Method for efficiently producing ergothioneine by metabolically engineering escherichia coli and application thereof

Abstract

The application discloses a kind of methods for efficiently producing ergothioneine of metabolically engineered escherichia coli and application, belong to the field of biotechnology.The recombinant escherichia coli EGT-1-B* provided by the application can realize the conversion of glucose into ergothioneine;Therefore the raw material of the present application is cheap and easy to obtain, no harmful additives to human health, heavy metal, for the product purification of later period lay good foundation, more can reach the requirement of commercial application.And in the recombinant escherichia coli EGT-1-B* of the application, further overexpresses the gene related to histidine, methionine, cysteine, S adenosyl methionine metabolism, to improve the content of precursor amino acid of ergothioneine synthesis, while after knocking out gene yqaB, it is found that the yield of ergothioneine is further improved, the utilization rate of glucose to ergothioneine can be further improved, and the yield of ergothioneine can reach 140.29 mg / L.

Description

Technical Field

[0001] This invention relates to a method and application of metabolic engineering to efficiently produce ergothionein from Escherichia coli, belonging to the field of biotechnology. Background Technology

[0002] Ergothioneine (ERG), also known as 2-thioimidazolium amino acid, is a sulfur-containing histidine derivative with a molecular weight of 229.3. The pure form is a white crystalline solid, a water-soluble compound, dissolving at 0.9 mol / L at room temperature. Due to its unique thione structure and high redox potential, ergothioneine does not oxidize at physiological pH or in strongly alkaline solutions. It possesses a thiol group at the C2 position of the imidazole ring, making it more stable than other thiol antioxidants (such as glutathione). It exists in two isomers: thiol and thione. Ergothioneine is a naturally occurring cell protectant and antioxidant in the human body. Compared to antioxidants like vitamin C, it is milder and safer, and has better antioxidant effects. Dietary intake may safely reduce hydroxyl and other free radicals, and prevent DNA damage and fatty acid peroxidation products, thereby inhibiting carcinogenic, lifestyle-related chronic diseases, and aging.

[0003] Ergothioneine is primarily found in plants such as mushrooms, broccoli, and Brussels sprouts, as well as certain bacteria and fungi. Since its discovery in animal organs and tissues, scientists have conducted extensive research, finding that ergothioneine effectively scavenge free radicals that cause skin aging, protects cell membranes and mitochondrial membranes from oxidative damage, and provides cellular protection and UV radiation protection, among other functions. Ergothioneine plays a crucial role in anti-oxidation and anti-aging in the body, primarily accumulating in organs, cells, and secretions susceptible to high levels of oxidative stress and inflammation. Tests have revealed that the liver and whole blood are the concentration sites of ERG, which is also present in the kidneys, spleen, heart, eyes, and brain tissue.

[0004] There are three methods for preparing ergothioneine: chemical synthesis, extraction, and bio-fermentation synthesis. Chemical synthesis of levorotatory ergothioneine is extremely difficult; several methods have failed to achieve the expected yield due to partial or complete racemization. Natural bio-extraction methods extract ergothioneine from the fruiting bodies of edible fungi, pig blood, animal tissues, ergot, and grains. However, the ergothioneine content in these raw materials remains very low, and problems such as high impurities, drug residues, and high extraction costs limit the application of ergothioneine and make it unsuitable for industrialization. Biosynthesis, through metabolic regulation and other fermentation process control techniques, can effectively improve the yield of ergothioneine, reduce production costs, and, more importantly, ensure product safety, thus broadening the application scope of ergothioneine.

[0005] In existing reports, ergothioneine biosynthesis involves multiple reactions. A complex regulatory network exists within *E. coli* to control ergothioneine synthesis, with biosynthetic pathways intertwined and influencing other metabolic pathways. Gene expression regulation is involved in the biosynthesis process, and predicting the expression level of the target gene in host cells can be difficult for heterologous gene expression systems. Furthermore, the supply of precursor amino acids such as methionine and histidine affects ergothioneine synthesis, as these precursors participate in other intracellular metabolic pathways, such as protein synthesis. In some cases, the intracellular content of these precursor amino acids is limited, insufficient to meet the demands of large-scale ergothioneine synthesis. However, directly adding additional amino acids increases the cost of raw material production. For example, in the literature (Gram-scalefermentative production of ergothioneine driven by overproduction of cysteinein *Escherichia coli*), adding the precursor amino acid methionine to shake-flask culture medium resulted in a yield of 98 mg / L of ergothioneine after 120 hours of fermentation, which is not very high and requires further improvement. Therefore, there is an urgent need to develop a safe, efficient, and inexpensive biological method for synthesizing ergothioneine. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides a method and application for the efficient production of ergothioneine by metabolic engineering of Escherichia coli. The aim is to solve the technical problems that the content of precursor amino acids in existing biosynthetic methods is limited, which cannot meet the demand for large-scale synthesis of ergothioneine. Furthermore, the direct addition of amino acids will increase the cost of raw material production, and the yield needs to be further improved.

[0007] The first technical solution provided by this invention is a recombinant Escherichia coli, which uses Escherichia coli as the host cell and recombinantly expresses ergothioneine synthase egtABCDE derived from Mycobacterium smegmatis, ergothioneine synthase egt1 derived from Neurospora crassa, and sulfoxide synthase Mp-egtB derived from Methylobacterium, and expresses one or more genes related to the metabolism of histidine, methionine, cysteine, and S-adenosylmethionine.

[0008] In some embodiments, the amino acid sequences of the ergothioneine synthase egtABCDE, ergothioneine synthase egt1, and sulfoxide synthase Mp-egtB are shown in SEQ ID NO:1 to 7, respectively.

[0009] In some embodiments, the histidine metabolism-related genes include the histG gene, which expresses the ATP phosphoribosyltransferase HisG, and mutant hisG. E271K ;

[0010] Genes related to methionine metabolism include the gene metA encoding the homoserine acyltransferase MetA, as well as its mutant metA. A881G ThrA (a truncated gene of aspartate kinase ThrA), methylenetetrahydrofolate reductase metF, and methionine adenosine transferase metK;

[0011] Genes related to cysteine ​​metabolism include the mutant gene cysE of the serine acetyltransferase CysE. T167A ;

[0012] Genes related to S-adenosylmethionine metabolism include the gene mtn encoding 5'-meththioadenosine / S-adenosylhomocysteine ​​nucleoside enzyme Mtn and the gene luxS encoding S-ribosylhomocysteine ​​lyase LuxS.

[0013] In some implementations, the GeneID of hisG is 1019477, and the hisG... E271K The nucleotide sequence is shown in SEQ ID NO:8.

[0014] In some implementations, the GeneID of metA is 948513, and the metA... A881G The nucleotide sequence is shown in SEQ ID NO:9;

[0015] The GeneID of the thrA is 945803, and the nucleotide sequence of the thrA is shown in SEQ ID NO:10;

[0016] The GeneID of metF is 948432, and the nucleotide sequence of metF is shown in SEQ ID NO:11;

[0017] The GeneID of metK is 945389, and the nucleotide sequence of metK is shown in SEQ ID NO:12;

[0018] In some implementations, the GeneID of the cysE is 946877. T167A The nucleotide sequence is shown in SEQ ID NO:13.

[0019] In some embodiments, the mtn has a GeneID of 948542; the nucleotide sequence of the mtn is shown in SEQ ID NO: 14.

[0020] In some embodiments, the GeneID of luxS is 947168. The nucleotide sequence of luxS is shown in SEQ ID NO: 15.

[0021] In some implementations, the egtA and egt1 genes are expressed using the plasmid pETDuet-1.

[0022] In some implementations, the egtB and egtD genes are expressed using the plasmid pCDFDuet-1.

[0023] In some implementations, the egtC and egtE genes are expressed using the plasmid pRSDuet-1.

[0024] In some implementations, the Mp-egtB gene, as well as genes related to the metabolism of histidine, methionine, cysteine, and S-adenosylmethionine, are expressed using the plasmid pACYCDuet-2.

[0025] In some implementations, the gene yqaB (Gene ID: 945776) that affects the degradation of the substrate glucose into products is knocked out in the host cell.

[0026] The second technical solution provided by this invention is a method for constructing recombinant Escherichia coli, the method comprising the following steps:

[0027] (1) Insert the egtA gene into plasmid pETDuet-1 to obtain recombinant plasmid pET-egtA;

[0028] (2) Insert the egtB and egtD genes into plasmid pCDFDuet-1 to obtain recombinant plasmid pCD-egtB-egtD; insert the egtC and egtE genes into plasmid pRSDuet-1 to obtain recombinant plasmid pRS-egtC-egtE; insert the egt1 gene into plasmid pET-egtA from step (1) to obtain recombinant plasmid pET-egtA-egt1;

[0029] (3) Insert the Mp-egtB gene into plasmid pACYCDuet-2 to obtain recombinant plasmid pACYC-egtB*;

[0030] (4) The mutant gene hisG E271K Gene insertion step (3) into plasmid pACYC-egtB* yields recombinant plasmid pACYC-egtB*-hisG*; the metA mutant metA A881GThe recombinant plasmid pACYC-egtB* was obtained by inserting the gene into the plasmid pACYC-egtB* in step (3); the truncated thrA gene was inserted into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-thrA; the metF gene was inserted into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-metF; the metK gene was inserted into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-metK; the mtn and luxS genes were inserted into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-mtn-luxS; the cysE mutant cysE T167A The gene was inserted into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-cysE*.

[0031] (5) Introduce all the recombinant plasmids from step (2) and one or more recombinant plasmids from step (4) into the Escherichia coli host to obtain recombinant Escherichia coli.

[0032] In some implementations, the yqaB gene of the *E. coli* host was knocked out using CRISPR / Cas9 gene editing technology.

[0033] Furthermore, the method for knocking out the yqaB gene includes the following steps:

[0034] (1) Construction of yqaB knockout frame: Design primers, use PCR to amplify and ligate fragments yqaB-U and yqaB-D, and ligate the above fragments to obtain yqaB knockout frame;

[0035] (2) Using the CRISPR-Cas9 method, the pCas plasmid was first transformed into Escherichia coli BL21(DE3) competent cells by electroporation. Then, the plasmid sg-pTarget containing sgRNA and the yqaB knockout box were electroporated into Escherichia coli BL21(DE3) / pCas competent cells. Transformants were picked for colony PCR verification. The verified strains were induced with IPTG to eliminate the sg-pTarget plasmid.

[0036] The third technical solution provided by the present invention is a method for producing ergothioneine, wherein the method is to produce ergothioneine by fermentation using recombinant Escherichia coli as described in the first technical solution.

[0037] In some embodiments, the method involves inoculating the recombinant Escherichia coli into a seed culture medium to prepare a seed solution; then inoculating the seed solution into a fermentation culture medium for fermentation to prepare ergothioneine.

[0038] In some embodiments, the recombinant Escherichia coli is inoculated onto LB solid medium and cultured to obtain single colonies. A single colony is then picked and inoculated into LB liquid medium and cultured at 37°C and 220 rpm for 10–12 h to prepare a seed culture.

[0039] In some embodiments, the recombinant Escherichia coli is streaked onto LB solid medium, and a single colony is picked and inoculated into a 100 mL Erlenmeyer flask containing 20 mL of LB liquid medium. The culture is then carried out at 37°C and 220 rpm for 10–12 h to prepare a seed culture.

[0040] In some embodiments, the prepared seed culture is inoculated into a fermentation liquid culture medium and cultured at 37°C and 180-220 rpm for 3-4 hours; then IPTG is added to induce fermentation to prepare ergothioneine.

[0041] In some embodiments, the seed culture is inoculated into a 250 mL Erlenmeyer flask containing 25 mL of LB liquid medium at a rate of 2% (v / v), and cultured at 37°C and 200 rpm for 3 h. Then, IPTG inducer is added for induction, and the culture is carried out at 25°C and 200 rpm for 96 h.

[0042] The fourth technical solution provided by this invention is the application of the recombinant Escherichia coli described in the first technical solution in the preparation of ergothioneine, products containing ergothioneine, and ergothioneine derivatives.

[0043] The technical effects of this invention are as follows:

[0044] The recombinant *E. coli* EGT-1-B* provided by this invention can convert glucose into ergothioneine. Therefore, the raw materials of this invention are inexpensive and readily available, free of harmful additives and heavy metals, laying a good foundation for subsequent product purification and meeting the requirements of commercial applications. Furthermore, this invention further overexpresses genes related to histidine, methionine, cysteine, and S-adenosylmethionine metabolism in the recombinant *E. coli* EGT-1-B* to increase the content of precursor amino acids for ergothioneine synthesis. Simultaneously, knocking out the yqaB gene further increased ergothioneine production, and the utilization rate of glucose to ergothioneine was further improved, with ergothioneine production reaching 140.29 mg / L. Attached Figure Description

[0045] Figure 1 The main metabolic pathway of the strain constructed in this invention has been modified.

[0046] Figure 2 The results show the ergothionein yields obtained by fermenting the strains EGT-ABCDE, EGT-ABCDE-1, EGT-ABCDE-B*, EGT-ABCDE1-B*, EGT-ABCDE-12, EGT,5-hisG*, EGT-5-metA*, EGT-5-thrA*, EGT-1-B*-metF, EGT-5-metK, EGT-5-mtn-luxS, EGT-5-cysE*, EGT-5-metK-metA*, and EGT-5-ΔyqaB-metK constructed in this invention. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, specific embodiments are described below, with reference to the appendix. Figures 1-2 The present invention will be further described in detail below.

[0048] Test method:

[0049] 1. Determination of bacterial cell concentration: After centrifuging 1 mL of bacterial fermentation broth to collect the bacterial cells, suspend and dilute them with deionized water. Use an ultraviolet-visible spectrophotometer (manufacturer) to measure the absorbance value (OD600) of the diluted bacteria at 600 nm. The data is more accurate when the measured bacterial cell OD600 value is kept between 0.2 and 0.8.

[0050] 2. Determination of ergothioneine content (HPLC method): The fermentation broth was centrifuged at 10,000×g, 4℃ for 2 min, and the supernatant was collected and filtered through a 0.22μm filter membrane. A Shimadzu HPLC system (LC-2050) (Shimadzu Corporation, Kyoto, Japan) was used: UV detector, C18 column (Diamonsil 5μm C18, 250×4.6mm). Mobile phase ratio: water / methanol = 95 / 5, column temperature: 30℃, flow rate: 0.7mL / min. UV absorption wavelength: 257nm, injection volume: 10μL, detection time: 20min.

[0051] 3. Perform enzyme digestion and ligation according to Table 1 and Table 2.

[0052] Table 1. Preparation of the enzyme digestion system

[0053]

[0054] Table 2 Preparation of enzyme ligation system

[0055]

[0056]

[0057] 4. Perform the PCR reaction according to Table 3.

[0058] Table 3. Preparation of PCR system

[0059]

[0060] Raw materials used in the examples:

[0061] 1. LB medium formula (g / L): yeast extract (OXOID) 5, peptone (OXOID) 10, NaCl (Guoyao) 10, and solid medium with an additional 1.5%-2.0% agar powder (BIOFROXX).

[0062] 2. Fermentation medium formula (g / L): glucose (Guoyao) 20, yeast powder (OXOID) 4, ammonium sulfate (Guoyao) 32, disodium hydrogen dodecyl phosphate (Guoyao) 32, potassium dihydrogen phosphate (Guoyao) 6, magnesium sulfate (Guoyao) 0.6, calcium chloride (Guoyao) 0.01.

[0063] 3. Plasmids pETDuet-1, pCDFDuet-1, pRSDuet-1, pACYCDuet-2, E. coli BL21(DE3), and E. coli JM109 are all commercially available plasmids and strains.

[0064] Example 1: Construction of recombinant Escherichia coli EGT

[0065] The specific steps are as follows:

[0066] (1) Preparation of E. coli BL21(DE3) competent cells: E. coli BL21(DE3) strain was inoculated onto LB solid medium and streaked to obtain single colonies. A single colony was picked and inoculated into a 100 mL Erlenmeyer flask containing 20 mL of LB liquid medium. The flask was then activated at 37℃ and 200 rpm for 12 h to prepare the seed culture. The prepared seed culture was inoculated at a rate of 1% (v / v) into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium and cultured at 37℃ and 200 rpm until OD (digestive activity) was reached. 600 =0.5 to obtain fermentation broth. After incubating the fermentation broth on ice for 30 min, centrifuge at 6000 rpm for 5 min at 4℃ to collect the cells. Wash the obtained cells once with pre-cooled ultrapure water and three times with pre-cooled 0.1 mol / L CaCl2 solution. Add 2 mL of 0.1 mol / L CaCl2 solution and 2 mL of 30% glycerol to the collected cells. Aliquot 100 μL into 1.5 mL EP tubes and store at -70℃ for later use to prepare E. coli BL21(DE3) competent cells.

[0067] (2) The egtA gene (nucleotide sequence as shown in SEQ ID NO:16) was synthesized from the company and inserted into the BamH I and HindIII sites of plasmid pETDuet-1 to obtain the recombinant plasmid pET-egtA. The egtB and egtD genes (nucleotide sequences as shown in SEQ ID NO:17 and 19) were synthesized from the company and inserted into the BamH I and HindIII sites and the Kpn I and NdeI sites of plasmid pCDFDuet-1, respectively, to obtain the recombinant plasmid pCD-egtB-egtD. The egtC and egtE genes (nucleotide sequences as shown in SEQ ID NO:18 and 20) were synthesized from the company and inserted into the BamH I and HindIII sites and the Kpn I and NdeI sites of plasmid pRSFDuet-1, respectively, to obtain the recombinant plasmid pRS-egtC-egtE.

[0068] (3) The recombinant plasmids pET-egtA, pCD-egtB-egtD, and pRS-egtC-egtE were chemically introduced into the E. coli BL21(DE3) competent cells obtained in step (1) to obtain transformation products. The transformation products were plated on LB solid medium containing kanamycin, ampicillin, and streptomycin and cultured at 37°C until transformants grew. The strain was preserved in glycerol tubes to obtain free recombinant E. coli EGT.

[0069] Example 2: Construction of free expression strains of egt1 and Mp-egtB

[0070] The specific steps are as follows:

[0071] (1) Preparation of E. coli JM109 competent cells: E. coli JM109 strain was inoculated onto LB solid medium and streaked to obtain single colonies. A single colony was picked and inoculated into a 100 mL Erlenmeyer flask containing 20 mL of LB liquid medium. The flask was then activated at 37℃ and 200 rpm for 12 h to prepare the seed culture. The prepared seed culture was inoculated at a rate of 1% (v / v) into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium and cultured at 37℃ and 200 rpm until OD... 600 =0.5 to obtain fermentation broth. After incubating the fermentation broth on ice for 30 min, centrifuge at 6000 rpm for 5 min at 4℃ to collect the bacterial cells. Wash the obtained bacterial cells once with pre-cooled ultrapure water, and wash three times with pre-cooled 0.1 mol / L CaCl2 solution. Add 2 mL of 0.1 mol / L CaCl2 solution and 2 mL of 30% glycerol to the collected bacterial cells. Aliquot 100 μL into 1.5 mL EP tubes and store at -70℃ for later use to prepare E. coli JM109 competent cells.

[0072] (2) The egt1 gene (nucleotide sequence shown in SEQ ID NO:21) was synthesized from the company and inserted into the Kpn I and NdeI sites of plasmid pET-egtA to obtain the recombinant plasmid pET-egtA-egt1. The Mp-egtB gene (nucleotide sequence shown in SEQ ID NO:22) was synthesized from the company and inserted into the BamH I and HindIII sites of plasmid pACYCDuet-2 to obtain the recombinant plasmid pACYC-egtB*.

[0073] (3) The recombinant plasmid pET-egtA-egt1 was chemically introduced into the E. coli JM109 competent cells obtained in step (1) to obtain the transformation product. The transformation product was plated on LB solid medium containing ampicillin and cultured at 37°C until transformants grew. The transformants were cultured to extract plasmids, and the plasmids were verified by Kpn I and NdeI enzyme digestion. The verified strains were stored in glycerol tubes.

[0074] (4) The recombinant plasmid pACYC-egtB* was chemically introduced into the E. coli JM109 competent cells obtained in step (1) to obtain the transformation product. The transformation product was plated on LB solid medium containing chloramphenicol and cultured at 37°C until transformants grew. The transformants were cultured to extract plasmids, and the plasmids were verified by BamHI and HindIII restriction enzyme digestion. The verified strains were stored in glycerol tubes.

[0075] (5) The recombinant plasmid pET-egtA-egt1 obtained in step (3) was chemically introduced into recombinant E. coli EGT competent cells to obtain the transformation product. The transformation product was plated on LB solid medium containing kanamycin, ampicillin, and streptomycin and cultured at 37°C until transformants grew. The strain was preserved in glycerol tubes to obtain free recombinant E. coli EGT-1.

[0076] (6) The recombinant plasmid pACYC-egtB* prepared in step (4) was chemically introduced into EGT competent cells to obtain the transformation product. The transformation product was plated on LB solid medium containing kanamycin, ampicillin, streptomycin, and chloramphenicol and cultured at 37°C until transformants grew. The strain was preserved in glycerol tubes to obtain free recombinant Escherichia coli EGT-B*.

[0077] (5) The recombinant plasmid pET-egtA-egt1 prepared in step (3) and the recombinant plasmid pACYC-egtB* prepared in step (4) were chemically introduced into EGT competent cells to obtain transformation products. The transformation products were plated on LB solid medium containing kanamycin, ampicillin, streptomycin, and chloramphenicol and cultured at 37°C until transformants grew. The strain was preserved in glycerol tubes to obtain free recombinant Escherichia coli EGT-1-B*.

[0078] (6) The mutant gene hisG E271K Insert the Kpn I and NdeI sites into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-hisG*; and insert the metA mutant metA A881G The gene was inserted into the Kpn I and Nde I sites of plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-metA*; the truncated thrA gene was inserted into the Kpn I and Nde I sites of plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-thrA; the metF gene was inserted into the Kpn I and Nde I sites of plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-metF; the metK gene was inserted into the Kpn I and Nde I sites of plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-metK; the mtn and luxS genes were inserted into the Kpn I and Nde I sites of plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-mtn-luxS; and the cysE mutant cysE was used... T167A The gene was inserted into the Kpn I and NdeI sites of plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-cysE*. The metA mutant metA... A881G The gene was inserted into the plasmid pACYC-egtB*-metK and linked to the metK gene with the RBS sequence to obtain the recombinant plasmid pACYC-egtB*-metK-metA*. The above plasmid was then verified by referring to the method in step (3) or (4).

[0079] (7) The recombinant plasmids pACYC-egtB*-hisG*, pACYC-egtB*-metA*, pACYC-egtB*-thrA, pACYC-egtB*-metF, pACYC-egtB*-mtn-luxS, and pACYC-egtB*-cysE* were transformed into recombinant Escherichia coli EGT-1 to obtain strains EGT-1-B*-hisG*, EGT-1-B*-metA*, EGT-1-B*-thrA*, EGT-1-B*-metF, EGT-1-B*-metK, EGT-1-B*-mtn-luxS, EGT-1-B*-serA*, EGT-1-B*-cysE*, and EGT-1-B*-metK-metA*.

[0080] Example 3: Knockout of gene yqaB

[0081] The specific steps are as follows:

[0082] (1) Using the CRISPR-Cas9 method, E. coli BL21(DE3) was prepared into electrotransfer competent cells. The plasmid pCas carrying the transport protein gene was electrotransformed into the competent cells and plated on kanamycin-resistant LB solid medium plates. The single colony that grew was E. coli BL21(DE3) / pCas.

[0083] (2) Design sgRNA primers sg-yqaB-F and sg-yqaB-R for the yqaB site (see Table 4). Use pTarget plasmid as a template for whole plasmid PCR. Purify the obtained gene fragments. Digest the template plasmid with DpnI and then transform it into E. coli JM109 competent cells by chemical transformation to obtain the transformation product. Spread the transformation product on spectinomycin LB solid medium plates. Pick single colonies that grow and extract plasmids for sequencing. The correct ones are the correct transformants.

[0084] (3) Construction of yqaB knockout frame: Design primers (see Table 4), PCR amplification was used to obtain fragments yqaB-U and yqaB-D, which were then ligated to obtain the yqaB knockout frame.

[0085] (4) Using the CRISPR-Cas9 method, the pCas plasmid was first transformed into E. coli BL21(DE3) competent cells by electroporation. Then, the plasmid sg-pTarget containing sgRNA and the knockout frame of yqaB were electroporated into E. coli BL21(DE3) / pCas competent cells. Transformants were picked for colony PCR verification. The verified strains were induced with IPTG to eliminate the sg-pTarget plasmid and construct recombinant E. coli, named E. coliΔyqaB.

[0086] (5) The recombinant plasmids pET-egtA-egt1, pCD-egtB-egtD, pRS-egtC-egtE, and pACYC-egtB*-metK were transformed into recombinant E. coli ΔyqaB to form strain EGT-5-ΔyqaB-metK.

[0087] Table 4 Primer sequences

[0088]

[0089] Example 4: Production of Ergothioneine by Shake Flask Fermentation

[0090] The specific steps are as follows:

[0091] (1) The recombinant E. coli EGT prepared in Example 1 and the strains EGT-1, EGT-B*, EGT-1-B*, EGT-1-B*-hisG*, EGT-1-B*-metA*, EGT-1-B*-thrA*, EGT-1-B*-metF, EGT-1-B*-metK, EGT-1-B*-mtn-luxS, EGT-1-B*-serA*, EGT-1-B*-cysE*, and EGT-1-B*-metK-metA* prepared in Example 2 were streaked on LB solid medium to obtain single colonies. A single colony was picked and inoculated into a 100mL Erlenmeyer flask containing 20mL of LB liquid medium and cultured at 37℃ and 200rpm for 12h to obtain seed culture.

[0092] (2) The seed liquid prepared in step (1) was inoculated into a 250 mL Erlenmeyer flask containing 25 mL of fermentation liquid culture medium at an inoculation rate of 2% (v / v). After incubation at 37℃ and 200 rpm for 3-4 h, IPTG was added for induction.

[0093] (3) Incubate at 25℃ and 200 rpm for 96 h. Results Figure 2 As shown in Tables 5-10.

[0094] Table 5. Effects of histidine module metabolic modification on ergothionein yield.

[0095] 0 0 0 12 5.785 7.83 24 18.33 19.76 36 22.15 32.89 48 29.84 51.91 60 29.96 77.62 72 34.27 86.4 84 40.42 89.24 96 43.71 90.53

[0096] Table 6. Effects of methionine module metabolic modification on ergothionein yield

[0097] 0 0 0 0 0 12 5.78 20.57 17.66 20.47 24 18.32 33.63 16.55 17.76 36 22.15 36.02 27.76 22.25 48 29.84 59.25 37.90 22.28 60 29.95 100.13 40.43 22.58 72 34.27 101.91 52.14 22.80 84 40.42 103.27 49.07 23.88 96 43.71 105.29 48.02 23.79

[0098] Table 7. Effects of S-adenosylmethionine module metabolic modification on ergothionein yield.

[0099]

[0100]

[0101] Table 8. Effects of cysteine ​​module metabolic modification on ergothionein yield

[0102] 0 0 0 12 5.79 17.61 24 18.33 40.57 36 22.15 57.91 48 29.84 71.50 60 29.96 89.17 72 34.27 87.36 84 40.42 92.57 96 43.71 89.54

[0103] Table 9. Effects of combined metabolic modifications between modules on ergothionein yield.

[0104] 0 0.00 0.00 12 20.59 8.80 24 29.16 45.48 36 34.79 79.83 48 71.97 86.11 60 98.07 89.58 72 90.51 94.82 84 99.19 98.82 96 107.31 111.07

[0105] Table 10. Effects of yqaB gene knockout on ergot production.

[0106] 0 0 0 12 20.59 15.92 24 29.16 37.87 36 34.79 62.46 48 71.97 74.60 60 98.07 79.37 72 90.51 102.56 84 99.19 114.03 96 107.31 140.29

[0107] Depend on Figure 2 As shown in Tables 5-10, the recombinant Escherichia coli EGT-ABCDE, EGT-ABCDE-1, EGT-ABCDE-1-2, EGT-ABCDE-B*, EGT-ABCDE-1-B*, EGT-5-hisG*, EGT-5-metA*, EGT-5-thrA*, EGT-5-metF, EGT-5-metK, EGT-5-mtn-luxS, EGT-5-cysE*, and EGT-5-metK-m provided by this invention etA*, EGT-5-ΔyqaB-metK were fermented in shake flasks for 96 h to produce ergothioneine at concentrations of 7.82 mg / L, 12.54 mg / L, 10.78 mg / L, 13.31 mg / L, 43.71 mg / L, 90.53 mg / L, 105.29 mg / L, 48.02 mg / L, 23.79 mg / L, 107.31 mg / L, 75.18 mg / L, 89.54 mg / L, 111.07 mg / L, and 140.29 mg / L, respectively.

[0108] Microbial fermentation for ergothioneine production still faces challenges and bottlenecks, such as the lack of precursor substances and insufficient methyl donors. Therefore, this study, building upon these limitations, aimed to synthesize ergothioneine de novo in *E. coli* using glucose as a substrate. By metabolically modifying the ergothioneine precursor amino acid module, the yield was further improved. The production of ergothioneine in *E. coli* requires numerous precursor substances, resulting in a complex and lengthy metabolic background. Key enzymes and regulatory mechanisms are not yet fully understood, necessitating analysis and research across different modules. Ultimately, the study revealed that increasing the supply of methyl donors led to greater ergothioneine production. Knocking out the yqaB gene further increased ergothioneine yield and improved glucose-to-ergothioneine utilization. Further research is needed to optimize the combination of modules; using glucose as a substrate for ergothioneine production is more economical when the direct addition of precursor amino acids is not possible.

[0109] 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, Using *Escherichia coli* as the host cell, recombinant expression was made of ergothioneine synthases egtA, egtB, egtC, egtD, and egtE derived from *Mycobacterium smegmatis*, ergothioneine synthase egt1 derived from *Neurospora crassa*, and sulfoxide synthase Mp-egtB derived from *Methylobacterium*. Genes related to methionine metabolism were also expressed. The *E. coli* host cell also had the gene yqaB knocked out. The amino acid sequences of the ergothioneine synthases egtA, egtB, egtC, egtD, and egtE, ergothioneine synthase egt1, and sulfoxide synthase Mp-egtB are shown in SEQ ID NO: 1-7, respectively. The gene related to methionine metabolism is the gene encoding methionine adenosine transferase metK, and the nucleotide sequence of the gene encoding metK is shown in SEQ ID NO:

12. The yqaB gene is shown in the NCBI database as GeneID:945776; The Escherichia coli mentioned is Escherichia coli BL21(DE3).

2. The recombinant Escherichia coli according to claim 1, characterized in that, The coding genes for egtA and egt1 were expressed using the plasmid pETDuet-1. The coding genes for egtB and egtD were expressed using plasmid pCDFDuet-1; The coding genes for egtC and egtE were expressed using the plasmid pRSDuet-1. The gene encoding Mp-egtB, as well as genes related to methionine metabolism, were expressed using the plasmid pACYCDuet-2.

3. A method for constructing recombinant Escherichia coli according to claim 1, characterized in that, The method includes the following steps: (1) Insert the coding gene of egtA into plasmid pETDuet-1 to obtain recombinant plasmid pET-egtA; (2) Insert the coding genes of egtB and egtD into plasmid pCDFDuet-1 to obtain recombinant plasmid pCD-egtB-egtD; insert the coding genes of egtC and egtE into plasmid pRSDuet-1 to obtain recombinant plasmid pRS-egtC-egtE; insert the coding gene of egt1 into plasmid pET-egtA from step (1) to obtain recombinant plasmid pET-egtA-egt1; (3) Insert the coding gene of Mp-egtB into plasmid pACYCDuet-2 to obtain recombinant plasmid pACYC-egtB*; (4) Insert the gene encoding metK into the plasmid pACYC-egtB* from step (3) to obtain the recombinant plasmid pACYC-egtB*-metK; (5) Introduce all the recombinant plasmids in step (2) and the recombinant plasmids in step (4) into the Escherichia coli host to obtain recombinant Escherichia coli; the Escherichia coli host is Escherichia coli BL21(DE3), and the gene yqaB of the Escherichia coli host is knocked out using CRISPR / Cas9 gene editing technology.

4. A method for producing ergothioneine, characterized in that, The method is to produce ergothionein by fermentation using recombinant Escherichia coli as described in any one of claims 1 to 2.

5. The use of the recombinant Escherichia coli according to any one of claims 1 to 2 in the preparation of ergothioneine.

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