Bacterial strain for producing ergothioneine, biological material, production method and application

Abstract

The invention provides a strain for producing ergothioneine, and belongs to the technical field of synthetic biology. The strain expresses and encodes a gene of EgtD methyltransferase, and the methyltransferase comprises an amino acid sequence as shown in SEQ ID NO. 15. According to the application, an efficient escherichia coli expression system is adopted, a plurality of EgtD gene sequences are screened from a database for in-vitro enzyme activity characterization by taking currently reported EgtD enzyme from Mycobacterium smegmatis with the highest activity as a contrast, and finally, an EgtD strain of which the activity is improved by 71% is successfully screened. The high-activity EgtD is a methyltransferase gene derived from Gammaproteobacteria bacterium, the enzyme activity is improved by 71% compared with that of a control group, and the yield of an engineering strain ergothioneine containing the enzyme is improved by 30%.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic biology, and in particular relates to a strain, a biological material, a production method and an application for producing ergothioneine. Background Art

[0002] Ergothioneine (EGT) is a natural chiral amino acid strong oxidant and the only natural 2-thioimidazole amino acid discovered so far. It is widely found in human daily diets such as mushrooms, black beans, red meat, and oats. It has many functions such as scavenging free radicals, detoxification, maintaining DNA biosynthesis, normal cell growth, anti-radiation, whitening and anti-aging. It can be widely used in the fields of medicine, food, health care products, and cosmetics. For example, it can be made into tablets, capsules, oral preparations, etc. in clinical practice; in the field of cosmetics, it can be used for anti-aging and can be made into products such as sunscreens. In 2014, ergothioneine was included in the list of cosmetic raw materials by the Food and Drug Administration. In 2018, the European Union approved the use of L-ergothioneine in some foods. At present, there are nutritional supplements containing ergothioneine and high-end chemicals containing ergothioneine on the market.

[0003] The synthetic method of thioneine at present mainly contains chemical synthesis and biosynthesis.Chemical synthesis is to take L-histidine methyl ester as starting material, and can indirectly realize the sulfurization of nitrogen heterocycle and the trimethylation of amino (reference patent US7767826B2) by multiple functional group selective protection and deprotection, and whole preparation technology is very loaded down with trivial details, and final yield is low, and environmental cost is high.And biosynthesis improves the output of thioneine, mainly by improving the activity of key enzyme in thioneine synthetic pathway to realize, and the in vitro enzyme catalysis preparation of thioneine also has relevant report, and it adopts thioneine Egt series synthase catalysis (Florian P.Seebeck, JACS, 2010,132,6632-6633) in organism.However, because this route is longer, EgtD enzyme and EgtB enzyme conversion efficiency are low, and the reasons such as adenosylmethionine consumption is large, enzyme synthesis prepares thioneine cost high.

[0004] There are many reports on the transformation of key enzymes for ergothioneine abroad: In 2010, Seebeck.FP recombined the genes of enzymes such as egtABCDE from Mycobacterium smegmatis in Escherichia coli, realizing the synthesis of ergothioneine in Escherichia coli (Florian P.Seebeck, JACS, 2010, 132, 6632-6633). The EgtD enzyme is responsible for transferring the methyl group of S-adenosylmethionine to histidine to generate histidine betaine, which is the first step in the synthesis of ergothioneine. In 2015, Vit et al. characterized EgtD, and the enzyme had a conversion number of only 0.58s for the substrate histidine. -1 (Misson, Laetitia, Chembiochem: A European journal of chemical biology, 2015.), is 2.3% of the most active EgtE enzyme activity in metabolic pathway. Therefore, EgtD is the key rate-limiting step of thioneine synthesis. In 2015, Vit etc. carried out structural analysis (PDB 4PIN) (Misson, Laetitia, Chembiochem: A European journal of chemical biology, 2015.), obtain the structural complex of EgtD and its substrate, transform EgtD enzyme active center, result shows that the activity of EgtD enzyme catalysis substrate histidine is reduced.

[0005] There are also some reports on the modification of ergothioneine-related enzymes in China: In 2020, Xie Xixian's research group at Tianjin University of Science and Technology used Escherichia coli to express egtBCDE from Mycobacterium smegmatis to produce ergothioneine (CN112251392A). In 2022, Kang Zhen's research group at Jiangnan University introduced egtABCDE from Mycobacterium smegmatis to construct and increase the production of ergothioneine (Wang Li, et al. Journal of Biotechnology, 2022(002):038.). However, there are few reports on the modification of EgtD and enzymes from other sources in China.

[0006] In the synthesis process of thioneine, the first step of the reaction, histidine, synthesizes histidine betaine under the catalysis of methyltransferase, which is an important rate-limiting step in the synthesis step of thioneine. However, the methyltransferase enzyme activity reported in the current literature and patents is relatively low, which does not meet the requirements of industrial production. Summary of the invention

[0007] In order to solve the above problems, the application is screened by the enzyme gene in the methyltransferase database, heterologous expression and in vitro enzyme activity determination, screens the EgtD enzyme gene sequence with higher enzyme activity, and is applied to engineering bacteria to produce thioneine. In addition, the application improves the generation efficiency of the key node histidine betaine in the synthesis process of thioneine by digging EgtD methyltransferase from the database, and improves the yield of thioneine.

[0008] On the one hand, the present application provides a strain for producing ergothioneine, wherein the strain expresses a gene encoding EgtD methyltransferase, and the methyltransferase comprises an amino acid sequence as shown in SEQ ID NO.15.

[0009] Optionally, the strain capable of expressing the methyltransferase having the amino acid sequence shown in SEQ ID NO.15 described in the present application may be a strain isolated from nature, or may be an artificially synthesized genetically engineered bacterium.

[0010] In one embodiment, the methyltransferase EgtD is derived from Gammaproteobacteriabacterium.

[0011] In one embodiment, the strain also expresses the EgtBCDE gene cluster.

[0012] Preferably, the EgtBCDE gene cluster is derived from Mycobacterium smegmatis, and is composed of amino acid sequences encoding EgtB, EgtC, EgtD, and EgtE.

[0013] More preferably, the EgtB encoding amino acid sequence is shown as SEQ ID NO.19; the EgtC encoding amino acid sequence is shown as SEQ ID NO.20; the EgtD encoding amino acid sequence is shown as SEQ ID NO.21; and the EgtE encoding amino acid sequence is shown as SEQ ID NO.22.

[0014] Preferably, the strain also expresses a nucleic acid molecule encoding the EgtBCDE gene cluster.

[0015] More preferably, the nucleotide molecule derived from the EgtBCDE gene cluster of Mycobacterium smegmatis (ie, the MsEgtBCDE gene cluster) is as shown in SEQ ID NO.17.

[0016] In one embodiment, the host bacteria of the strain is selected from one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae, preferably Escherichia coli.

[0017] Optionally, the host of the Escherichia coli and the recombinant microorganism may be Escherichia coli EGT33 and / or Escherichia coli BL21 (DE3).

[0018] Among them, Escherichia coli BL21 (DE3) can be selected from commercial products; the construction method of Escherichia coli EGT33 has been disclosed in the patent document with application number ZL202211546541.5, and the construction method recorded in the patent can be adopted.

[0019] On the other hand, the present application also provides a biomaterial, wherein the biomaterial comprises any of the following:

[0020] B1) a nucleic acid molecule encoding a methyltransferase according to any one of claims 1 to 3;

[0021] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] B4) a whole cell catalyst containing the nucleic acid molecule described in B1), or a whole cell catalyst containing the expression cassette described in B2), or a whole cell catalyst containing the recombinant vector described in B3).

[0024] In one embodiment, the nucleic acid molecule B1) is any one of the following:

[0025] C1) a DNA molecule encoding the amino acid sequence shown in SEQ ID No.15;

[0026] C2) a DNA molecule whose nucleotide sequence is shown in SEQ ID No.18;

[0027] C3) a DNA molecule that has 98% or more identity with the nucleotide sequence defined in any one of C1) to C2) and encodes the methyltransferase;

[0028] C4) is a genomic DNA molecule that hybridizes with any one of the nucleotide sequences defined in C1) to C2) under stringent conditions and encodes the methyltransferase.

[0029] On the other hand, the present application also provides the use of the strain and / or the biomaterial in producing ergothioneine or food, medicine and / or cosmetics containing ergothioneine.

[0030] Optionally, the dosage form of the medicine, food or cosmetic includes but is not limited to powder, tablet, capsule, gel, or external ointment such as lotion, emulsion, cream, mask, etc., or daily necessities such as shampoo, conditioner, hair mask, etc.

[0031] On the other hand, the present application also provides a method for producing ergothioneine, comprising the step of producing ergothioneine using the strain and / or the biomaterial.

[0032] Preferably, the substrate used in the method is histidine.

[0033] Preferably, the production method comprises: culturing the strain to obtain a fermentation broth, and obtaining ergothioneine from the fermentation broth.

[0034] Preferably, the culture includes but is not limited to shake flask culture and fermenter culture.

[0035] On the other hand, the present application also provides a method for a recombinant microorganism, wherein the recombinant microorganism is capable of producing ergothioneine using the methyltransferase expressed by the strain, and the method comprises:

[0036] The ergothioneine production of the microorganism is regulated by regulating the expression of the gene encoding the methyltransferase, or regulating the activity or content of the methyltransferase, so as to obtain a microorganism with a changed ergothioneine production.

[0037] Preferably, the method for regulating the expression of the gene encoding the methyltransferase or regulating the activity or content of the methyltransferase is any one of the following:

[0038] D1) introducing the gene encoding the methyltransferase into the target microorganism;

[0039] D2) Introducing a gene encoding the amino acid sequence of SEQ ID NO.15 into the target microorganism.

[0040] On the other hand, the present application also provides any of the following applications of the strain and / or the biological material:

[0041] E1) Application in regulating the production of ergothioneine from microorganisms;

[0042] E2) application in the preparation of ergothioneine;

[0043] E3) Application in constructing genetically engineered microorganisms of ergothioneine.

[0044] On the other hand, the present application also provides any of the following uses of a methyltransferase comprising the amino acid sequence shown in SEQ ID NO.15:

[0045] F1) Application in regulating the production of ergothioneine of microorganisms;

[0046] F2) application in the preparation of ergothioneine;

[0047] F3) is used in constructing genetically engineered microorganisms of ergothioneine.

[0048] In one embodiment, the biological cell of the recombinant microorganism is yeast, bacteria, algae, fungi, plant cells or animal cells, preferably bacteria, more preferably Escherichia coli.

[0049] It is to be understood that the "vector" mentioned in the present application refers to a vector that can carry exogenous DNA or target gene into host cells for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, phage (such as λ phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid) or viral vector.

[0050] In one embodiment, the gene expression method of the strain described in the present application includes but is not limited to plasmid expression, genome multi-copy expression, strong promoter expression or fusion protein expression.

[0051] In a preferred embodiment, the strain described in the present application uses a recombinant plasmid as a vector to express a gene encoding EgtD methyltransferase, and the recombinant plasmid includes pET series, pQE series, pRSET series, pGEX series, pBV series, pUC series, pTrc99A, pTwin1, pEZZ18, pKK232-18, and pACYC series, preferably pET series and pACYC series.

[0052] More preferably, the recombinant plasmid is pACYC-PSC101-Ptac, which is obtained by replacing the promoter T7-1 in the plasmid pACYCDuet-1 with Ptac and the replicon with PSC101.

[0053] The "microorganism" described herein may be yeast, bacteria, algae or fungi. Among them, the bacteria may be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., Brevibacterium sp., Corynebacterium sp., Aerobacter sp., Enterobacteria sp., Micrococcus sp., Serrati sp., Salmonella sp., Streptomyces sp., Providencia sp., etc., but are not limited thereto. Further, the recombinant microorganism is one or more of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium pekinense, Brevibacterium ammoniagenes, Corynebacterium crenatum or Pantoea, but is not limited thereto.

[0054] This application has at least the following beneficial effects:

[0055] 1. The application adopts efficient Escherichia coli expression system, with the EgtD enzyme in the source of the most active Mycobacterium smegmatis (Mycolicibacterium smegmatis) reported at present as control, 15 EgtD gene sequences are screened from the gene database for in vitro enzyme activity characterization, and finally successfully screens the EgtD strain with 71% activity improvement. The highly active EgtD is the methyltransferase gene in Gammaproteobacteria bacterium source, and the enzyme activity is improved by 71% over the control. Moreover, after the gene is imported into Escherichia coli, compared with the control strain EGT33, the OD of C14 strain increases by 2%, and the output of thioneine reaches 244.63mg / L, increases by 16%, with significantly improved thioneine output. DETAILED DESCRIPTION

[0056] In order to more clearly explain the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided so as to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0057] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0058] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0059] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are performed in accordance with the kit instructions.

[0060] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0061] Technical terms:

[0062] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules in molecular evolution research.

[0063] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0064] Expression cassette: An expression cassette is a group of DNA sequences consisting of a promoter, a target gene, and a reporter gene, which can be expressed in specific tissues and easily detected.

[0065] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0066] Whole-cell catalyst: Whole-cell biocatalysis refers to the process of using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst for chemical transformation. The corresponding complete biological organism involved in the catalytic process is the whole-cell catalyst.

[0067] Engineered strains (recombinant microorganisms): Fungal cell strains that use genetic engineering methods to efficiently express exogenous genes.

[0068] Recombinant cell: The term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. , Bacterial species: microorganisms used as living cell catalysts in fermentation processes.

[0069] Inoculation size: Inoculation size refers to the ratio of the volume of seed solution transferred in to the volume of culture solution after inoculation.

[0070] Example 1 Construction of genetically engineered bacteria

[0071] The EgtD gene from Mycolicibacterium smegmatis was selected, the restriction sites NdeⅠ and XhoⅠ in the gene base sequence were removed, the NdeⅠ site was inserted at the N-terminus of the gene, the 6×His tag, the stop codon and the XhoⅠ site were connected to the C-terminus, and then connected to the vector pET30a to obtain the recombinant plasmid pET30a-E0, which was transferred into the E. coli BL21 (DE3) host to obtain the genetically engineered bacteria E. coli BL21 (DE3) / pET30a-E0, i.e., the E0 strain.

[0072] Through the screening of genes in the NCBI and Brenda databases, a total of 15 gene sequences from different host sources were selected for gene synthesis. The synthesized genes were connected to the Nde I and Xho I sites of the pET30a expression vector to obtain plasmids pET30a-E1, pET30a-E2, pET30a-E3, pET30a-E4, pET30a-E5, pET30a-E6, pET30a-E7, pET30a-E8, pET30a-E9, pET30a-E10, pET30a-E11, pET30a-E12, pET30a-E13, pET30a-E14, and pET30a-E15. The above 16 plasmids were respectively transformed into Escherichia coli BL21 (DE3) to obtain genetically engineered strains E1, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11, E12, E13, E14, and E15.

[0073] Example 2 Establishment of high-throughput enzyme activity detection method

[0074] The methyltransferase activity determination method in the reference (A method for detecting S-adenosylmethionine-dependent methyltransferase activity [J]. Gu Jinsong. Journal of Chemistry in Higher Education Institutions. 2012, 3: 512-525.) was used to synthesize the S-adenosyl-L-homocysteine ​​nucleosidase SAHN (EC 3.2.2.9) and S-ribosylhomocysteine ​​enzyme SRHH (EC 4.4.1.21) genes, a hexahistidine tag was inserted into the N-terminus of the SAHN gene, and a hexahistidine tag was inserted into the C-terminus of the SRHH gene to obtain recombinant plasmids pET30-SAHN and pET30-SRHH. The plasmids were transferred into the E. coli BL21 (DE3) host to obtain genetically engineered bacteria E. coli BL21 (DE3) / pET30-SAHN and E. coli BL21 (DE3) / pET30-SRHH.

[0075] Single colonies of E. coli BL21(DE3) / pET30-SAHN and E. coli BL21(DE3) / pET30-SRHH were picked from the plates and inoculated into 5 mL of LB medium (containing 50 μg / mL Kan) and cultured overnight at 37°C and 220 rpm. The above culture solution was transferred to 50 mL of LB medium (containing 50 μg / mL Kan) at a ratio of 1:100 and cultured at 37°C and 220 rpm. When the OD reached 0.6-0.8, 0.2 mM isopropylthiogalactoside (IPTG) was added and cultured at 16°C and 220 rpm for 16 h. 120OD of bacteria were collected and suspended with 1mL buffer (100mM Tris-HCl, pH 8.0), and then the bacterial cells were ultrasonically disrupted. The precipitate was removed by centrifugation at 12000rpm / min for 60min, and the supernatant was taken for enzyme protein purification. The supernatant was added to a Ni2+ agarose affinity chromatography column and washed with Binding buffer (50mmol / L Tris-HCl, 50mmol / L NaCl and 5mmol / L imidazole, pH=8.0) for 3 to 4 column volumes. Finally, it was eluted with Elution buffer (50mmol / LTris-HCl, 150mmol / L NaCl and 250mmol / L imidazole, pH=8.0) solution to finally obtain pure enzyme protein. The protein concentration was determined using Nanodrop, and the SAHN enzyme solution was diluted 100 times for standby use.

[0076] The enzyme-coupled analysis method is as follows: S-adenosyl-L-homocysteine ​​(AdoHcy) is used as a standard substance, and the concentration range of the standard substance is 0-200 μmol / L. The enzyme reaction mixture (0.1 μmol / L SAHN, 10 μmol / L SRHH and 100 mmol / L Hepes buffer (pH = 8.0)) is added, and after reacting at 37°C for 30 minutes, 4 volumes of 5,5'-dithio-2,2'-dinitrobenzoic acid (DTNB) (133 μmol / L)-guanidine hydrochloride (8 mol / L) solution are added to terminate the reaction, and the amount of TNB generated is measured colorimetrically at 412 nm. The blank control uses the enzyme reaction system without SAHN and 4 volumes of DTNB-guanidine hydrochloride mixture.

[0077] Example 3 Detection of enzyme activity of genetically engineered strains

[0078] The experimental method is as follows:

[0079] (1) Single colonies of 16 engineered strains (E0-E15) were picked from the plates and inoculated into 5 mL of LB medium (containing 50 μg / mL Kan) and cultured overnight at 37°C and 220 rpm.

[0080] (2) The above culture solution was transferred to 50 mL of LB medium (containing 50 μg / mL Kan) at a ratio of 1:100 and cultured at 37°C and 220 rpm. When the OD reached 0.6-0.8, 0.2 mM isopropylthiogalactoside (IPTG) was added and cultured at 16°C and 220 rpm for 20 h.

[0081] (3) Collect 120OD of bacterial cells, suspend them in 1mL buffer (100mM Tris-HCl, pH 8.0), and then ultrasonically disrupt the bacterial cells. Centrifuge at 12000rpm for 60min to remove the precipitate. Take the supernatant for enzyme protein purification. Apply the supernatant to a Ni2+ agarose affinity chromatography column and wash it for 3 to 4 column volumes with a binding buffer (50mmol / L Tris-HCl, 50mmol / L NaCl and 5mmol / L imidazole, pH=8.0). Finally, elute it with an elution buffer (50mmol / L Tris-HCl, 150mmol / L NaCl and 250mmol / L imidazole, pH=8.0) to obtain pure enzyme protein. The protein concentration was determined using Nanodrop, and the enzyme solution was diluted 50 times for later use.

[0082] Enzyme activity detection system and method: To ensure the accuracy of enzyme activity detection, the final enzyme activity detection system is 200 μL, of which the final concentrations of L-histidine and S-adenosylmethionine are 400 μmol / L. According to the enzyme activity detection method, 0.1 μmol / L SAHN and 10 μmol / L SRHH are added. The amount of methyltransferase (EgtD) added is uniformly 5 μg / mL. Hepes buffer (pH = 8.0) is used to make up the system. After reacting at 37°C for 30 minutes, 4 times the volume of DTNB-guanidine hydrochloride solution is added to terminate the reaction. The amount of TNB generated is colorimetrically determined at 412 nm, and E0 is used as the control to compare the changes in enzyme activity.

[0083] The relative enzyme activities of the engineered bacterial methyltransferases from different sources constructed above are compared in Table 1.

[0084] Table 1 Comparison of gene numbers, corresponding sequences, sources and relative activities involved in strain construction

[0085]

[0086]

[0087] From the content of Table 1, it can be seen that the enzyme activity of the engineered bacteria E. coli BL21 (DE3) / pET30-E14 derived from Gammaproteobacteria bacteria was increased the most, by about 71%, which is the highest level reported so far.

[0088] Embodiment 4 Construction of thioneine genetic engineering bacteria

[0089] use DNA Polymerase (purchased from takara, R045A) was used as the amplification enzyme, and plasmid pET30a-E14 was used as the template to amplify the target gene EgtD. The primers used for PCR amplification are shown in Table 2.

[0090] Table 2 Primers for PCR amplification

[0091]

[0092] Agarose gel electrophoresis is carried out to detect PCR product after amplification is completed. Target band is cut from agarose gel, and gel recovery kit (Omega, D2500-02) is used to carry out DNA purification and recovery. The fragment NcoI-HF (purchased from NEB, R3193S) and XhoI (purchased from NEB, R0146V) recovered are double digested, (the applicant builds preservation by being connected into carrier pACYC-PSC101-Ptac, construction method is that the T7-1 promoter in plasmid pACYCDuet-1 (Excellent Treasure Biology) is replaced by Ptac promoter, replicon is replaced by PSC101, plasmid pACYC-PSC101-Ptac is obtained), after obtaining plasmid pACYC-PSC101-Ptac-E14 to be sequenced correctly, proceed to the Escherichia coli EGT33 that can produce thioneine, obtain engineering strain C14.

[0093] Among them, the construction method of Escherichia coli EGT33 is shown in Patent 202211546541.5, which specifically includes the following steps:

[0094] The genetically engineered bacteria uses Escherichia coli as a host, heterologously overexpresses the egtD gene from Microcoleus sp. PCC 7113, the egtB gene from Methylobacterium pseudosasicola, the egt2 gene from Neu rospora crassa, the ATP phosphoribosyl transferase mutant encoding gene hisG* of Corynebacterium glutamicum; and overexpresses the hisDBCHAFI gene cluster, the serine transacetylase mutant CysE (M201R) encoding gene, the phosphoglycerate dehydrogenase enzyme mutant SerA (H344A / N364A) encoding gene, and the adenosylmethionine synthase mutant MetK (I303V) encoding gene; and does not express the tryptophanase gene tnaA. The strain systematically transforms the ergothioneine synthesis module, the precursor histidine, cysteine ​​and adenosylmethionine synthesis module, and realizes the efficient and stable production of ergothioneine in Escherichia coli.

[0095] The information of the above-mentioned engineered bacteria is shown in Table 3.

[0096] Table 3 Engineering bacteria information

[0097] Name of engineered bacteria Plasmid / Host antibiotic EGT33 Comparison (stored by the company) none C14 pACYC-PSC101-Ptac-E14 / EGT33 Cm(30μg / mL)

[0098] Example 5 Engineering bacteria shake flask culture and thioneine production

[0099] The engineered strain EGT33, C14 was streaked on the LB plate containing the corresponding resistance and cultured in a 37°C incubator overnight. Each engineered bacterium picked 3 monoclones and inoculated them into 4mL of LB medium containing the corresponding resistance, and cultured overnight at 37°C and 200rpm. Take the above-mentioned seed solution 3mL respectively and add it to a 500ml triangular flask containing 30mL of the corresponding resistance fermentation medium (formula as shown in Table 4), culture for 0.5h at 37°C and 200rpm, add the inducer isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2mM, induce at 37°C, and synthesize the target product ergothioneine. During the culture process, ammonia water was used to regulate pH so that pH was maintained at about 7.0. Shake flask culture ended for 10-12h, and fermentation supernatant was collected by centrifugation at 12000rpm for 2min to measure the yield of biomass OD and ergothioneine.

[0100] Table 4 Fermentation medium formula

[0101]

[0102]

[0103] The concentration of ergothioneine in the fermentation broth was detected by high performance liquid chromatography. Detection conditions: chromatographic column Kromasil C18 (250 mm × 4.60 mm, 5 μm); mobile phase: acetonitrile: water = 2:98 (volume ratio); flow rate 0.7 mL / min; column temperature 30 ° C; ultraviolet detection wavelength 257 nm. The results are shown in Table 5.

[0104] Table 5 The output of engineering strain thioneine

[0105] OD Yield (mg / L) EGT33 12.79±0.44 210.92±8.63 C14 13.09±0.46 244.63±1.37

[0106] As shown in Table 5, compared with the control strain EGT33, the OD of the C14 strain increased by 2%. The yield of ergothioneine was 16% higher, reaching 244.63 mg / L.

[0107] Example 6 Effect detection of EgtD from different sources in BL21 (DE3) host

[0108] (1) Target gene amplification and recovery

[0109] In this example, the genome of Mycobacterium smegmatis (purchased from CGMCC, No. 1.562) was used as a template. DNA Polymerase (purchased from takara, R045A) was used as the amplification enzyme, and PCR amplification was performed using the primers listed in Table 6 below to amplify the MsEgtBCDE gene cluster.

[0110] Table 6 Primers for PCR amplification

[0111]

[0112] After amplification, PCR products were detected by agarose gel electrophoresis, the target band was cut out from the agarose gel, and the DNA was purified and recovered using a gel recovery kit (Omega, D2500-02).

[0113] (2) Enzyme digestion and recovery of expression vector

[0114] The expression vector pETDuet-1 (purchased from Youbao Bio, VT1237, Amp resistance) was double-digested with restriction endonucleases NcoI-HF (purchased from NEB, R3193S) and XhoI (purchased from NEB, R0146V), reacted at 37°C for 2h, and then the DNA was purified and recovered using a recovery kit (purchased from Tiangen, DP214-03).

[0115] (3) Gibson connection

[0116] The purified and recovered single DNA fragment and the expression vector after enzyme digestion have some repetitive sequences at the end, which can be connected to form a circular plasmid by Gibson kit (Full Gold, CU201-03). After the reaction system is prepared, the connection can be completed by reacting in a 50℃ water bath for 15 minutes, and the subsequent transformation experiment can be carried out.

[0117] (4) Chemical transformation of Escherichia coli

[0118] Take the competent E. coli Trans10 cells (purchased from Quanshijin, CD101-02) and place them in an ice bath. After the competent cells melt, add 4-10 μL of Gibson ligation product for chemical transformation. After recovery, take 100 μL of bacterial solution and spread it on the LB solid medium plate containing 100 mg / mL Amp, and culture it in a 37°C incubator overnight to obtain transformants.

[0119] (5) Sequencing verification

[0120] Green Taq Mix (purchased from Novazon, P131-01) was used for PCR screening and verification, and T7ter (nucleotide sequence GCTAGTTATTGCTCAGCGG) was selected as the screening primer. 24 single clones were selected for each construction for screening and verification, and positive clones were screened.

[0121] Three single clones were picked and added to 4 mL of LB liquid culture medium containing 100 mg / mL Amp, and cultured overnight at 37°C and 200 rpm. The bacteria were then collected and the plasmid was extracted using the Tiangen Plasmid Extraction Kit DP103-03 for sequencing verification.

[0122] (6) Chemical transformation to prepare engineered bacteria

[0123] Take 1uL of the correctly sequenced plasmid pETDuet-MsEgtBCDE and pET-30a-E0 and pET-30a-E14 (obtained by gene synthesis by Jin Weizhi) and transform them into Escherichia coli BL21 (DE3) competent cells (purchased from Quanshijin, CD601-02). After recovery, spread the bacterial solution on a LB solid culture medium plate containing 100μg / mL Amp+50μg / mLKan resistance, and culture it in a 37°C incubator overnight. Select single clones and inoculate them in 4mL of LB liquid culture medium containing 100μg / mL Amp+50μg / mLKan resistance, culture them overnight at 37°C and 200rpm to obtain strains D0 and D14. Then store the bacterial solution in more than 30% glycerol and store it at -80°C.

[0124] The information of the engineered bacteria constructed in this example is shown in Table 7 below.

[0125] Table 7 Construction of engineering bacteria information

[0126] Name of engineered bacteria Plasmid / Host Resistance D0 pETDuet-MsEgtBCDE+pET-30a-MsEgtD / BL21(DE3) Amp+Kan D14 pETDuet-MsEgtBCDE+pET-30a-E14 / BL21(DE3) Amp+Kan

[0127] (7) Shake flask culture verification

[0128] Engineering strains D0 and D14 were streaked on LB plates containing corresponding resistance and cultured in 37°C incubators overnight. Each engineering bacterium picked 3 monoclonal inoculations into 4mL LB medium containing corresponding resistance, and cultured overnight at 37°C and 200rpm. Take the above-mentioned seed solution 3mL respectively and add it to a 500ml triangular flask equipped with 30mL of corresponding resistance fermentation medium (formula as shown in Table 4), culture 0.5h at 37°C and 200rpm, add the inducer isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2mM, induce at 37°C, and synthesize the target product thioneine. During the culture process, ammonia was used to regulate pH so that pH was maintained at about 7.0. Shake flask culture ended for 10-12h, and fermentation supernatant was collected at 12000rpm for 2min to measure the yield of biomass OD and thioneine.

[0129] The concentration of ergothioneine in the fermentation broth was detected by high performance liquid chromatography. Detection conditions: chromatographic column Kromasil C18 (250 mm × 4.60 mm, 5 μm); mobile phase: acetonitrile: water = 2:98 (volume ratio); flow rate 0.7 ml / min; column temperature 30 ° C; ultraviolet detection wavelength 257 nm. The results are shown in Table 8.

[0130] Table 8 Genetically engineered bacteria ergothioneine production

[0131] Engineered strains OD Ergothioneine yield (mg / L) D0 2.19±0.08 19.77±1.37 D14 3.52±0.59 26.16±4.70

[0132] As shown in Table 8, compared with the control strain D0, the OD of the D14 strain increased by 61%, and the yield of ergothioneine was 30% higher.

[0133] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A bacterial strain for producing ergothioneine, characterized in that, The strain expresses a gene encoding EgtD methyltransferase, and the methyltransferase comprises an amino acid sequence as shown in SEQ ID NO.

15.

2. The strain according to claim 1, characterized in that The host bacteria of the strain is selected from one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, and Saccharomyces cerevisiae, preferably Escherichia coli.

3. Biomaterial, characterized in that The biological material includes any of the following: B1) a nucleic acid molecule encoding the methyltransferase according to claim 1 or 2; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a whole cell catalyst containing the nucleic acid molecule described in B1), or a whole cell catalyst containing the expression cassette described in B2), or a whole cell catalyst containing the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that B1) The nucleic acid molecule is any of the following: C1) a DNA molecule encoding the amino acid sequence shown in SEQ ID No.15; C2) a DNA molecule whose nucleotide sequence is shown in SEQ ID No.18; C3) a DNA molecule that has 98% or more identity with the nucleotide sequence defined in any one of C1) to C2) and encodes the methyltransferase; C4) is a genomic DNA molecule that hybridizes with any one of the nucleotide sequences defined in C1) to C2) under stringent conditions and encodes the methyltransferase.

5. The method according to claim 1 or 2, and / or the method according to claim 3 or 4, wherein the biomaterial is used in the production of thioneine or in food, medicine and / or cosmetics containing thioneine.

6. A method for producing ergothioneine, characterized in that, The method comprises the step of producing ergothioneine by utilizing the strain as claimed in claim 1 or 2, and / or the biomaterial as claimed in claim 3 or 4.

7. A method for a recombinant microorganism, wherein the recombinant microorganism is capable of producing ergothioneine using a methyltransferase expressed by a strain as described in claim 1 or 2, characterized in that, The method comprises: The ergothioneine production of the microorganism is regulated by regulating the expression of the gene encoding the methyltransferase, or regulating the activity or content of the methyltransferase, so as to obtain a microorganism with a changed ergothioneine production.

8. The method according to claim 7, characterized in that The method for regulating the expression of the gene encoding the methyltransferase or regulating the activity or content of the methyltransferase is any one of the following: D1) introducing the gene encoding the methyltransferase into the target microorganism; D2) Introducing a gene encoding the amino acid sequence of SEQ ID NO.15 into the target microorganism.

9. Any of the following uses of the strain according to claim 1 or 2, and / or the biological material according to claim 3 or 4; E1) Application in regulating the production of ergothioneine from microorganisms; E2) application in the preparation of ergothioneine; E3) Application in constructing genetically engineered microorganisms of ergothioneine.

10. Any of the following uses of a methyltransferase comprising the amino acid sequence shown in SEQ ID NO.15: F1) Application in regulating the production of ergothioneine of microorganisms; F2) application in the preparation of ergothioneine; F3) is used in constructing genetically engineered microorganisms of ergothioneine.

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