Method for efficiently producing ergothioneine by metabolic engineering modified escherichia coli and application

Abstract

The invention discloses a method for efficiently producing ergothioneine by metabolic engineering modified escherichia coli and application, and belongs to the technical field of biology. By adopting the recombinant escherichia coli EGT-1-B * provided by the invention, glucose can be converted into ergothioneine; therefore, the raw materials are cheap and easy to obtain, additives and heavy metals which are harmful to human health are avoided, a good foundation is laid for later product purification, and the requirements of commercial application can be met. Furthermore, genes related to metabolism of histidine, methionine, cysteine and S-adenosylmethionine are further overexpressed in the recombinant escherichia coli EGT-1-B *, so that the content of precursor amino acid synthesized by the ergothioneine is increased, and meanwhile, after the gene yqaB is knocked out, the yield of the ergothioneine is further increased; 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] The invention relates to a method for metabolically engineering Escherichia coli to efficiently produce ergothioneine and application thereof, belonging to the field of biotechnology. Background Art

[0002] Ergothioneine (ERG), also known as 2-thioimidazole amino acid, is a sulfur-containing histidine derivative with a molecular weight of 229.3. The pure product is a white crystal and a water-soluble compound that can dissolve 0.9 mol / L at room temperature. Because ergothioneine has a special thione structure and a high redox potential, it will not oxidize itself at physiological pH and in strong alkaline solutions. It has a thiol group at the C2 position of the imidazole ring, so it is more stable than other thiol antioxidants (such as glutathione). It exists in two isomers, namely thiol and thione. Ergothioneine is a naturally occurring cell protector and antioxidant in the human body. Compared with antioxidants such as vitamin C, it is mild and safe enough, and has a better antioxidant effect. Its dietary intake may safely reduce hydroxyl and other free radicals, and avoid DNA damage and fatty acid peroxidation products, thereby inhibiting carcinogenicity, lifestyle-related chronic diseases and aging.

[0003] Ergothioneine is mainly found in plants such as mushrooms, broccoli, and Brussels sprouts, and in certain bacteria and fungi. Since ergothioneine was also found in animal organs and tissues, scientists have conducted a lot of research and found that ergothioneine can effectively remove free radicals that cause skin aging, protect cell membranes and mitochondrial membranes from oxidative damage, protect cells and resist ultraviolet radiation, and other functions. Ergothioneine plays an important role in the body in antioxidant and anti-aging, mainly guarding organs, cells, and secretions that are susceptible to high levels of oxidative stress and inflammation. Tests have found that the liver and whole blood are the concentrations of ERG, and it also exists in the kidneys, spleen, heart, eyes, and brain tissues.

[0004] There are three preparation methods of thioneine: chemical synthesis, extraction and bio-fermentation synthesis. It is very difficult to synthesize left-handed thioneine by chemical method, and several synthesis methods do not reach the expected output due to partial or complete racemization. The natural biological extraction method is to extract thioneine from the fruiting body of edible fungi, pig blood, animal tissue, ergot and cereals, but the content of thioneine in the above-mentioned raw materials is still very low, and there are many impurities in the raw materials, drug residues, and the problems of high extraction cost, which limit the application of thioneine and is not suitable for industrialization. The biosynthesis method can effectively improve the yield of thioneine by fermentation process control means such as metabolic regulation, reduce production costs, and more importantly, can ensure the safety of the product and widen the application space of thioneine.

[0005] In existing reports, thioneine biosynthesis involves multiple reactions, and there is a complex regulatory network in Escherichia coli organisms to control the synthesis of thioneine, and biosynthetic pathways are intertwined with other metabolic pathways and affect each other. In the biosynthetic process, it will be related to the expression regulation of genes, and for heterologous gene expression systems, the expression level of target genes in host cells may be difficult to predict. At the same time, the precursor substances such as methionine, the supply of amino acids such as histidine and the like of ergothioneine synthesis will affect its synthesis, because the precursor substances will also participate in other metabolic pathways such as protein synthesis in the cell in addition to participating in the synthesis, in some cases, the content of these precursor amino acids in the cell is limited, it is impossible to meet the demand for the synthesis of thioneine in large quantities, but if additional direct addition of amino acids will increase the cost of raw material production, such as in the literature (Gram-scalefermentative production of ergothioneine driven by overproduction of cysteinein Escherichia coli), additionally add precursor amino acid methionine in shake flask medium, fermentation 120h produces 98mg / L thioneine, production is not very high, and further improvement is needed. Therefore, it is urgent to develop a safe, efficient and low-cost method for synthesizing ergothioneine by biological method. Summary of the invention

[0006] In view of the deficiencies in the above-mentioned prior art, the present invention provides a method and application of metabolically engineered Escherichia coli to efficiently produce thioneine, the purpose is to solve the limited content of precursor amino acids in the existing biosynthesis method, the demand for a large amount of thioneine synthesis cannot be met, and the additional direct addition of amino acids will increase the cost of raw material production, and the output has a technical problem to be further improved.

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

[0008] In certain embodiments, the amino acid sequences of the ergothioneine synthase egtABCDE, ergothioneine synthase egt1 and sulfoxide synthase Mp-egtB are shown in SEQ ID NOs: 1 to 7, respectively.

[0009] In certain embodiments, the genes related to histidine metabolism include the gene hisG expressing ATP phosphoribosyltransferase HisG and a mutant hisG E271K ;

[0010] Genes related to methionine metabolism include metA, a gene encoding homoserine acyltransferase MetA, and metA mutants. A881G , truncated gene thrA of aspartate kinase ThrA, methylenetetrahydrofolate reductase metF, methionine adenosyltransferase metK;

[0011] Mutations in genes related to cysteine ​​metabolism, including serine acetyltransferase CysE T167A ;

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

[0013] In certain embodiments, the hisG GeneID is 1019477, the hisG E271K The nucleotide sequence is shown in SEQ ID NO:8.

[0014] In certain embodiments, the metA has GeneID: 948513, the metA A881G The nucleotide sequence is shown in SEQ ID NO:9;

[0015] The GeneID of thrA is 945803, and the nucleotide sequence of 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 certain embodiments, the GeneID of cysE is 946877, the cysE T167A The nucleotide sequence is shown in SEQ ID NO:13.

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

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

[0021] In certain embodiments, the plasmid pETDuet-1 is used to express the egtA gene and the egt1 gene.

[0022] In certain embodiments, plasmid pCDFDuet-1 is used to express the egtB gene and the egtD gene.

[0023] In certain embodiments, the plasmid pRSDuet-1 is used to express the egtC gene and the egtE gene.

[0024] In certain embodiments, plasmid pACYCDuet-2 is used to express the Mp-egtB gene, as well as genes related to histidine, methionine, cysteine, and S-adenosylmethionine metabolism.

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

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

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

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

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

[0030] (4) The mutant gene hisG E271K The gene was inserted into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-hisG*; the metA mutant metA A881GThe gene is inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-metA*; the truncated gene of thrA is inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-thrA; the metF gene is inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-metF; the metK gene is inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-metK; the mtn and luxS genes are inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-mtn-luxS; the cysE mutant cysE T167A Insert the gene into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-cysE*;

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

[0032] In certain embodiments, the gene yqaB of the Escherichia coli host is knocked out using CRISPR / Cas9 gene editing technology.

[0033] Further, the method for knocking out the gene yqaB comprises the following steps:

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

[0035] (2) Using the CRISPR-cas9 method, the pCas plasmid was first transformed into the Escherichia coli BL21 (DE3) competent cells by electroporation, and then the plasmid sg-pTarget with sgRNA and the yqaB knockout box were electroporated into the Escherichia coli BL21 (DE3) / pCas competent cells; the transformants were picked for colony PCR verification, and the verified correct strains were induced with IPTG to eliminate the sg-pTarget plasmid.

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

[0037] In certain embodiments, the method comprises inoculating the recombinant Escherichia coli into a seed culture medium for cultivation to prepare a seed solution; and inoculating the seed solution into a fermentation medium to ferment and prepare ergothioneine.

[0038] In certain embodiments, the recombinant Escherichia coli is inoculated on LB solid culture medium to obtain a single colony, and the single colony is picked and inoculated into LB liquid culture medium, and cultured at 37° C. and 220 rpm for 10 to 12 hours to prepare a seed solution.

[0039] In certain embodiments, the recombinant Escherichia coli is inoculated onto LB solid culture medium and streaked, a single colony is picked and inoculated into a 100 mL conical flask containing 20 mL LB liquid culture medium, and cultured at 37° C. and 220 rpm for 10 to 12 h to prepare a seed solution.

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

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

[0042] The fourth technical solution provided by the present invention is the use 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 the present invention are as follows:

[0044] Adopting the recombinant Escherichia coli EGT-1-B* provided by the present invention, it is possible to realize that glucose is converted into thioneine; Therefore, the raw material of the present invention is cheap and easy to obtain, without additives and heavy metals that are harmful to human health, and lays a good foundation for the purification of products in the later stage, and it is more possible to meet the requirements of commercial applications. And in the present invention, further overexpression of genes related to histidine, methionine, cysteine, and S-adenosylmethionine metabolism is carried out in recombinant Escherichia coli EGT-1-B*, to improve the precursor amino acid content of thioneine synthesis, and it is found that thioneine production is further improved after knocking out gene yqaB, and the utilization rate of glucose to thioneine can be further improved, and thioneine production can reach 140.29mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The main metabolic pathway of the strain constructed by the present invention is transformed.

[0046] Figure 2 The present invention discloses ergothioneine production results 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 by the present invention. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the following is a detailed description of the present invention in conjunction with the following specific embodiments. Figures 1-2 , the present invention is further described in detail.

[0048] Test method:

[0049] 1. Determination of bacterial concentration: Take 1 mL of bacterial fermentation broth and centrifuge to collect the bacteria, then suspend and dilute with deionized water, and use a UV-visible spectrophotometer (manufacturer) to measure the absorbance OD600 of the diluted bacteria at 600 nm. When the OD600 value of the bacteria is kept between 0.2-0.8, the data is more accurate.

[0050] 2. Detection of thioneine content (HPLC method): fermented liquid was centrifuged at 10,000 × g, 4 ° C for 2 min, supernatant was taken, and filtered with 0.22 μm filter membrane. Shimadzu high performance liquid chromatograph (LC-2050) (Shimadzu Corporation, Kyoto, Japan) was used: UV detector, C18 column (Diamonsil 5 μm C18, 250 × 4.6 mm). Mobile phase ratio: water / methanol = 95 / 5, column temperature: 30 ° C, flow velocity: 0.7 mL / min. Ultraviolet absorption wavelength: 257 nm, injection volume: 10 μL, detection time: 20 min.

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

[0052] Table 1 Preparation of enzyme digestion system

[0053]

[0054] Table 2 Preparation of enzyme-linked system

[0055]

[0056]

[0057] 4. Carry out PCR reaction according to Table 3.

[0058] Table 3 Preparation of PCR system

[0059]

[0060] The raw materials used in the examples are:

[0061] 1. LB medium formula (g / L): yeast powder (OXOID) 5, peptone (OXOID) 10, NaCl (Sinopharm) 10, solid medium plus 1.5%-2.0% agar powder (BIOFROXX).

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

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

[0064] Example 1: Construction of recombinant E. coli EGT

[0065] The specific steps are as follows:

[0066] (1) Preparation of E. coli BL21 (DE3) competent cells: Inoculate the E. coli BL21 (DE3) strain onto LB solid medium and streak to obtain a single colony. Pick a single colony and inoculate it into a 100 mL conical flask containing 20 mL LB liquid medium. Perform activation culture at 37°C and 200 rpm for 12 h to prepare a seed solution. Inoculate the prepared seed solution into a 250 mL conical flask containing 50 mL LB liquid medium at an inoculum volume of 1% (v / v), and culture at 37°C and 200 rpm until the OD reaches 0. 600 =0.5 to obtain fermentation liquid, ice-bath the fermentation liquid for 30 minutes, centrifuge at 6000 rpm for 5 minutes at 4°C to collect the cells, wash the obtained cells once with precooled ultrapure water, wash three times with 0.1 mol / L precooled CaCl2 solution, add 2 mL of 0.1 mol / L CaCl2 solution and 2 mL of 30% glycerol to the collected cells. Each 100 μL portion was packaged and stored in a 1.5 mL EP tube at -70°C for future 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 Hind III sites of the 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 Hind III sites and Kpn I and Nde I sites of the 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 Hind III sites and Kpn I and Nde I sites of the 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 introduced into the E. coli BL21 (DE3) competent cells obtained in step (1) by chemical method to obtain transformation products, which were spread on LB solid medium containing kanamycin, ampicillin, and streptomycin, and cultured at 37° C. until transformants grew out. The strains were stored in glycerol tubes to obtain free-type recombinant E. coli EGT.

[0069] Example 2: Construction of egt1, Mp-egtB free expression strain

[0070] The specific steps are as follows:

[0071] (1) Preparation of E. coli JM109 competent cells: Inoculate the E. coli JM109 strain onto LB solid medium and streak culture to obtain a single colony, pick a single colony and inoculate it into a 100 mL conical flask containing 20 mL LB liquid medium, and activate and culture it at 37°C and 200 rpm for 12 h to prepare a seed solution. Inoculate the prepared seed solution into a 250 mL conical flask containing 50 mL LB liquid medium at an inoculum of 1% (v / v), and culture it at 37°C and 200 rpm until the OD reaches 0. 600 =0.5 to obtain fermentation liquid, ice-bath the fermentation liquid for 30 minutes, centrifuge at 6000 rpm for 5 minutes at 4°C to collect the cells, wash the obtained cells once with precooled ultrapure water, wash three times with 0.1 mol / L precooled CaCl2 solution, add 2 mL of 0.1 mol / L CaCl2 solution and 2 mL of 30% glycerol to the collected cells. Each 100 μL portion was packaged and stored in a 1.5 mL EP tube at -70°C for future use, to prepare E. coli JM109 competent cells.

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

[0073] (3) The recombinant plasmid pET-egtA-egt1 was introduced into the competent cells of E. coli JM109 obtained in step (1) by chemical method to obtain a transformation product, which was spread on LB solid medium containing ampicillin and cultured at 37°C until transformants grew out. The transformants were cultured to extract plasmids, and the plasmids were verified by Kpn I and Nde I restriction enzyme digestion. The verified correct strain was stored in a glycerol tube.

[0074] (4) The recombinant plasmid pACYC-egtB* was introduced into the competent cells of E. coli JM109 obtained in step (1) by chemical method to obtain a transformation product, which was spread on LB solid medium containing chloramphenicol and cultured at 37°C until transformants grew out. The transformants were cultured to extract plasmids, and the plasmids were verified by BamH I and Hind III restriction enzyme digestion. The verified correct strain was stored in a glycerol tube.

[0075] (5) The recombinant plasmid pET-egtA-egt1 prepared in step (3) is introduced into the competent cells of recombinant E. coli EGT by chemical method to obtain a transformation product, which is spread on LB solid medium containing kanamycin, ampicillin, and streptomycin, and cultured at 37° C. until transformants grow out. The strain is stored in a glycerol tube to obtain a free-type recombinant E. coli EGT-1.

[0076] (6) The recombinant plasmid pACYC-egtB* prepared in step (4) is introduced into EGT competent cells by chemical method to obtain transformation products, and the transformation products are spread on LB solid medium containing kanamycin, ampicillin, streptomycin, and chloramphenicol, and cultured at 37° C. until transformants grow. The strain is preserved in a glycerol tube to obtain free recombinant E. 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) are introduced into EGT competent cells by chemical method to obtain transformation products, and the transformation products are spread on LB solid culture medium containing kanamycin, ampicillin, streptomycin and chloramphenicol, and cultured at 37°C until transformants grow. The strain is preserved in a glycerol tube to obtain free-type recombinant E. coli EGT-1-B*.

[0078] (6) The mutant gene hisG E271K The gene was inserted into the Kpn I and Nde I sites of the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-hisG*; the metA mutant metA A881G The gene was inserted into the Kpn I and Nde I sites of the plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-metA*; the truncated gene of thrA was inserted into the Kpn I and Nde I sites of the 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 the 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 the 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 the plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-mtn-luxS; the cysE mutant cysE T167A The gene was inserted into the Kpn I and Nde I sites of the plasmid pACYC-egtB* to obtain the recombinant plasmid pACYC-egtB*-cysE*. A881G The gene is inserted into the plasmid pACYC-egtB*-metK and connected to the metK gene using the RBS sequence to obtain the recombinant plasmid pACYC-egtB*-metK-metA* and the extraction of the above plasmid is verified by referring to the method of 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 an electroporation competent state, and the plasmid pCas carrying the transporter gene was electroporated into the competent cells, and then spread on a kanamycin-resistant LB solid culture medium plate. The single colony that grew was E. coli BL21 (DE3) / pCas.

[0083] (2) Design yqaB site sgRNA primers sg-yqaB-F and sg-yqaB-R (see Table 4), perform whole plasmid PCR using pTarget plasmid as template, purify the obtained gene fragment, digest the template plasmid with DpnⅠ and then transfer it into E. coli JM109 competent cells by chemical transformation to obtain transformation products, spread the transformation products on LB solid culture medium plates containing spectinomycin, pick out single colonies, extract plasmids and send them for sequencing, and the correct ones are the correct transformants.

[0084] (3) Construction of yqaB knockout cassette: Design primers (see Table 4), use PCR amplification to obtain fragments yqaB-U and yqaB-D, and connect the above fragments to obtain the yqaB knockout cassette;

[0085] (4) Using the CRISPR-cas9 method, the pCas plasmid was first transformed into E. coli BL21 (DE3) competent cells by electroporation, and then the plasmid sg-pTarget with sgRNA and the knockout frame of yqaB were electroporated into E. coli BL21 (DE3) / pCas competent cells; the transformants were picked for colony PCR verification, and the verified correct strains were induced with IPTG to eliminate the sg-pTarget plasmid, and the recombinant E. coli was constructed and named E. coliΔyqaB.

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

[0087] Table 4 Primer sequences

[0088]

[0089] Embodiment 4: shake flask fermentation produces ergothioneine

[0090] The specific steps are as follows:

[0091] (1) The recombinant E. coli EGT prepared in Example 1 and the 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* strains prepared in Example 2 were streaked on LB solid culture medium to obtain single colonies, and a single colony was picked and inoculated into a 100-mL conical flask containing 20 mL of LB liquid culture medium, and cultured at 37° C. and 200 rpm for 12 h to prepare a seed solution.

[0092] (2) The seed solution prepared in step (1) was inoculated into a 250 mL conical flask containing 25 mL of fermentation liquid medium at a rate of 2% (v / v), and the culture was cultured at 37° C. and 200 rpm for 3-4 h, followed by induction with IPTG.

[0093] (3) Cultured at 25°C and 200 rpm for 96 h. Figure 2 As shown in Tables 5 to 10.

[0094] Table 5 Effect of histidine module metabolic modification on ergothioneine production

[0095] Time (h) EGT-ABCDE-1-B* EGT-5-hisG* 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 Effect of methionine module metabolic modification on ergothioneine production

[0097] Time (h) EGT-ABCDE-1-B* EGT-5-metA* EGT-5-thrA EGT-5-metF 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 Effect of metabolic modification of S-adenosylmethionine module on ergothioneine production

[0099]

[0100]

[0101] Table 8 Effect of cysteine ​​module metabolic modification on ergothioneine production

[0102] Time (h) EGT-ABCDE-1-B*(mg / L) EGT-5-cysE*(mg / L) 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 Effect of combined metabolic modification between modules on ergothioneine production

[0104] Time (h) EGT-5-metK(mg / L) EGT-5-metK-metA(mg / L) 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 Effect of knocking out yqaB gene on ergothioneine production

[0106] Time (h) EGT-5-metK(mg / L) EGT-5-ΔyqaB-metK(mg / L) 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 to 10, the present invention provides 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*, EGT-5-metK-m etA*, EGT-5-ΔyqaB-metK produced 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 of ergothioneine respectively in shake flask fermentation for 96 h.

[0108] There are still problems and bottlenecks in the production of ergothioneine by microbial fermentation, such as the lack of precursor substances and the shortage of important methyl donors. Therefore, based on this, this study uses glucose as a substrate to synthesize ergothioneine from scratch in Escherichia coli. Through the metabolic transformation of the ergothioneine precursor amino acid module, the ergothioneine production is further improved. The production of ergothioneine in Escherichia coli requires many precursor substances, and its metabolic background is complex and lengthy. The key enzymes and regulatory mechanisms are not yet fully understood, so it is necessary to analyze and study between various modules. Finally, it was found in the study of each module that more ergothioneine can be produced when the supply of methyl donors is increased. After knocking out the gene yqaB, it was found that the production of ergothioneine was further improved, and the utilization rate of glucose to ergothioneine could be further improved. At the same time, the combination optimization between modules also needs further study. In the absence of direct addition of precursor amino acids, it is more economical to produce ergothioneine using glucose as a substrate.

[0109] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli, characterized in that Using Escherichia coli as the host cell, the ergothioneine synthase egtABCDE from Mycobacterium smegmatis, the ergothioneine synthase egt1 from Neurospora crassa and the sulfoxide synthase Mp-egtB from Methylobacterium are recombinantly expressed, and one or more genes related to the metabolism of histidine, methionine, cysteine ​​and S-adenosylmethionine are expressed.

2. The recombinant Escherichia coli according to claim 1, characterized in that The amino acid sequences of the ergothioneine synthase egtABCDE, ergothioneine synthase egt1 and sulfoxide synthase Mp-egtB are shown in SEQ ID NOs: 1 to 7, respectively.

3. The recombinant Escherichia coli according to claim 1, characterized in that The genes related to histidine metabolism include the gene hisG expressing ATP phosphoribosyltransferase HisG and the mutant hisG E271K ; Genes related to methionine metabolism include metA, a gene encoding homoserine acyltransferase MetA, and metA mutants. A881G , truncated gene thrA of aspartate kinase ThrA, methylenetetrahydrofolate reductase metF, methionine adenosyltransferase metK; Mutations in genes related to cysteine ​​metabolism, including serine acetyltransferase CysE T167A ; Genes related to S-adenosylmethionine metabolism include the mtn gene encoding the 5'-methylthioadenosine / S-adenosylhomocysteine ​​nucleosidase Mtn and the luxS gene encoding the S-ribosylhomocysteine ​​lyase LuxS.

4. The recombinant Escherichia coli according to claim 3, characterized in that The hisG GeneID: 1019477, the hisG E271K The nucleotide sequence is shown in SEQ ID NO:8; The metA GeneID: 948513, the metA A881G The nucleotide sequence is shown in SEQ ID NO:9; The GeneID of thrA is 945803, and the nucleotide sequence of thrA is shown in SEQ ID NO: 10; The GeneID of metF is 948432, and the nucleotide sequence of metF is shown in SEQ ID NO: 11; The GeneID of metK is 945389, and the nucleotide sequence of metK is shown in SEQ ID NO: 12; The GeneID of cysE: 946877, the cysE T167A The nucleotide sequence is shown in SEQ ID NO:13; The GeneID of the mtn is 948542; the nucleotide sequence of the mtn is shown in SEQ ID NO: 14; The GeneID of the luxS is 947168. The nucleotide sequence of the luxS is shown in SEQ ID NO:

15.

5. The recombinant Escherichia coli according to claim 1, characterized in that Plasmid pETDuet-1 was used to express the egtA gene and the egt1 gene; Plasmid pCDFDuet-1 was used to express egtB gene and egtD gene; Plasmid pRSDuet-1 was used to express egtC gene and egtE gene; Plasmid pACYCDuet-2 was used to express the Mp-egtB gene and genes related to histidine, methionine, cysteine, and S-adenosylmethionine metabolism.

6. The recombinant Escherichia coli according to claim 1, characterized in that The host cell also has the gene yqaB knocked out.

7. A method for constructing a recombinant Escherichia coli, characterized in that: The method comprises the following steps: (1) Inserting the egtA gene into the plasmid pETDuet-1 to obtain the recombinant plasmid pET-egtA; (2) inserting egtB and egtD genes into plasmid pCDFDuet-1 to obtain recombinant plasmid pCD-egtB-egtD; inserting egtC and egtE genes into plasmid pRSDuet-1 to obtain recombinant plasmid pRS-egtC-egtE; inserting egt1 gene into plasmid pET-egtA of step (1) to obtain recombinant plasmid pET-egtA-egt1; (3) Insert the Mp-egtB gene into the plasmid pACYCDuet-2 to obtain the recombinant plasmid pACYC-egtB*; (4) The mutant gene hisG E271K The gene was inserted into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-hisG*; the metA mutant metA A881G The gene is inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-metA*; the truncated gene of thrA is inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-thrA; the metF gene is inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-metF; the metK gene is inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-metK; the mtn and luxS genes are inserted into the plasmid pACYC-egtB* of step (3) to obtain the recombinant plasmid pACYC-egtB*-mtn-luxS; the cysE mutant cysE T167A Insert the gene into the plasmid pACYC-egtB* in step (3) to obtain the recombinant plasmid pACYC-egtB*-cysE*; (5) Introducing all the recombinant plasmids in step (2) and one or more recombinant plasmids in step (4) into an Escherichia coli host to obtain recombinant Escherichia coli.

8. The method according to claim 7, characterized in that The gene yqaB of the Escherichia coli host was knocked out using CRISPR / Cas9 gene editing technology.

9. A method for producing ergothioneine, characterized in that, The method comprises the step of using the recombinant Escherichia coli according to any one of claims 1 to 6 to ferment and produce ergothioneine.

10. Use of the recombinant Escherichia coli according to any one of claims 1 to 6 in the preparation of ergothioneine, products containing ergothioneine and ergothioneine derivatives.

Images