by means of CRISPR gene editing; preferably, the

[0014] Further, the promoter Pfic is a highly efficient promoter after saturation mutation screening, and the mutated promoters are Pfic-ECT1, Pfic-ECT5 and Pfic-ECT14 respectively, and their nucleic acid sequences are SEQ ID NO:8-10.

[0015] Further, the combination mode of the enzymes mediated by the promoter is one of the following:

[0016] Pfic-ECT1_EgtD_Egt1_EgtE,

[0017] Pfic-ECT5_EgtD_Egt1_EgtE,

[0018] Pfic-ECT14_EgtD_Egt1_EgtE,

[0019] Pfic-ECT1_EgtD_Pfic-ECT5_Egt1_Pfic-ECT14_EgtE,

[0020] Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE,

[0021] Pfic-ECT5_EgtD_Pfic-ECT14_Egt1_Pfic-ECT1_EgtE, or

[0022] Pfic-ECT14_EgtD_Pfic-ECT5_Egt1_Pfic-ECT1_EgtE;

[0023] Preferably, the combination mode is Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE; its nucleotide sequence is shown as SEQ ID NO:17;

[0024] Furthermore, the expression is to construct the promoter and the coding sequence of the enzyme into a plasmid vector, and then transform the starting bacterium; preferably, the plasmid vector is pSC101; preferably, the plasmid sequence containing the combination mode of Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE is shown as SEQ ID NO:18.

[0025] The second aspect of the present invention is the application of the microorganism described in the first aspect in the preparation of ergothioneine, which is characterized in that the microorganism in the first aspect is separated to obtain ergothioneine through seed culture and fermentation culture.

[0026] Furthermore, the medium for the seed culture is: LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 25 mg / L; the conditions for the seed culture are: 35-39 °C, rotation speed 180-240 rpm, constant temperature shaking culture overnight; preferably, it is constant temperature shaking culture overnight at 37 °C with a rotation speed of 220 rpm;

[0027] Furthermore, the fermentation culture is carried out in a 5-30 L fermenter;

[0028] Preferably, the medium for fermentation culture is as follows: glucose 15 - 25 g / L, yeast extract 3 - 8 g / L, ammonium sulfate 3 - 8 g / L, dipotassium hydrogen phosphate 5 - 10 g / L, citric acid monohydrate 1 - 3 g / L, magnesium sulfate heptahydrate 0.5 - 1.5 g / L, ferrous sulfate heptahydrate 100 - 200 mg / L, manganese sulfate monohydrate 5 - 15 mg / L, vitamin B1 1 - 3 mg / L, methionine 1 - 3 g / L, trace elements 0.5 - 1.5 mL / L; wherein, the trace elements include: sodium molybdate dihydrate 2.5 g / L, nickel sulfate hexahydrate 2.5 g / L, calcium chloride dihydrate 2 g / L, copper sulfate pentahydrate 0.75 g / L, aluminum sulfate octadecahydrate 2.25 g / L, cobalt chloride hexahydrate 2.5 g / L, zinc chloride 0.5 g / L, boric acid 3 g / L; kanamycin 20 - 30 mg / L;

[0029] Preferably, the conditions for fermentation culture are aerobic fermentation for 60 - 100 hours at pH = 6.5 - 7.5 and 25 - 32°C, with dissolved oxygen controlled at 18 - 22%; more preferably, the fermentation culture adopts controlled glucose zero - sugar fermentation, feeding is started after 11 hours of fermentation, and histidine, cysteine, and methionine are fed at a uniform rate of 0.5 g / L, and the feeding medium is: 600 g / L glucose, 5 g / L histidine, 5 g / L cysteine, 5 g / L methionine; the feeding time is 25 - 35 hours.

[0030] The third aspect of the present invention is to provide a preparation method of the microorganism described in the first aspect, characterized in that the method is: constructing the encoding genes of histidine methyltransferase EgtD, sulfoxide synthase Egt1 from Trichoderma reesei, and PLP - dependent C - S lyase EgtE from Mycobacterium into a vector, transforming into Escherichia coli, and screening to obtain positive clones;

[0031] The Escherichia coli uses promoter Pfic to mediate the expression of EgtD, Egt1, and EgtE; preferably, the nucleotide sequence of Pfic is as shown in SEQ ID NO:7;

[0032] Furthermore, the amino acid sequence of EgtD is as shown in SEQ ID NO:1;

[0033] Furthermore, the amino acid sequence of Egt1 is as shown in SEQ ID NO:2;

[0034] Furthermore, the amino acid sequence of EgtE is as shown in SEQ ID NO:3;

[0035] Furthermore, the coding sequences of EgtD, Egt1, and EgtE are codon-optimized to be suitable for transcription and translation in Escherichia coli; preferably, the optimized coding sequence of EgtD is as shown in SEQ ID NO:4; preferably, the optimized coding sequence of Egt1 is as shown in SEQ ID NO:5; preferably, the optimized coding sequence of EgtE is as shown in SEQ ID NO:6;

[0036] Furthermore, the Escherichia coli is Escherichia coli K12 MG1655 strain;

[0037] Furthermore, the endogenous promoter HisJ of the Escherichia coli is replaced by the promoter Ptac; preferably, the nucleotide sequence of Ptac is as shown in SEQ ID NO:12;

[0038] Furthermore, the endogenous gene yhaM of the Escherichia coli is inactivated; preferably, the inactivation is achieved by CRISPR gene editing;

[0039] Furthermore, the promoter Pfic is a highly efficient promoter after saturation mutation screening, and the mutated promoters are Pfic-ECT1, Pfic-ECT5, and Pfic-ECT14, and their nucleic acid sequences are SEQ ID NO:8 - 10 respectively;

[0040] Furthermore, the combination mode of the promoters mediating the enzymes is one of the following:

[0041] Pfic-ECT1_EgtD_Egt1_EgtE,

[0042] Pfic-ECT5_EgtD_Egt1_EgtE,

[0043] Pfic-ECT14_EgtD_Egt1_EgtE,

[0044] Pfic-ECT1_EgtD_Pfic-ECT5_Egt1_Pfic-ECT14_EgtE,

[0045] Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE,

[0046] Pfic-ECT5_EgtD_Pfic-ECT14_Egt1_Pfic-ECT1_EgtE, or

[0047] Pfic-ECT14_EgtD_Pfic-ECT5_Egt1_Pfic-ECT1_EgtE;

[0048] Preferably, the combination mode is Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE; its nucleotide sequence is shown in SEQ ID NO:17;

[0049] Furthermore, the expression is to construct the promoter and the coding sequence of the enzyme into a plasmid vector, and then transform the starting bacterium; preferably, the plasmid vector is pSC101; preferably, the plasmid sequence containing the combination mode of Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE is shown in SEQ ID NO:18.

[0050] The beneficial effects of the present invention include:

[0051] In the present invention, by knocking out the cysteine lyase originally contained in Escherichia coli, the exogenously supplemented cysteine can only be converted into ergothioneine through ergothioneine synthase. At the same time, by overexpressing the amino acid transporter, the exogenously supplemented histidine and methionine can be better transported into Escherichia coli for the synthesis of ergothioneine. Under this background, the yield of ergothioneine is increased. Meanwhile, by using saturation mutation and screening with a fluorescence microscope, a promoter with quorum sensing properties (a derivative sequence of fic) is artificially created. This promoter can reduce the expression of ergothioneine synthase in the early stage of cell growth, allowing more energy to be used for strain growth in the early stage, and further releasing the expression ability in the later stage, enabling the strain in the stationary phase to utilize more carbon sources to convert into the target product ergothioneine. By combining the above strategies, the present invention provides inspiration and a commercial production idea for the efficient synthesis of ergothioneine, and lays a foundation for reducing the synthesis cost of ergothioneine. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Schematic diagram of the ergothioneine synthesis pathway;

[0053] Figure 2 HPLC detection peak time graph of ergothioneine standard;

[0054] Figure 3 Growth of the engineered Escherichia coli strain for ergothioneine fermentation culture;

[0055] Figure 4 Optimal fermentation yield trend graph of the engineered Escherichia coli strain for ergothioneine;

[0056] Figure 5 Schematic diagram of the exogenous expression plasmid for reference. DETAILED DESCRIPTION OF THE INVENTION

[0057] The following further elaborates on the concept and technical effects of the present invention in combination with specific embodiments to fully understand the purpose, features, and effects of the present invention. Unless otherwise specified, the methods are all conventional methods. Unless otherwise specified, the materials can all be obtained from public commercial channels. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0058] Example 1 Mutation and Screening of High-Efficiency Promoter Pfic

[0059] 1) Use the plasmid backbone Psc101 to construct a green fluorescent protein (the coding gene is shown in SED ID NO: 11) expression vector Psc101_Pfic_gfp expressed using the wild-type Pfic promoter (SEQ ID NO: 7).

[0060] 2) Perform whole-plasmid PCR amplification on the above Psc101_Pfic_gfp, and use a fusion PCR kit to integrate the above linearized plasmid into a circular plasmid, and then electrotransform it into Escherichia coli competent cell DH5α.

[0061] 3) The above primers are PrimerFicF (SEQ ID NO: 15) and the saturation mutation primer PrimerMutation1 (SEQ ID NO: 16).

[0062] 4) Subsequently, spread the Escherichia coli competent cell DH5α containing the mutant plasmid on an LB + kan plate for screening multiple monoclonal colonies, and enrich and culture them in an LB + kan liquid medium respectively. Then, use a microscope to screen different fluorescence intensities. After screening, the strains with fluorescence intensities 1.5 times / 2 times and 2.5 times that of the fluorescence intensity expressed by the original Pfic promoter are selected for promoter sequencing, and these three promoter mutants are named Pfic-ECT1, Pfic-ECT5, and Pfic-ECT14; their nucleic acid sequences are SEQ ID NOs: 8 - 10 respectively.

[0063] Example 2 Obtaining of Codon-Optimized Genes

[0064] The metabolic pathway of ergothioneine is as Figure 1 shown. According to the key enzymes for its synthesis and metabolism, the present invention codon-optimizes the proteases EgtD (WP_060849908.1), Egt1 (XP_006968620), and EgtE (WP_011731155.1) from Methylobacterium, Trichoderma reesei, and Mycobacterium in the protein database for Escherichia coli, and conducts gene synthesis sequencing through Nanjing Genscript Biotech Corporation.

[0065] Among them, EgtD is a methylobacterium histidine methyltransferase with an amino acid sequence of SEQ ID NO:1. Its codons are optimized for Escherichia coli, and its coding sequence is SEQ ID NO:4; Egt1 is a sulfoxide synthase from Trichoderma reesei with an amino acid sequence of SEQ ID NO:2. Its codons are optimized for Escherichia coli, and its coding sequence is SEQ ID NO:5; EgtE is a PLP-dependent C-S lyase from Mycobacterium with an amino acid sequence of SEQ ID NO:3. Its codons are optimized for Escherichia coli, and its coding sequence is SEQ ID NO:6.

[0066] Example 3 Combinations of Codons and Genes

[0067] 1) The combinations of promoters used in the three key genes expressing ergothioneine synthase are respectively:

[0068] Pfic-ECT1_EgtD_Egt1_EgtE,

[0069] Pfic-ECT5_EgtD_Egt1_EgtE,

[0070] Pfic-ECT14_EgtD_Egt1_EgtE,

[0071] Pfic-ECT1_EgtD_Pfic-ECT5_Egt1_Pfic-ECT14_EgtE,

[0072] Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE,

[0073] Pfic-ECT5_EgtD_Pfic-ECT14_Egt1_Pfic-ECT1_EgtE,

[0074] Pfic-ECT14_EgtD_Pfic-ECT5_Egt1_Pfic-ECT1_EgtE.

[0075] 2) Multiple permutations and combinations of different inducible promoters are respectively constructed into the pSC101 plasmid backbone and electrotransformed into Escherichia coli K12 MG1655;

[0076] Taking the construction of pSC101 plasmid containing Pfic-ECT1_EgtD_Egt1_EgtE as an example, primers were used to introduce Pfic-ECT1, and Pfic-ECT1_EgtD, Egt1 and EgtE fragments were amplified respectively. Then, the three fragments were fused using the overlap PCR principle to form a large fragment Pfic-ECT1_EgtD_Egt1_EgtE. The backbone of the pSC101 plasmid and the large fragment Pfic-ECT1_EgtD_Egt1_EgtE were then Gibson fused at 50° for 20 minutes, and then electroporated into commercial competent DH5a. The electroporation conditions were: 5 μL of Gibson transformation solution was transplanted into competent cells, ice bathed for 30 minutes, followed by heat shock at 42° for 90 seconds, and ice bathed for 2 minutes. 900 μL of LB liquid was added, and the shaking table was cultured at 37°, 250rpm for 60 minutes. Then centrifuge at 5000rpm for 3min, discard the supernatant, pipette the remaining liquid repeatedly, and transfer 100 μl to the LB plate containing Kan and culture at 37° overnight. Select a single clone for PCR verification tomorrow. If the verification is correct, extract the plasmid for later use (the schematic diagram of the expression plasmid is shown in the figure below). Figure 5 shown).

[0077] 2) The above combination was transformed into Escherichia coli K12 MG1655 competent cells, and positive clones were screened to obtain an Escherichia coli engineered strain producing ergothioneine.

[0078] 3) Seed cultivation

[0079] The fermentation seed medium was LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 25 mg / L;

[0080] The conditions for seed fermentation are: 37°C, 220 rpm constant temperature shaker overnight culture

[0081] 4) Fermentation culture

[0082] The fermentation was carried out by inoculating the culture medium with glucose as the carbon source, and fermenting was carried out for 80 hours at pH=6.5-7.5 and 30°C with aeration, and the dissolved oxygen was controlled at 20%. The fermentation of glucose was controlled to be 0 sugar, and feeding was started after 11 hours of fermentation, and histidine, cysteine ​​and methionine were fed at a uniform rate, and the feeding rate was 0.5 g / L.

[0083] The fermentation medium described above is as follows: The composition of the 5L fermentation medium is: glucose 20g / L, yeast extract 5g / L, ammonium sulfate 5g / L, dipotassium hydrogen phosphate 8g / L, citric acid monohydrate 2g / L, magnesium sulfate heptahydrate 1g / L, ferrous sulfate heptahydrate 150mg / L, manganese sulfate monohydrate 10mg / L, vitamin B1 2mg / L, methionine 2g / L, trace elements 1mL / L; among them, the trace elements include: sodium molybdate dihydrate 2.5g / L, nickel sulfate hexahydrate 2.5g / L, calcium chloride dihydrate 2g / L, copper sulfate pentahydrate 0.75g / L, aluminum sulfate octadecahydrate 2.25g / L, cobalt chloride hexahydrate 2.5g / L, zinc chloride 0.5g / L, boric acid 3g / L, kanamycin 25mg / L.

[0084] One group of the feeding medium is: 600g / L glucose, 5g / L histidine, 5g / L cysteine, 5g / L methionine.

[0085] 5) Detect the production of ergothioneine

[0086] The HPLC detection method is as follows: Agilent 1260 high performance liquid chromatograph; the chromatographic column is Agilent SB-Aq, the detection wavelength is 260nm, the mobile phase is 0.1% perchloric acid: acetonitrile = 95:5, the flow rate is 1ml / min, the column oven temperature is 40°C; the RT of ergothioneine is 3.5min.

[0087] Example 4 Replacement of the HisJ promoter of Escherichia coli

[0088] Replace the 300bp fragment upstream of HisJ with the promoter Ptac; the method is as follows:

[0089] Using fusion PCR, a fragment of 300 - 800 bp upstream of HisJ, the Ptac promoter, and a 1 - 500 bp fragment of HisJ were fused to obtain large fragment A; the plasmid pTargetF was constructed using the N20 HisJ original expression promoter sequence (SEQ ID NO:13) (reference: Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas / pTargetF system for CRISPR - Cas9 - assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin(Shanghai). 2021 Apr 15;53(5):620 - 627. doi:10.1093 / abbs / gmab036. PMID:33764372.). According to the method in the literature, pTargetF and large fragment A were co - electroporated into Escherichia coli, and then positive clones were screened using Spec and kan antibiotics. Plasmid curing was performed by reverse selection using 10 mM rhamnose and 10 g / L sucrose; a strain with the HisJ promoter replaced by the Ptac promoter was obtained; the sequence of Ptac is shown in SEQ ID NO:12.

[0090] Example 5: Knockout of yhaM in Escherichia coli

[0091] Using the method as in Example 4 and its cited literature, the native yhaM was knocked out: the 500 bp fragments upstream and downstream of yhaM were fused together to form large fragment B, and pTargetF was constructed using the N20 yhaM knockout sequence (SEQ ID NO:14), and then it was electroporated into the target host. Positive clones were screened using SPEC and KAN, and the knockout plasmid was reverse - screened using 10 mM rhamnose and 10 g / L sucrose.

[0092] Example 6: Effects of different combinations on the expression level of ergothioneine

[0093] 1) The combinations of the promoter and the target gene obtained in Example 3 were transformed into the competent cells prepared from wild - type Escherichia coli K12MG1655, the Escherichia coli obtained in Example 4, and the Escherichia coli obtained in Example 5. Positive clones were screened to obtain Escherichia coli engineering strains for producing ergothioneine.

[0094] 3) Seed culture

[0095] The fermentation seed medium was LB medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 25 mg / L; cultured overnight in a constant - temperature shaker at 37 °C with a rotation speed of 220 rpm.

[0096] 4) Fermentation culture

[0097] Inoculate the above into a sterile medium with glucose as the carbon source for fermentation. Carry out aeration fermentation at pH = 6.5 - 7.5 and 30 °C for 80 hours, and control the dissolved oxygen at 20%. Among them, control the glucose fermentation at 0 sugar, start feeding after 11 hours of fermentation, and feed histidine, cysteine, and methionine at a uniform rate, with a feeding rate of 0.5 g / L, which lasts for 25 - 35 hours continuously.

[0098] The fermentation medium is as follows: The composition of the 5L fermentation medium is: glucose 20 g / L, yeast extract 5 g / L, ammonium sulfate 5 g / L, dipotassium hydrogen phosphate 8 g / L, citric acid monohydrate 2 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 150 mg / L, manganese sulfate monohydrate 10 mg / L, vitamin B1 2 mg / L, methionine 2 g / L, trace elements 1 mL / L; among them, the trace elements include: sodium molybdate dihydrate 2.5 g / L, nickel sulfate hexahydrate 2.5 g / L, calcium chloride dihydrate 2 g / L, copper sulfate pentahydrate 0.75 g / L, aluminum sulfate octadecahydrate 2.25 g / L, cobalt chloride hexahydrate 2.5 g / L, zinc chloride 0.5 g / L, boric acid 3 g / L, kanamycin 25 mg / L.

[0099] One group of the feeding medium is: 600 g / L glucose, 5 g / L histidine, 5 g / L cysteine, 5 g / L methionine.

[0100] 5) Detect the ergothioneine yield

[0101] The HPLC detection method is as follows: Agilent 1260 high-performance liquid chromatograph; the chromatographic column is Agilent Agilent SB-Aq, the detection wavelength is 260 nm, the mobile phase is 0.1% perchloric acid: acetonitrile = 95:5, the flow rate is 1 ml / min, and the column oven temperature is 40 °C; the RT of ergothioneine is 3.5 min. Among them, the detection of the standard product is as Figure 2 shown; check the yield of ergothioneine at the end of fermentation as shown in Table 1 and Figures 3 - 4 shown, among which the bacteria have the highest OD at 55 h and then slowly decrease, so stop fermentation at 80 h.

[0102] Table 1 Yield of the engineered Escherichia coli strain in a 5L fermenter at 80 h

[0103]

[0104]

[0105] From Table 1 and Figure 4As shown, after transforming Escherichia coli with HisJ replaced and yhaM knocked out by Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE (SEQ ID NO:17), the highest concentration of ergothioneine, which can reach 13.1 g / L, was obtained after 80 hours of fermentation.

[0106] The embodiments described above are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

Claims

1. A method for preparing a microorganism for efficiently producing ergothioneine, characterized in that, The method is as follows: The coding genes of histidine methyltransferase EgtD, sulfoxide synthase Egt1 from Trichoderma reesei, and PLP-dependent C-S lyase EgtE from Mycobacterium are constructed into a vector, transformed into Escherichia coli, and positive clones are screened; the Escherichia coli uses promoter Pfic to mediate the expression of EgtD, Egt1, and EgtE; the nucleotide sequence of Pfic is shown as SEQ ID NO:

7.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of the said EgtD is shown as SEQ ID NO:1, the amino acid sequence of the said Egt1 is shown as SEQ ID NO:2, and the amino acid sequence of the said EgtE is shown as SEQ ID NO:

3.

3. The preparation method according to claim 2, characterized in that, The coding sequences of EgtD, Egt1, and EgtE are codon-optimized to adapt to the transcription and translation of Escherichia coli; the optimized coding sequence of EgtD is shown as SEQ ID NO:4; the optimized coding sequence of Egt1 is shown as SEQ ID NO:5; the optimized coding sequence of EgtE is shown as SEQ ID NO:

6.

4. The preparation method according to claim 3, characterized in that, The endogenous promoter HisJ of the said Escherichia coli is replaced by promoter Ptac; the nucleotide sequence of Ptac is shown as SEQ ID NO:

12.

5. The preparation method according to claim 4, characterized in that, The endogenous gene yhaM of the said Escherichia coli is inactivated.

6. The preparation method according to claim 5, characterized in that, The said promoter Pfic is a highly efficient promoter after saturation mutation screening. The mutated promoters are Pfic-ECT1, Pfic-ECT5, and Pfic-ECT14, and their nucleic acid sequences are SEQ ID NOs:8-10 respectively; the combination modes of the promoter mediating the expression of the said enzymes are one of the following: Pfic-ECT1_EgtD_Egt1_EgtE, Pfic-ECT5_EgtD_Egt1_EgtE, Pfic-ECT14_EgtD_Egt1_EgtE, Pfic-ECT1_EgtD_Pfic-ECT5_Egt1_Pfic-ECT14_EgtE, Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE, Pfic-ECT5_EgtD_Pfic-ECT14_Egt1_Pfic-ECT1_EgtE, or Pfic-ECT14_EgtD_Pfic-ECT5_Egt1_Pfic-ECT1_EgtE.

7. The preparation method according to claim 6, characterized in that, The said combination mode is Pfic-ECT5_EgtD_Pfic-ECT1_Egt1_Pfic-ECT14_EgtE; its nucleotide sequence is shown as SEQ ID NO:

17.

8. A microorganism capable of highly efficiently fermenting and producing ergothioneine prepared by the method according to any one of claims 1-7.

9. A method for fermentatively producing ergothioneine using claim 8, characterized in that, The said method is to separate ergothioneine after seed culture and fermentation culture of the microorganism according to claim 8.

10. The method according to claim 9, characterized in that, The fermentation culture is as follows: culture in a 5-30 L fermenter; the conditions for the fermentation culture are aerobic fermentation for 60-100 hours at pH = 6.5-7.5 and 25-32 °C, with dissolved oxygen controlled at 18-22%.

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