A genetically engineered fungus with high ergothioneine production and a construction method and application thereof

Abstract

The application discloses a genetically engineered bacterium with high ergothioneine yield and a construction method and application thereof. The genetically engineered bacterium is a recombinant microorganism with the following characteristics or changes in a host for ergothioneine production: (1) overexpression of betaine homocysteine methyltransferase BHMT; (2) overexpression of ergothioneine synthase EgtE, EgtD and Egt1; and (3) knockout of spermidine synthase gene spec and tRNA methyltransferase gene trmB in the genome. In the fermentation production of ergothioneine, the genetically engineered bacterium is supplemented with betaine, and the addition of methionine is reduced. The utilization rate of methionine reaches more than 70%, and the yield of ergothioneine reaches more than 10 g / L, thereby providing a new scheme with improved economy for the industrial production of ergothioneine.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, specifically, it relates to a method for constructing and applying a genetically engineered bacterium that produces high levels of ergothionein. Background Technology

[0002] Ergothioneine (EGT), also known as 2-thio-L-histidine trimethyl inner salt, is a rare, naturally occurring chiral histidine-derived thiol compound, first isolated by Tamcet from *Claviceps purpurea* in 1909. Ergothioneine is an important physiologically active substance in the body, participating in the regulation of intracellular redox reactions, scavenging free radicals, preventing UV radiation damage, regulating intracellular energy metabolism, and anti-fatigue functions, thus showing broad application prospects in cosmetics, functional foods, pharmaceuticals, and biomedicine.

[0003] To promote the industrial production of ergothioneine, we have conducted a series of studies on microbial synthesis technology in recent years, as detailed in patent documents with publication numbers CN111534535A, CN118360172A, CN112011464A, CN112094750A, CN120310764A, CN120310863A, and CN122103257A.

[0004] In the microbial synthesis pathway of ergothioneine, S-adenosylmethionine (SAM) is a key methyl donor in the methyltransferase (such as EgtD) catalytic reaction, and the metabolic supply and regeneration efficiency of its precursor methionine directly determine the upper limit of the final product synthesis. However, using methionine / SAM as a synthetic precursor has several inherent limitations in actual industrial production, mainly manifested in the following bottlenecks: 1. Insufficient intracellular supply and metabolic imbalance of SAM. SAM synthesis strictly depends on methionine and ATP, and its intracellular concentration is precisely regulated. The continuous and large consumption of SAM by high-yield ergothioneine engineered strains can easily lead to rapid depletion of the intracellular pool. This not only directly limits the methylation reaction rate, but also interferes with other core life activities of the host cell that depend on SAM (such as methylation modification of nucleic acids, proteins, and phospholipids), causing global pressure on normal cell growth and metabolism, and thus significantly inhibiting cell growth and product synthesis. 2. Competition for precursor metabolic flux and obstacles to regeneration cycle. Methionine, as a basic amino acid, faces multiple competitive challenges in its metabolic flux within engineered bacteria: it needs to be used for its own protein synthesis, and it also needs to be efficiently converted into SAM for use in synthetic pathways. More importantly, S-adenosylhomocysteine ​​(SAH), generated from SAM via methyltransferase, must be efficiently cleared and regenerated back into methionine to maintain the SAM cycle. This regeneration pathway involves multiple enzymatic steps, and inefficiency in any step leads to SAH accumulation. SAH is a potent competitive inhibitor of many methyltransferases, and its accumulation creates severe negative feedback inhibition, further reducing the catalytic efficiency of key enzymes such as EgtD, forming a vicious cycle that limits product accumulation. 3. Cost and toxicity limitations of exogenous supplementation strategies. To compensate for precursor deficiencies, directly supplementing methionine during fermentation is a common strategy. However, this amino acid is expensive, significantly increasing production costs and hindering industrial application. Furthermore, high concentrations of methionine or its metabolic intermediates often exhibit cytotoxic or growth-inhibiting effects on commonly used production hosts (such as *E. coli* and *Pichia pastoris*), making it difficult to implement methods that directly increase yield through high-concentration feeding. 4. Metabolic network complexity and systemic regulatory challenges. The synthesis, utilization, and regeneration of SAM involve a complex and highly interconnected metabolic network, subject to multi-level feedback regulation. Simply overexpressing a single or a few genes (such as the methionine synthase gene *metH* or the SAM synthase gene *metK*) often fails to effectively increase SAM flux due to inherent cellular feedback inhibition, imbalanced metabolic flux distribution, or accumulation of toxic intermediates. Therefore, how to systematically modify the methionine-SAM metabolic subnetwork, including removing feedback inhibition, balancing synthesis and regeneration modules, and achieving synergy with central carbon metabolism, remains a significant challenge that has not yet been fully resolved in current technologies.

[0005] In summary, methionine and SAM, as essential precursors for ergothioneine synthesis, face fundamental limitations in achieving efficient and economical ergothioneine production through existing microbial synthesis systems. These limitations include the instability of their dynamic intracellular supply, the efficiency bottleneck of metabolic regeneration cycles, the trade-off between the economy and biocompatibility of exogenous supplementation, and the complex regulatory characteristics of the overall metabolic network. Overcoming these limitations requires a deeper, more systematic reconstruction and precise regulation of the host metabolic network, going beyond conventional pathways. Summary of the Invention

[0006] To address the aforementioned technical problems, the inventors devised a novel approach to reduce ergothioneine production costs and improve economic efficiency by modifying the biosynthetic / metabolic pathways of ergothioneine based on engineered Escherichia coli hosts, such as strain SH2428 reported in CN120310863A or strain ETTE reported in CN120310764A. This includes introducing an exogenous betaine homocysteine ​​methyltransferase (BHMT) gene to avoid or reduce the addition of methionine during fermentation, thereby saving raw material costs. Furthermore, by optimizing the metabolic flux of the SAM pathway, ergothioneine yield can be increased while reducing methionine usage. Experiments have confirmed the feasibility of the above-mentioned novel approach. Specifically, this invention includes the following technical solutions.

[0007] The first aspect of this invention provides a genetically engineered bacterium that produces ergothioneine, wherein the genetically engineered bacterium is a recombinant microorganism whose ergothioneine-producing host has the following characteristics or has undergone the following changes:

[0008] (1) Overexpression of betaine homocysteine ​​methyltransferase (BHMT).

[0009] (2) Overexpression of ergothioneine synthase E (EgtE), ergothioneine synthase D (EgtD), and ergothioneine synthase 1 (Egt1); and / or

[0010] (3) Knock out the spermine synthase gene spec and the tRNA methyltransferase gene trmB, which negatively regulate the synthesis of S-adenosylmethionine (SAM) in the genome.

[0011] In one embodiment, further preferably, the aforementioned betaine homocysteine ​​methyltransferase BHMT is a human betaine homocysteine ​​methyltransferase BHMT (UniProt ID: Q93088 or GeneID: 635 version) with an amino acid sequence as shown in SEQ ID NO.1, or a conserved variant polypeptide thereof. The conserved variant polypeptide is a polypeptide whose amino acid sequence has more than 80% homology, preferably more than 85% homology, more preferably more than 90% homology, more preferably more than 92% homology, more preferably more than 95% homology, more preferably more than 98% homology, more preferably more than 99% homology, and exhibits increased enzyme activity.

[0012] MPPVGGKKAKKGILERLNAGEIVIGDGGFVFALEKRGYVKAGPWTPEAAVEHPEAVRQLHREFLRAGSNVMQTFTFYASEDKLENRGNYVLEKISGQEVNEAACDIARQVADEGDALVAGGVSQTPSYLSCKSETEVKKVFLQQLEVFMKKNVDFLIAEYFEHVEEAVWAVETLIASGKPVAATMCIGPEGDLHGVPPGECAVRL VKAGASIIGVNCHFDPPTISLKTVKLMKEGLEAAQLKAHLMSQPLAYHTPDCNKQGFIDLPEFPFGLEPRVATRWDIQKYAREAYNLGVRYIGGCCGFEPYHIRAIAEELAPERGFLPPASEKHGSWGSGLDMHTKPWVRARARKEYWENLRIASGRPYNPSMSKPDGWGVTKGTAELMQQKEATTEQQLKELFEKQKFKSQ (SEQ ID NO.1).

[0013] Preferably, the aforementioned conserved variant polypeptide can be the N69D mutant of wild-type BHMT, i.e., BHMT. N69D Its amino acid sequence is shown in SEQ ID NO.2:

[0014] MPPVGGKKAKKGILERLNAGEIVIGDGGFVFALEKRGYVKAGPWTPEAAVEHPEAVRQLHREFLRAGSDVMQTFTFYASEDKLENRGNYVLEKISGQEVNEAACDIARQVADEGDALVAGGVSQTPSYLSCKSETEVKKVFLQQLEVFMKKNVDFLIAEYFEHVEEAVWAVETLIASGKPVAATMCIGPEGDLHGVPPGECAVRL VKAGASIIGVNCHFDPPTISLKTVKLMKEGLEAAQLKAHLMSQPLAYHTPDCNKQGFIDLPEFPFGLEPRVATRWDIQKYAREAYNLGVRYIGGCCGFEPYHIRAIAEELAPERGFLPPASEKHGSWGSGLDMHTKPWVRARARKEYWENLRIASGRPYNPSMSKPDGWGVTKGTAELMQQKEATTEQQLKELFEKQKFKSQ (SEQ ID NO.2).

[0015] The host of the ergothioneine-producing bacteria mentioned above can be bacteria, preferably *Escherichia coli*, more preferably *Escherichia coli* MG1655 or its derivatives, such as strain SH2428 reported in patent document CN120310863A, deposited by Huarui Biotechnology (Chuzhou) Co., Ltd., with genotype MG1655 ΔhisGL::hisD-I 3hisG*. The nucleotide sequence of the coding gene of the human BHMT is shown in SEQ ID NO.3; the mutant BHMT N69D The nucleotide sequence of the encoding gene is shown in SEQ ID NO.4; the spermidine synthase gene spec is the E. coli endogenous spec gene (2026 UniProt ID: P21169 or GeneID: 947457 version); the tRNA methyltransferase gene trmB is the E. coli endogenous trmB gene (2026 UniProt ID: P0A815 or GeneID: 947448 version).

[0016] Further, EgtE is MSegtE, which is derived from Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) with an amino acid sequence as shown in SEQ ID NO.5; EgtD is EgtD, which is derived from Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) with an amino acid sequence as shown in SEQ ID NO.6; and Egt1 is TtrEgt1, which is derived from Trichoderma reesei (strain QM6a) with an amino acid sequence as shown in SEQ ID NO.7.

[0017] Furthermore, the host of the ergothioneine-producing bacteria is Escherichia coli, and the CDS nucleotide sequence of the encoding gene of MSegtE is SEQ ID NO.8; the CDS nucleotide sequence of the encoding gene of EgtD is SEQ ID NO.9; and the CDS nucleotide sequence of the encoding gene of TtrEgt1 is SEQ ID NO.10.

[0018] A second aspect of the present invention provides a method for constructing genetically engineered bacteria as described above, comprising the following steps:

[0019] A. Using ergothionein-producing bacteria as the chassis bacteria, the exogenous betaine homocysteine ​​methyltransferase BHMT gene was integrated into the strain genome. Through resistance screening and genotype verification, strain A was obtained.

[0020] B. Knock out the endogenous spec and trmB genes in strain A, and obtain strain B through resistance selection and genotype verification;

[0021] C. By integrating the exogenous ergothionein synthesis gene cluster EgtE+EgtD+Egt1 into the genome of strain B, and through resistance screening and genotype verification, strain C, a recombinant microorganism with an increased ergothionein fermentation level compared to the host strain, was obtained.

[0022] It should be understood that in specific genetic engineering implementation plans, the order of steps A, B, and C is not fixed according to the alphabetical order. They can be performed in a cross-cutting or reversed manner, as long as each step can achieve its respective function and complete the directional change of the host cell genotype.

[0023] In one embodiment, the gene integration method in steps A and C is selected from gene editing technology or gene overexpression plasmid transformation to obtain positive transformants.

[0024] The gene editing technologies used for gene integration described above may be selected from the following group: homologous double crossover, Red homologous recombination, TALEN system, CRISPR-Cpf1 system, CRISPR-Cas12a system, MuGENT (multiplex genome editing by natural transformation), and MUCICAT (multi-copy chromosomal integration by CRISPR-associated transposase, a bacterial chromosomal multicopy integration technology based on CRISPR-associated transposases (CASTs)).

[0025] The plasmid transformation used for the integration of the BHMT coding gene and / or gene cluster EgtE+EgtD+Egt1 in steps A and C above is selected from traditional chemical transformation, heat shock transformation, or electrotransformation methods, and the plasmid may include two or more copies of the coding gene.

[0026] Preferably, the number of copies of the BHMT encoding gene and / or gene cluster EgtE+EgtD+Egt1 integrated into the host bacterium genome is two or more, preferably four or more, six or more, or eight or more.

[0027] In one embodiment, the knockout of genes spec and trmB in step B above is carried out by gene editing technology, wherein the gene editing technology is selected from the group consisting of: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT, CRISPRi, RAD system, and preferably CRISPR-Cas9 gene editing system.

[0028] Preferably, the gene knockout in step B above is implemented using the CRISPR-Cas9 system, i.e., by designing sgRNAs targeting the genes spec and trmB and homologous repair templates.

[0029] A third aspect of the present invention provides the application of the genetically engineered bacteria described above in the production of ergothioneine.

[0030] In one application implementation, ergothioneine is produced by fermentation of the genetically engineered bacteria.

[0031] Optionally, the method for producing ergothioneine by fermentation of the above-mentioned genetically engineered bacteria includes the following steps: fermenting the genetically engineered bacteria in a culture medium containing a carbon source, betaine, homocysteine ​​and histidine, adding betaine during the fermentation process to partially replace methionine, and collecting ergothioneine from the fermentation broth.

[0032] Furthermore, the above fermentation method is a fed-batch fermentation. For example, the specific process is: the recombinant *E. coli* strain is cultured at 37°C for 12 hours, and the OD... 600 When the bacterial culture reaches a concentration of 3-5, it is transferred to a fermentation tank for fermentation, with the total amount of bacterial culture added being 35% of the tank volume. In one embodiment, the engineered strain initially ferments at 37°C. Once the OD600 reaches 30, amino acids are added sequentially. Histidine is not added, and the feed carbon source is 80% glucose monohydrate. The added amino acids are a mixture of betaine, cysteine, and methionine, with a concentration ratio of approximately 30 g / L:30 g / L:30 g / L. The pH is controlled at approximately 7.0. In the above fermentation, the amount of methionine added is reduced to 30-50% of the conventional amount, the methionine utilization rate reaches over 70%, and the ergothioneine yield reaches over 10 g / L.

[0033] This invention, through gene mining, screened a human BMHT gene with the potential to enhance ergothioneine synthesis. This enzyme can efficiently convert betaine into methionine, a precursor for ergothioneine synthesis. Furthermore, through codon optimization and point mutation screening, we obtained the N69D mutant, BHMT, with significantly enhanced enzyme activity. N69D This invention increased the expression level of BHMT enzyme in host cells. Furthermore, to increase the intracellular supply of the methyl donor S-adenosylmethionine (SAM), the invention knocked out the genes spec and trmB related to SAM biosynthesis in microorganisms, thereby enhancing the metabolic flux of the ergothioneine synthesis pathway. The engineered strain constructed using this invention supplemented with betaine during fermentation, reducing the need for expensive methionine. Methionine utilization reached over 70%, and ergothioneine yield reached over 10 g / L, significantly reducing ergothioneine production costs and providing a new, more economically efficient solution for the industrial production of ergothioneine. Attached Figure Description

[0034] Figure 1 This is the structural map of the plasmid pDonor-BHMT1 constructed in this invention.

[0035] Figure 2 This is an electrophoretic image of BHMT integration in Escherichia coli strain SH2428.

[0036] Figure 3This is a comparison chart of the results of SH2428 strains integrating BHMT from different sources in a 1.5 L fermenter.

[0037] Figure 4 This is a comparison chart of ergothionein yield results from shake-flask fermentation of different BHMT1 mutants.

[0038] Figure 5 This is an electrophoretic image of the strain in which the speC gene has been knocked out.

[0039] Figure 6 This is an electrophoretic image of the strain in which the trmB gene has been knocked out.

[0040] Figure 7 This is the fermentation curve of the engineered strain SH2428::BHMT*ΔspeCΔtrmB+ETTE-pSC101 in a 1.5L tank.

[0041] Figure 8 This is the structural map of the plasmid ETTE-pDonor constructed in Example 1.

[0042] Figure 9 This is an electrophoretic detection image of the ETTE bands in Example 1.

[0043] Figure 10 This is a map of the plasmid ETTE-pSC101 constructed in this invention for transfecting the fusion protein EgtD+Egt1+EgtE gene. Detailed Implementation

[0044] Betaine homocysteine ​​methyltransferase (BHMT) can utilize betaine to recover homocysteine ​​into methionine, forming an internal cycle independent of de novo synthesis or large-scale exogenous addition, thus improving the sustainability of the methylation reaction. After codon-optimized insertion of the human BHMT gene into the ergothioneine-producing bacterial host strain SH2428 (the strain reported in CN120310863A), we significantly increased ergothioneine expression levels. Building upon this, based on metabolic pathways, we further knocked out / inactivated the speC and trmB genes to optimize the metabolic flow of S-adenosylmethionine (SAM). Combining this with the yield-enhancing strategy reported in patent document CN120310863A, we further overexpressed the ergothioneine synthase genes EgtE, EgtD, and Egt1 in the strain. The resulting recombinant microorganism was able to efficiently convert the relatively inexpensive betaine into ergothioneine even with reduced methionine addition in the culture medium. The economic advantage of using the inexpensive substrate betaine to replace the expensive substrate methionine is obvious.

[0045] As used herein, the terms “(methionine dosage / addition) reduction,” “lower,” or “decrease” can mean a reduction of at least 10% relative to a reference level (such as the base strain / originating strain), for example, a reduction of at least about 20%, or at least about 50%, or at least about 80%, or at least about 100%, or at least about 2 times the reference level.

[0046] BHMT can be used as a gene resource to construct ergothionein engineered bacteria. Its multi-site insertion in the genome of ergothionein-producing bacteria can be performed simultaneously or in one step, for example, by using the MUCICAT system for gene editing; or it can be inserted in stages.

[0047] In this document, for the sake of simplicity, the name of a protein, such as tRNA methyltransferase trmB, and its encoding gene (DNA) name trmB are sometimes used interchangeably. Those skilled in the art should understand that they represent different types of substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or category of tRNA methyltransferase, trmB refers to a protein; when described as an encoding gene, it refers to the gene encoding that protein.

[0048] Given that the wild-type BHMT enzyme typically exhibits low activity, we employed mutagenesis to modify its amino acid sequence in order to enhance its activity. Through multiple rounds of mutagenesis experiments, we screened for mutants with improved enzyme activity, including the mutant BHMT enzyme with the amino acid sequence shown in SEQ ID NO.2. N69D That is, the 69th N is mutated to D, but it is not limited to this.

[0049] It should be understood that the aforementioned “mutation” includes, but is not limited to, the substitution, deletion, insertion, or chemical modification of amino acid residues, preferably a positive mutation, i.e., a mutation that increases enzyme activity. The substitution can be a non-conservative substitution, a conserved substitution, or a combination of both. A “conservative” amino acid substitution or mutation refers to the interchangeability of residues with similar side chains, and therefore generally includes the substitution of amino acids in a polypeptide with amino acids from the same or similar amino acid definition class. However, as used herein, if a conserved mutation can alternatively be an aliphatic to aliphatic, nonpolar to nonpolar, polar to polar, acidic to acidic, basic to basic, aromatic to aromatic, or restriction residue to restriction residue substitution, then a conserved mutation does not include hydrophilic to hydrophilic, hydrophobic to hydrophobic, hydroxyl-containing to hydroxyl-containing, or small residue to small residue substitution. As is known in this technical field, common examples of conservative substitutions include: substitutions between aromatic amino acids F, W, and Y; substitutions between hydrophobic amino acids L, I, and V; substitutions between polar amino acids Q and N; substitutions between basic amino acids K, R, and H; substitutions between acidic amino acids D and E; and substitutions between hydroxyl amino acids S and T. Furthermore, A, V, L, or I can be conservatively mutated into another aliphatic residue or another nonpolar residue.

[0050] "Non-conservative substitution" refers to the substitution or mutation of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitution can be performed between, rather than within, the amino acids defined above. In one embodiment, a non-conservative mutation affects (a) the structure of the peptide backbone in the substituted region (e.g., proline replacing glycine), (b) charge or hydrophobicity, or (c) side chain volume.

[0051] "Deletion" refers to a modification of a peptide by removing one or more amino acids from a reference peptide. Deletion can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids constituting the reference enzyme, while preserving enzyme activity and / or the modified properties of engineered BHMT. Deletion can target the interior and / or ends of the peptide. In several embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0052] "Insertion" refers to a modification of a peptide by adding one or more amino acids to a reference peptide. In some embodiments, modified engineered BHMTs include inserting one or more amino acids into naturally occurring BHMTs and inserting one or more amino acids into other modified BHMT peptides. Insertion can be internal to the peptide, or at the carboxyl or amino terminus. Insertions as used herein include fusion proteins as known in the art. Insertion can be a continuous amino acid segment or separated by one or more amino acids in a naturally occurring peptide.

[0053] By using exogenous BHMT and its mutant BHMT*, ergothionein synthesis genes (EgtE, EgtD, Egt1), and knocking out the genes spec and trmB that negatively regulate SAM synthesis, it is possible to construct ergothionein-engineered bacteria that fully utilize the substrate betaine, efficiently utilize methionine, and enhance ergothionein expression.

[0054] In this article, the terms "(ergothioneine) genetically engineered bacteria", "engineered bacteria (strain)", "reconstructed bacteria" and "recombinant bacteria (strain)" have the same meaning and can be used interchangeably.

[0055] Correspondingly, for ease of description, Escherichia coli MG1655 derivatives, such as strain SH2428, can be referred to as the "originating strain" or "original strain".

[0056] In one specific embodiment, the construction of a high-yield ergothionein engineered strain includes the following steps: using the histidine recombinant strain SH2428 reported in patent document CN120310863A as the starting strain, the BHMT encoding gene (SEQ ID NO.3) or its N69D mutant gene (SEQ ID NO.4) with the amino acid sequence shown in SEQ ID NO.1 is integrated into the Escherichia coli genome; the speC gene and trmB gene are knocked out; and a fusion plasmid containing the ergothionein synthase (EgtE, EgtD, Egt1) gene cluster reported in patent document CN120310863A is introduced, and positive clones are screened to obtain a high-yield ergothionein strain.

[0057] In this invention, the high-yield ergothionein strain constructed according to the above method can reduce the addition of methionine during fermentation, and the yield of ergothionein is also significantly improved, thus promoting the feasibility of industrial fermentation production of ergothionein.

[0058] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0059] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0060] Example

[0061] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0062] The embodiments in this article involve the addition amount, content and concentration of various substances. Unless otherwise specified, the percentage content mentioned refers to the mass percentage content.

[0063] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0064] Materials and methods

[0065] This study is based on the invention disclosed in patent document CN120310863A, therefore, some of its experimental content is incorporated into this paper by way of citation.

[0066] In this embodiment, the whole genome synthesis, primer synthesis and sequencing were outsourced to Sangon Biotech (Shanghai) Co., Ltd. (hereinafter referred to as Shanghai Sangon Biotech).

[0067] The molecular biology experiments in the examples included plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., mainly referring to "Molecular Cloning: A Laboratory Manual" (4th edition), edited by M.R. Green and J. Sambrook (USA), translated by He Fuchu, Science Press, Beijing, 2017.

[0068] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0069] Plasmids pTargetF and pCassacB, and recombinant Escherichia coli SH2428 were deposited in the laboratory of Huarui Biotechnology (Chuzhou) Co., Ltd.

[0070] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2 (LB solid medium with an additional 20 g / L agar powder).

[0071] Metal ions: 20 mg / L ferrous sulfate heptahydrate, 20 mg / L manganese sulfate monohydrate, pH adjusted to <2 with hydrochloric acid.

[0072] Vitamins: 2 mg / L thiamine, 2 mg / L hydrochloric acid, 2 mg / L cobalamin, 2 mg / L pantothenic acid.

[0073] Trace elements: 2.5 g / L sodium molybdate dihydrate, 1.6 g / L nickel chloride hexahydrate, 10 g / L calcium chloride, 0.4 g / L copper sulfate pentahydrate, 2.25 g / L aluminum sulfate, 1.8 g / L cobalt chloride hexahydrate, 0.5 g / L NiCl2 hexahydrate, zinc sulfate heptahydrate, 0.14 g / L boric acid.

[0074] Final antibiotic concentrations: 50 mg / L kanamycin, 50 mg / L spectinomycin, 35 mg / L chloramphenicol, 50 mg / L bleomycin.

[0075] Final concentration of inducer: 10 mM rhamnose, 10 mM arabinose, 0.001 mM anhydrous tetracycline.

[0076] In the following examples, the seed culture conditions for the strain were: 37°C, 220 rpm, 500ml shake flask, 80ml culture medium (LB + antibiotic*), cultured for 12h, OD>3.

[0077] The composition of the culture medium for fermentation in the upper tank is shown in Tables 1 and 2:

[0078] Table 1. Fermentation medium in the upper tank (1.6L / tank, adjusted to 1400ml)

[0079]

[0080] Table 2, Preparation of T019-1 and V019-2

[0081]

[0082] The trace elements in the above culture media T019-1 and V019-2 were all sterilized by high temperature (except for iron, all other elements were sterilized together).

[0083] During fermentation in the upper tank, amino acids (cysteine:methionine:betaine concentration = 30 g / L: 30 g / L: 30 g / L) are added, but histidine is not added. The feed is 80% glucose monohydrate. The pH is maintained at 7.0 during fermentation. Tank pressure: initial 0.005 MPa, maximum 0.02 MPa. Aeration rate: initial 1.5, maximum 3.

[0084] The steps for fermentation in the tank are as follows:

[0085] (1) Inoculate 80ml of seed culture into 1600ml of fermentation broth (final volume), incubate at 37℃, and after OD600=30, cool down and supplement amino acids at an initial rate of about 12ml H. -1 L -1 .

[0086] (2) After the initial sugar is consumed, replenish with glucose solution (80%) (before replenishing amino acids, dissolved oxygen > 25%; after replenishing amino acids, dissolved oxygen is maintained at 20%. If the dissolved oxygen is low in the later stage, reduce the feeding rate to maintain dissolved oxygen above 5% and control pH = 7.0). The glucose feeding rate is about 16-18 ml / h.

[0087] (3) Detect the amino acid concentration during the fermentation process. If there is no residue, increase the feeding speed appropriately.

[0088] When using a culture medium containing kanamycin (KAN), the final concentration of kanamycin in the culture medium is 50 µg / ml; when using a culture medium containing chloramphenicol (CM), the final concentration of chloramphenicol in the culture medium is 30 µg / ml; when using a culture medium containing bleomycin (Zoe), the final concentration of bleomycin in the culture medium is 50 µg / ml.

[0089] The HPLC detection method for ergothioneine is as follows:

[0090] Ergothionein was detected by high-performance liquid chromatography (HPLC) using an Agilent 1260II instrument. An Agilent SB-Aq column (5 μm, 4.6 x 250 mm) was used, with a UV detector at 258 nm. The mobile phase was 0.1% perchloric acid:acetonitrile = 95:5 (v / v), the flow rate was 1 mL / min, the injection volume was 5 μL, and the column temperature was 40 °C. Ergothionein standards were dissolved in ddH₂O at concentrations ranging from 0.05 g / L to 1 g / L. The prepared standard solutions and sample solutions were analyzed by HPLC, and the peak areas were recorded. The ergothionein content was calculated using the external standard method. Diluent: ultrapure water; Blank solution: ultrapure water.

[0091] The calculation formula is as follows:

[0092] .

[0093] The detection method for histidine is referenced in patent document CN118147236A.

[0094] Some of the PCR primers used in the examples are shown in Table 3:

[0095] Table 3. PCR Primer Information

[0096] Primer Name Sequence (5'-3') EgtD-F ATGACCCTGAGCctggccaacta Linker-EgtD-R gggcattcctccacccctccgcaccgccagcgacaac linker-TtrEgt1-F gtgcggaggggtggaggaatgcccttccccaggctccg TtrEgt1-R GGTATATCTCCTTCTTAAAGTTAAACAttaaacatctcgcaccaaccttgcc EgtE-F ggttggtgcgagatgtttaaTGTTTAACTTTAAGAAGGAGATATACCatgctcgcgcagcagtggcg EgtE-R tcagggcgcctcacgcaac V-F cgttgcgtgaggcgccctgacgatcccctcggatccaattggtc V-R ttggccaggctcagggtcatAGATCAGTCTCCTTAATTGTTATCCGCTC ACAAT EgtD(wt)-seqF1 cagacgcattggtggaccgatc pDonor-V-R2 gactctaaacgcgcctccatcgc Vpsc101-F CGATCCCCTCGGATCCAATTGGTC Vpsc101- R gcatgtcgaccatggaattcccg IETTE-F cgggaattccatggtcgacatgc IETTE-R GACCAATTGGATCCGAGGGGATCG pdonor-GJ-F CGATCCCCTCGGATCCAATTGGTC pdonor-GJ-R tgtatatctccttcttaaacaaaaTTatttctagaAATTGTTATCCGCTCACAATtcca BHMT1-F tgtttaagaaggagatatacatATGCCTCCTGTGGGCGG BHMT1-R ccaattggatccgaggggatcgTTACTGACTTTTGAATTTCTGCTTTTCAAACAGTTCT BHMT2-F ttgtttaagaaggagatatacatATGACCGATGATCTGCGTACCCG BHMT2-R ccaattggatccgaggggatcgTTAGGCTTTATCGGCAAAATCGCGA BHMT3-F tgtttaagaaggagatatacatATGGCAGATAGCCAGAATACCGG BHMT3-R ccaattggatccgaggggatcgTTACAGTTCATCGGCAAAACTCTTATTAATATCGCG BHMT4-F tgtttaagaaggagatatacatATGCTGCCTATTACCGATAGTCGC BHMT4-R ccaattggatccgaggggatcgTTACTGCTGCTGAATTTTGGCTGCA 67-69-F GTGATGCAGACCTTTACCTTTTATGCCA F74C-F ACCTTTTATGCCAGCGAAGATAAACTG F74C-R TATCTTCGCTGGCATAAAAGGTACAGGTCTGCATCACATTGCTACCG G67V-R TGGCATAAAAGGTAAAGGTCTGCATCACATTGCTAACGGCACGCAGAAATTCGCG N69D-R TGGCATAAAAGGTAAAGGTCTGCATCACATCGCTACCGGCACGCAGAAATTCGCG S68T-R TGGCATAAAAGGTAAAGGTCTGCATCACATTCGTACCGGCACGCAGAAATTCGCG S68V-R TGGCATAAAAGGTAAAGGTCTGCATCACATTGACACCGGCACGCAGAAATTCGCG psc101-seq-R gcatgtcgaccatggaattcccg trmB-CF gagaaagcgagagaatcgcgcc trmB-CR gcatttgctggccccagatt trmB-LF gctgcaacttcctcaaaggtttcc trmB-LR aaaggtttagtcgcttgtgaaagtgt trmB-RF tttcacaagcgactaaacctttatggcaaagaaccgtagccg trmB-RR agtacgcgttttccatgccg trmB-SG-F TAGTgcgcgtgatgtgtcacgatg trmB-SG-R AAACcatcgtgacacatcacgcgc speC-CF taccttcctgcacagcttcgac speC-CR gttggcacatttcaccaccag speC-LF catgtcgatttcgcagatctctg speC-LR cgactttcttctcatcgtcgcgcggttgtacggttatgtgttgaag speC-RF cttcaacacataaccgtacaaccgcgcgacgatgagaagaaagtcg speC-RR ggatacgttccggtaagcgttg speC-SG-F ttgttcataaccgacccacg speC-SG-R cgtgggtcggttatgaacaa pQCas-1-F cctcttgctgagattgataaggtaattaaccttat pQCas1-R ggcaagaactgaatgaaaaaggcttcc pQCas-2-F ctggtacaagtatgttggtctgaaaggg pQCas-2-R ggcgggacatgtaggccg pQCas-3-F gccgctggcctacggtaagag pQCas-3-R agtaaacgtcattcgtttaaaatgagaaagcc pQCas-4-F atcccgcagtttggtcagcaa pQCas-4-R ttgcaacatgttgaatcttcatgcgttt pQCas-5-F tgggggttgattatctgattaaccgtg pQCas-5-R ctgagaaatttttaattcgctctgaaactgatgg pQCas-6-F tgcttaaaagcacagaatctaagatccctgc pQCas-6-R tcacctggatgataagagattcactgtgt pQCas-7-F gtgttgttgacgccattgaacgc pQCas-7-R gtttctgcccgacgggtggtattt pQCas-8-F taaagcgaaggccaaaccgaaaaca pQCas-8-R caacgcctgagattttcactcgtca

[0097] In primer names, "-F" indicates forward and "-R" indicates reverse.

[0098] It should be noted that, for the sake of convenience, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number may share the same number. This is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0099] Example 1: Construction of a plasmid expressing the EgtD+Egt1+EgtE fusion protein

[0100] Referring to patent document CN120310863A, a plasmid expressing the EgtD+Egt1+EgtE fusion protein gene was constructed. The specific steps are as follows:

[0101] (1) Constructing a fusion protein expression plasmid with EgtD, Egt1 and EgtE catalytic functions

[0102] Fragments with EgtD, Egt1, and EgtE catalytic functions, as well as the component pDonor of the expression plasmid, were amplified by PCR and then recovered by gel extraction. EgtD was derived from *Mycobacterium smegmatis* (strain ATCC 700084 / mc(2)155), Egt1 from *Trichoderma reesei* (strain QM6a, TtrEgt1), and EgtE from *Mycobacterium smegmatis* (strain ATCC700084 / mc(2)155, MSegtE). The nucleotide sequence of the gene encoding MSegtE is shown in SEQ ID NO.8; the nucleotide sequence of the gene encoding EgtD is shown in SEQ ID NO.9; and the nucleotide sequence of the gene encoding TtrEgt1 is shown in SEQ ID NO.10.

[0103] The primers for amplifying the EgtD gene fragment are EgtD-F and Linker-EgtD-R; the primers for amplifying the Egt1 gene fragment are linker-TtrEgt1-F and TtrEgt1-R; the primers for amplifying the EgtE gene fragment are EgtE-F and EgtE-R; and the primers for amplifying pDonor, a component of the expression plasmid, are VF and VR.

[0104] A vector was constructed using the pEASY-Basic Seamless Cloning and Assembly Kit homologous recombinase. Multiple gene fragments with homologous arms were then homologously recombinated to form a circular plasmid, ETTE-pDonor (e.g., ...). Figure 8 (As shown in the table). The specific reaction system and reaction conditions are shown in Table 4.

[0105] Table 4. Homologous recombination reaction system and reaction conditions

[0106]

[0107] The constructed plasmid ETTE-pDonor was validated by PCR using primers EgtD(wt)-seqF1 and pDonor-V-R2. The PCR products were identified by 1% agarose gel electrophoresis, and colonies with the correct band size (e.g., ...) were examined. Figure 9 The ETTE bands shown were preserved, plasmids were extracted, and the samples were sent for testing to confirm their correctness.

[0108] (2) Using Psc101-plasmid as a template, amplification was performed using primer pair Vpsc101-F / R; using the plasmid ETTE-pDonor constructed in step (1) as a template, the ETTE gene portion was amplified using primer pair IETTE-F / R, and the two amplified fragments were recovered. A vector was constructed using the pEASY-Basic Seamless Cloning and Assembly Kit homologous recombinase, and the two gene fragments with homologous arms were homologously recombined to construct a circular plasmid ETTE-pSC101, as shown below. Figure 10 As shown in the figure. The specific reaction system and reaction conditions are shown in Table 5.

[0109] Table 5. Reaction system and reaction conditions for homologous recombination

[0110]

[0111] The constructed plasmid ETTE-pSC101 was verified by PCR according to the method in step (1), and the sequencing verification was correct.

[0112] Example 2: Screening of exogenous BHMT enzymes

[0113] Using the histidine recombinant strain SH2428 constructed in patent document CN120310863A as the starting strain, BHMT enzymes from different sources were integrated and transformed into the fusion protein particle ETTE-pSC101, which has EgtD, Egt1, and EgtE catalytic functions. The specific steps are as follows:

[0114] (1) Integration of BHMT

[0115] The pDonor plasmid was specifically amplified using primer pair pdonor-GJ-F / R to obtain plasmid vector fragment 1. The coding genes of different BHMT sources, optimized with codons, were amplified using primer BHMT-F / R to obtain target gene fragment 2. Taking human BHMT (numbered BHMT1 in this example) as an example, the codon-optimized BHMT1 gene sequence SEQ ID NO.3 was amplified using primer BHMT1-F / R to obtain target gene fragment 2. Fragments 1 and 2 were then assembled using a Gibson assembly kit to construct the plasmid pDonor-BHMT1 for multi-copy gene integration (see [link to Gibson assembly kit]). Figure 1 The specific reaction system and reaction conditions are shown in Table 6 below:

[0116] Table 6. Homologous recombination reaction system and reaction conditions

[0117]

[0118] Recombinant plasmids pDonor-BHMT1 and pQCas8 were electroporated into the substrate strain SH2824 and plated onto LB agar plates containing kanamycin and chloramphenicol, and incubated at 30°C for 12–14 h. The positive transformants obtained in the above steps were resuspended in sterile LB broth and then plated onto LB agar plates containing 0.001 mM anhydrous tetracycline, kanamycin, and chloramphenicol. Anhydrous tetracycline was used to induce transposase expression. The incubation conditions were 30°C for 16 h. The positive transformants on the above plates were diluted a certain factor with sterile LB broth, and the diluted bacterial solution was plated onto LB agar plates containing 0.01 mM anhydrous tetracycline, kanamycin, and chloramphenicol. Anhydrous tetracycline was used to induce transposase expression. The incubation conditions were 30°C for 16 h.

[0119] To increase the gene copy number, repeated streaking can extend the transposition time. Positive transformants from the above plates are appropriately diluted and resuspended in sterile LB broth, then streaked on new LB solid plates containing 0.01 mM anhydrous tetracycline, kanamycin, and chloramphenicol to isolate single colonies. Colony PCR is used to verify the gene copy number of the positive transformants on the plates, thus obtaining engineered *E. coli* with different copy numbers of the BHMT1 gene. Colony PCR is performed on the same positive transformant using primer pairs pQcas-1-F / R, pQcas-2-F / R, pQcas-3-F / R, pQcas-4-F / R, pQcas-4-F / R, pQcas-5-F / R, pQcas-6-F / R, pQcas-7-F / R, and pQcas-8-F / R. The specific integration site of the BHMT gene is determined based on the nucleic acid electrophoresis bands of different primer pairs. The final result was a recombinant *E. coli* strain with the BHMT1 gene integrated at position 6 (i.e., position YsaE), containing one copy number. See also... Figure 2 .

[0120] After successful single-colony validation, the integrated gene in the genome was sequenced, with the sequencing work completed by Shanghai Sangon Biotech Co., Ltd. The removal of the two plasmids pDonor and pQCas8 in the above system required plasmid removal via pfree, resulting in plasmid-free *E. coli* strains. The integrated host obtained in the above steps was prepared into electrocompetent cells, electrotransformed with pfree plasmid, activated, and then plated onto LB agar containing bleomycin, incubated overnight at 30°C. Several positive transformants from the plates were selected, 1.0 mM rhamnose was added, and the culture was incubated overnight at 30°C. A portion of the bacterial culture was streaked onto LB agar plates, and then validated on LB agar plates containing kanamycin, chloramphenicol, or bleomycin as a single antibiotic. Single colonies without pDonor-BHMT1 and pQCas8 were screened, and pfree plasmid was simultaneously removed, resulting in plasmid-free engineered *E. coli*, named SH2428::BHMT1.

[0121] The remaining integrated strains were named SH2428::BHMT2, SH2428::BHMT3, and SH2428::BHMT4, respectively.

[0122] (1) Transplantation of fusion protein particles with EgtD, Egt1 and EgtE catalytic functions

[0123] The plasmid ETTE-pSC101, containing the ergothioneine synthase gene clusters EgtD, Egt1, and EgtE, was introduced into strains SH2428::BHMT1, SH2428::BHMT2, SH2428::BHMT3, and SH2428::BHMT4, respectively. PCR confirmation using primers EgtD(wt)-seqF1 and pSC101-seq-R confirmed successful plasmid transformation. Recombinant ergothioneine strains were obtained: SH2428::BHMT1+ETTE-pSC101, SH2428::BHMT2+ETTE-pSC101, SH2428::BHMT3+ETTE-pSC101, and SH2428::BHMT4+ETTE-pSC101, and were stored in glycerol tubes.

[0124] (2) Detection of ergothionein fermentation capacity of the above strains

[0125] The above-mentioned strains SH2428::BHMT1+ETTE-pSC101, SH2428::BHMT2+ETTE-pSC101, SH2428::BHMT3+ETTE-pSC101, and SH2428::BHMT4+ETTE-pSC101 were inoculated into 50 mL shake flasks of LB broth and cultured at 37 °C and 220 rpm. After 10–12 h of culture, the seed culture in the shake flasks was transferred to a 1.5 L fermenter at an inoculation rate of 35% (v / v) and fermented at 30 °C. Samples of the fermentation broth were taken at fixed time intervals, diluted appropriately, and used to prepare samples for OD and product detection. The results are as follows: Figure 3 As shown.

[0126] Among the strains constructed according to this embodiment, SH2428::BHMT1+ETTE-pSC101 can produce 9.4 g / L of ergothioneine after 70 hours of fermentation. Compared with the other three different sources of BHMT, it has the highest ability to convert betaine into cysteine ​​and then synthesize ergothioneine. It is named SH2844.

[0127] Example 3: Mutation screening of BHMT enzyme

[0128] (1) Mutual integration of BHMT

[0129] Furthermore, point mutations were performed on the screened BHMT1 to enhance BHMT enzyme activity, thereby increasing betaine utilization efficiency and ergothioneine expression levels. Using pDonor-BHMT1 as a template, loop amplification was performed using primers F / R. Taking the mutant BHMT1-N69D as an example, pDonor-BHMT1 was amplified using primers N69D-F / R, followed by digestion with DpnI. The specific reaction system and conditions are shown in Table 7 below.

[0130] Table 7. Digestion Reaction System and Reaction Conditions

[0131]

[0132] The digestion product was then transferred into DH5α competent cells, and the subsequent steps were the same as step (1) in Example 2. The resulting recombinant plasmid was named pDonor-BHMT1(N69D). Referring to step (1) in Example 2, the pDonor-BHMT1(N69D) plasmid was integrated into the SH2428 chassis strain, resulting in strains SH2428::BHMT1(G67V), SH2428::BHMT1(S68V), SH2428::BHMT1(S68T), SH2428::BHMT1(N69D), and SH2428::BHMT1(F74C).

[0133] (2) Transplantation of fusion protein particles with EgtD, Egt1 and EgtE catalytic functions

[0134] The plasmid ETTE-pSC101, containing the ergothioneine synthase gene clusters EgtD, Egt1, and EgtE, was introduced into strains SH2428::BHMT1(G67V), SH2428::BHMT1(S68V), SH2428::BHMT1(S68T), SH2428::BHMT1(N69D), and SH2428::BHMT1(F74C), respectively. PCR was then performed using primers EgtD(wt)-seqF1 and pSC101-seq-R to confirm successful plasmid transformation into the strains. Recombinant ergothionein strains of SH2428::BHMT1(G67V)+ETTE-pSC101, SH2428::BHMT1(S68V)+ETTE-pSC101, SH2428::BHMT1(S68T)+ETTE-pSC101, SH2428::BHMT1(N69D)+ETTE-pSC101, and SH2428::BHMT1(F74C)+ETTE-pSC101 were obtained and preserved in glycerol tubes.

[0135] (3) Detection of ergothionein fermentation capacity of the above strains

[0136] The above-mentioned recombinant ergothioneine strain and strain SH2844 were inoculated into 5 mL LB medium in test tubes and cultured at 37℃ and 220 rpm. After 12-14 h of culture, the seed culture in the test tubes was transferred at 5% (v / v) to a 250 mL shake flask containing 25 mL of fermentation medium for fermentation at 30℃ and 220 rpm for 24 hours. The fermentation broth was then analyzed, and the specific results are as follows: Figure 4 As shown.

[0137] Among the strains constructed according to this embodiment, the ergothioneine production of strain SH2428::BHMT1(N69D) can reach 0.17 g / L at the shake-flask level after 24 hours, which is significantly higher than that of wild-type strain SH2428.

[0138] Example 4: Optimization of the SAM pathway in recombinant bacteria

[0139] Starting with SH2428::BHMT1(N69D), the speC and trmB genes were knocked out, and the fusion protein plasmid ETTE-pSC101, which possesses EgtD, Egt1, and EgtE catalytic functions, was introduced. The specific steps are as follows:

[0140] (1) Knockout of the SpeC gene

[0141] The gene speC was knocked out using CRISPR-Cas9.

[0142] The constructed plasmid pCassac containing the Cas9 protein was transformed into SH2428::BHMT1(N69D) competent cells via electroporation. The plasmid was plated on Kana-resistant plates and incubated overnight at 37°C for 18 h. Single colonies were selected and inoculated into liquid LB containing Kana, and incubated at 37°C and 220 rpm for 18 h. A 1% (v / v) inoculum was then transferred to fresh LB, along with 10 g / L arabinose. The cells were incubated at 37°C for 1.5-2 h until OD (digestive growth) was achieved. 600 When the pH reaches 0.6-0.8, collect the cells to prepare electrocompetent cells. The electrocompetent cells need to be washed twice with 10% sterile glycerol, then resuspended in 10% glycerol and stored at -80℃.

[0143] Using primers speC-sg-F and speC-sg-R, the speC-sgRNA plasmid was seamlessly integrated into the sgRNA expression plasmid pTarget, resulting in the speC-sgRNA plasmid. Using primers speC-LF and speC-LR, speC-up was amplified by PCR, and using primers speC-RF and speC-RR, speC-dn was amplified by PCR. Then, using primers speC-LF and speC-RR, the homologous fragments speC-donor and speC-sgRNA plasmid were added to competent cells, gently mixed by pipetting, and incubated on ice for 20 min. The mixture was then added to a 1 mm electroporation cuvette (pre-chilled on ice), electroporated at 1.8 kV for 5 ms, and 800 μL of pre-chilled LB broth was added. After recovery at 37°C and 220 rpm for 1 h, the mixture was plated onto LB agar plates supplemented with the appropriate antibiotics and incubated at 37°C for 24 h.

[0144] Colony PCR verification was performed using primers speC-CF / speC-CR, see [link to documentation]. Figure 5 Positive transformants were screened to obtain strain SH2428::BHMT1(N69D)ΔspeC.

[0145] (2) Knockout of the trmB gene

[0146] Following the SpeC knockout method, we obtained the homologous fragments trmB-donor and trmB-sgRNA plasmids that knocked out trmB.

[0147] First, starting with strain SH2428::BHMT1(N69D)ΔspeC, the pCassac plasmid was transformed into strain SH2428::BHMT1(N69D)ΔspeC via electroporation. The plasmid was then plated on plates containing Kana resistance and incubated overnight at 37°C for 18 h. Single colonies were selected and inoculated into liquid LB containing Kana and incubated at 37°C and 220 rpm for 18 h. 1% by volume was then transferred to fresh LB and 10 mM arabinose was added to induce the expression of the λRed recombinase system. Homologous fragments trmB-donor and trmB-sgRNA were electroporated into the pCassac-containing SH2428::BHMT1(N69D)ΔspeC and plated on LB plates containing spectinomycin and kanamycin. The plates were incubated at 37°C for 15–24 h, and single colonies on the plates were verified by colony PCR. After successful colony PCR validation, samples were sent for sequencing to verify the integrity of the integrated genome. This sequencing work was handled by Shanghai Sangon Biotech Co., Ltd. Successfully validated single colonies were transferred to 5 mL LB liquid tubes, and 10 mM rhamnose was added to induce the removal of the pTarget plasmid. After overnight shaking, a portion of the bacterial culture was streaked and incubated overnight, followed by plate testing to verify the removal of the pTarget plasmid. Single colonies with successfully removed pTarget plasmids were inoculated into antibiotic-free LB liquid tubes and incubated overnight at 37°C and 200 rpm. A portion of the bacterial culture was streaked onto LB solid plates containing sucrose, and single colonies were picked to verify the removal of the pCassac plasmid. (See [link to relevant documentation]). Figure 6 After successful verification, the recombinant Escherichia coli SH2428::BHMT1(N69D)ΔspeCΔtrmB strain was obtained.

[0148] (3) Transfusion of fusion proteins with EgtD, Egt1 and EgtE catalytic functions

[0149] The plasmid ETTE-pSC101, containing the ergothioneine synthesis gene cluster, was introduced into strain SH2428::BHMT1(N69D)ΔspeCΔtrmB. PCR identification using primers EgtD(wt)-seqF1 and pSC101-seq-R confirmed successful plasmid transformation. A recombinant ergothioneine strain, SH2428::BHMT1(N69D)ΔspeCΔtrmB+ETTE-pSC101, was obtained, stored in glycerol tubes, and named SH2844*ΔspeCΔtrmB+ETTE-pSC101.

[0150] Example 4: Fermentation of high-yield ergothionein strain

[0151] Take 50 µL of SH2844*ΔspeCΔtrmB+ETTE-pSC101 bacterial suspension from glycerol bacteria, spread it on a 7cm×7cm plate (Cannabidiol resistant), and incubate overnight at 37°C. Take 1 / 5000 of the bacterial cells from the plate and inoculate into a seed LB shake flask (80 / 500ml), incubate at 37°C for 12 hours, and wait for OD. 600 =3~5, top fermenter. Fermentation volume 1.6L, inoculated into the fermenter at 35% (v / v). Ferment at 30°C, OD... 600 After reaching 30°C, amino acids (betaine:cysteine:methionine concentration = 30 g / L: 30 g / L: 30 g / L) were added, without histidine. The feed consisted of 80% glucose monohydrate. The pH was maintained at 7.0.

[0152] Figure 7 The fermentation curve of the engineered strain in a 1.5L tank was shown. After 66 hours of fermentation, the yield of ergothioneine reached 11.48 g / L, which is significantly higher than the fermentation level of existing industrial strains, indicating its potential for industrial application.

[0153] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium that produces ergothioneine, characterized in that, The genetically engineered bacteria are recombinant microorganisms whose ergothioneine-producing host has the following characteristics or has undergone the following changes: (1) Overexpression of betaine homocysteine ​​methyltransferase (BHMT). (2) Overexpression of ergothioneine synthase E (EgtE), ergothioneine synthase D (EgtD), and ergothioneine synthase 1 (Egt1); and / or (3) Knock out the spermine synthase gene spec and the tRNA methyltransferase gene trmB in the genome.

2. The genetically engineered bacterium according to claim 1, characterized in that, The betaine homocysteine ​​methyltransferase BHMT is the human betaine homocysteine ​​methyltransferase BHMT (UniProtID: Q93088 or GeneID: 635 version) with an amino acid sequence as shown in SEQ ID NO.1, or a conserved variant polypeptide thereof. The conserved variant polypeptide is a polypeptide with more than 80% homology to wild-type betaine homocysteine ​​methyltransferase (BHMT) in amino acid sequence and with enhanced enzyme activity.

3. The genetically engineered bacteria according to claim 2, characterized in that, The conserved variant polypeptide is the N69D mutant of wild-type BHMT, i.e., BHMT. N69D Its amino acid sequence is shown in SEQ ID NO.

2.

4. The genetically engineered bacteria according to claim 2, characterized in that, The ergothioneine-producing bacteria host is bacteria, preferably *Escherichia coli*, more preferably *Escherichia coli* MG1655 or its derivatives, such as strain SH2428 reported in patent document CN120310863A. The nucleotide sequence of the coding gene for the human BHMT is shown in SEQ ID NO.3; the mutant BHMT N69D The nucleotide sequence of the encoding gene is shown in SEQ ID NO.4; the spermidine synthase gene spec is the E. coli endogenous spec gene (2026 UniProt ID: P21169 or GeneID: 947457 version); the tRNA methyltransferase gene trmB is the E. coli endogenous trmB gene (2026 UniProt ID: P0A815 or GeneID: 947448 version). Further, EgtE is MSegtE, which is derived from Mycobacterium smegmatis (strainATCC 700084 / mc(2)155) with an amino acid sequence as shown in SEQ ID NO.5; EgtD is EgtD, which is derived from Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) with an amino acid sequence as shown in SEQ ID NO.6; and Egt1 is TtrEgt1, which is derived from Trichoderma reesei (strain QM6a) with an amino acid sequence as shown in SEQ ID NO.

7.

5. A method for constructing the genetically engineered bacteria according to any one of claims 1-4, characterized in that, Includes the following steps: A. Using ergothionein-producing bacteria as the chassis bacteria, the betaine homocysteine ​​methyltransferase BHMT gene was integrated into the strain genome. Through resistance screening and genotype verification, strain A was obtained. B. Knock out the endogenous spec and trmB genes in strain A, and obtain strain B through resistance selection and genotype verification; C. The ergothioneine synthesis gene cluster EgtE+EgtD+Egt1 was integrated into the genome of strain B. Through resistance screening and genotype verification, strain C, a recombinant microorganism with an increased ergothioneine fermentation level compared to the host strain, was obtained.

6. The method according to claim 5, characterized in that, The gene integration methods in steps A and C are selected from gene editing technology or gene overexpression plasmid transformation.

7. The method according to claim 5, characterized in that, In step B, the knockout of genes spec and trmB is carried out using gene editing technology selected from the group consisting of homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MuGENT, CRISPRi, RAD system, with CRISPR-Cas9 gene editing system being preferred.

8. The use of the genetically engineered bacteria according to any one of claims 1-4 in the production of ergothioneine.

9. The application according to claim 8, characterized in that, Ergothioneine is produced through fermentation of the genetically engineered bacteria.

10. The application according to claim 9, characterized in that, The method for producing ergothioneine by fermentation of genetically engineered bacteria includes the following steps: fermenting the genetically engineered bacteria in a culture medium containing a carbon source, betaine, homocysteine ​​and histidine, adding betaine during the fermentation process to partially replace methionine, and collecting ergothioneine from the fermentation broth.

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