Enzymes, genetically engineered bacteria, and methods for their catalytic production of ergothionein

Abstract

This application provides a method for producing ergothioneine, comprising the following steps: obtaining ergothioneine by catalysis of histidine betaine with truncated or untruncated L-histidine betaine sulfonase. It also provides a truncated L-histidine betaine sulfonase, biological materials, an enzyme combination, genetically engineered bacteria, and a whole-cell catalyst.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to enzymes, genetically engineered bacteria, and methods for catalytic production of ergothioneine, specifically to a method for enzyme-catalyzed production of ergothioneine, a truncated L-histidine betaine sulfonase and its application, a nucleotide sequence encoding L-histidine methyltransferase and its application, the application of truncated L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase in enzyme-catalyzed production of ergothioneine, and combinations of enzymes. Background Technology

[0002] Ergothioneine (ERT) is a sulfur-containing histidine derivative. Ergothioneine possesses antioxidant properties, scavenges free radicals, and participates in intracellular energy regulation. Compared to known antioxidants such as vitamin C and glutathione, it is chemically more stable and has a more significant antioxidant capacity, thus showing broad prospects for biomedical applications.

[0003] Currently, ergothioneine is mainly obtained through chemical synthesis, extraction, and bio-fermentation. Chemical synthesis of potent L-ergothioneine is extremely difficult, involving complex processes and low yields. Extraction methods, primarily involving the chemical extraction of edible fungi, are costly, have low yields, and leave chemical residues. Traditional microbial fermentation methods utilize edible fungi such as mushrooms to synthesize ergothioneine, but due to the long fermentation cycle, yields are only in the milligram range, and separation and purification are difficult, making it unsuitable for large-scale industrial production.

[0004] In recent years, with the elucidation of the ergothioneine synthesis pathway in aerobic and anaerobic organisms, the development of genetically engineered strains for high ergothioneine production using genetic engineering has gradually emerged. In prokaryotes, ergothioneine is synthesized by five enzymes encoded by the egtABCDE gene cluster: egtD, egtB, egtA, egtC, and egtE, using histidine and cysteine ​​as precursors. In eukaryotes, histidine undergoes a two-step reaction catalyzed by egt1 to generate histidine betaine cysteine ​​sulfoxide, which is then catalyzed by egt2 to produce ergothioneine. In anaerobic organisms, the precursor histidine requires only two enzymes, eanA and eanB, to generate ergothioneine in two steps. Currently, ergothioneine-producing genetically engineered bacteria mainly use Escherichia coli, yeast, or fungi as chassis cells, constructing cell factories by introducing heterologous ergothioneine synthases. Patent document CN107250347B discloses a genetically engineered Aspergillus strain with a fermentation yield of up to 438 mg / L. Patent document CN111534535B discloses a genetically engineered Rhodotorula glutinis strain. By introducing egt1 from Neurospora crassa, the engineered strain achieved a maximum yield of 82.1 mg / L during shake-flask fermentation. Patent document CN106661585B discloses a genetically engineered Escherichia coli strain. By introducing the ergothioneine synthesis gene cluster egtABCDE from mycobacteria, the engineered strain achieved a fermentation yield of 12 mg / L. Patent document CN112251392B discloses a genetically engineered Escherichia coli strain. By modifying the histidine synthesis pathway to increase the precursor histidine content and introducing an ergothioneine synthesis gene from Mycobacterium smegmatis, the engineered strain achieved a yield of 84.7 mg / L after 26 hours of shake-flask fermentation. Patent document CN104854245B discloses a novel ergothioneine synthesis pathway, using L-histidine or histidine betaine as substrates and catalyzing the synthesis of ergothioneine through two enzymes, egt1 and egtE. The yield of ergothionein genetically engineered bacteria currently reported still does not meet the requirements for industrial production. Moreover, using a heterologous host to synthesize ergothionein increases the metabolic burden on the host cells, making them prone to metabolic imbalance and growth inhibition or death during industrial scale-up.

[0005] In vitro enzymatic catalysis for ergothioneine preparation has also been reported, which has the advantages of high efficiency and high product purity. Patent document CN112301013A discloses a composite enzyme for ergothioneine preparation, which uses three enzymes to achieve the synthesis of histidine to ergothioneine through two-step in vitro catalysis. Due to the poor stability of the intermediate products histidine betaine and thiotransferase, the enzymes need to be removed by filtration and nitrogen purging after the first step of the reaction before the next step can be carried out. Moreover, some key technical problems have not been solved, which limits the high-yield production of ergothioneine. These problems are mainly twofold: (1) the activity of the egtD enzyme used is low, resulting in insufficient supply of the intermediate product histidine betaine; (2) the soluble expression level of the most important thiotransferase in the reaction is low, requiring the preparation of a large amount of enzyme for the reaction, which increases the industrialization cost. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this application provides a method for enzyme-catalyzed production of ergothioneine, a truncated L-histidine betaine sulfonase and its application, a nucleotide sequence encoding L-histidine methyltransferase and its application, the application of truncated L-histidine betaine sulfonase, L-histidine methyltransferase and S-adenosylmethionine synthase in enzyme-catalyzed production of ergothioneine, and combinations of enzymes.

[0007] Specifically, this application relates to the following aspects:

[0008] 1. A truncated L-histidine betaine thiolase, which is an N-terminal truncated sequence of the amino acid sequence shown in SEQ ID NO.12, preferably truncated by 1-50 amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO.12, and the sequence is further preferably shown in SEQ ID NO.4.

[0009] 2. Biomaterials, wherein the biomaterials are any one of the following:

[0010] A1) The nucleic acid molecule encoding the L-histidine betaine sulfonase of claim 1, preferably, the nucleic acid molecule of the L-histidine betaine sulfonase is shown in SEQ ID NO.18;

[0011] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0012] A3) A recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2);

[0013] A4) Recombinant microorganisms containing the nucleic acid molecules described in A1), or recombinant microorganisms containing the expression cassette described in A2), or recombinant microorganisms containing the recombinant vector described in A3).

[0014] Preferably, the recombinant microorganism is Escherichia coli.

[0015] 3. The application of the L-histidine betaine thiosulfate enzyme described in item 1 or the biological material described in item 2 in the production of ergothionein.

[0016] 4. A method for producing ergothioneine, comprising the following steps:

[0017] Ergothioneine is obtained by catalysis of truncated or untruncated L-histidine betaine sulfonase, wherein the truncated L-histidine betaine sulfonase is the truncated L-histidine betaine sulfonase as described in claim 1.

[0018] 5. The method according to item 4, further comprising:

[0019] L-histidine and S-adenosylmethionine are catalyzed by L-histidine methyltransferase to obtain histidine betaine.

[0020] 6. According to the method described in item 5,

[0021] The amino acid sequence of the L-histidine methyltransferase is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5.

[0022] 7. According to the method described in item 6,

[0023] The nucleic acid molecules encoding the L-histidine methyltransferase are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15.

[0024] 8. The method according to item 7, further comprising:

[0025] Methionine and adenosine triphosphate are catalyzed by S-adenosylmethionine synthase to obtain S-adenosylmethionine.

[0026] 9. According to the method described in item 8,

[0027] The amino acid sequence of the S-adenosylmethionine synthase is shown in SEQ ID NO.3.

[0028] 10. The method described in item 9,

[0029] The nucleic acid molecule of the S-adenosylmethionine synthase is shown in SEQ ID NO.16.

[0030] 11. According to the method in item 10,

[0031] The mass ratio of the S-adenosylmethionine synthase, the L-histidine methyltransferase, and the L-histidine betaine sulfonase is (0.5–1):(0.8–1.2):(0.2–0.6).

[0032] 12. A biomaterial, said biomaterial being any of the following:

[0033] B1) A nucleic acid molecule encoding L-histidine methyltransferase, wherein the nucleic acid molecule encoding L-histidine methyltransferase is shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15;

[0034] B2) An expression cassette containing the nucleic acid molecule described in B1):

[0035] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0036] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3), preferably Escherichia coli.

[0037] 13. The application of the biomaterial described in item 12 in the production of ergothionein.

[0038] 14. Application of truncated L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase in the production of ergothioneine.

[0039] Preferred,

[0040] The truncated L-histidine betaine sulfonase is the truncated L-histidine betaine sulfonase as described in claim 1.

[0041] The amino acid sequence of the L-histidine methyltransferase is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5;

[0042] The amino acid sequence of the S-adenosylmethionine is shown in SEQ ID NO.3.

[0043] 15. A combination of enzymes, said combination of enzymes comprising:

[0044] Untruncerated or truncated L-histidine betaine sulfonase,

[0045] L-histidine methyltransferase; and

[0046] S-adenosylmethionine synthase.

[0047] 16. The combination described in item 15,

[0048] The truncated L-histidine betaine sulfonase is as described in claim 1; the amino acid sequence of the untruncated L-histidine betaine sulfonase is shown in SEQ ID NO. 12.

[0049] The amino acid sequence of the L-histidine methyltransferase is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5;

[0050] The amino acid sequence of the S-adenosylmethionine synthase is shown in SEQ ID NO.3.

[0051] 17. According to the combination described in item 16,

[0052] The untruncated L-histidine betaine thiosulfate nucleic acid molecule is shown in SEQ ID NO.17;

[0053] The truncated L-histidine betaine thiosulfate nucleic acid molecule is shown in SEQ ID NO.18;

[0054] Nucleic acid molecules encoding L-histidine methyltransferases are shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15;

[0055] The nucleic acid molecule of the S-adenosylmethionine synthase is shown in SEQ ID NO.16.

[0056] 18. The combination according to any one of items 15-17, wherein the mass ratio of the S-adenosylmethionine synthase, the L-histidine methyltransferase and the L-histidine betaine sulfonase is (0.5-1):(0.8-1.2):(0.2-0.6).

[0057] 19. A method for producing ergothioneine, comprising using a combination of enzymes as described in any one of items 15-18.

[0058] 20. A genetically engineered bacterium, said engineered bacterium comprising:

[0059] The L-histidine betaine sulfonase shown in SEQ ID NO.4,

[0060] L-histidine methyltransferases shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.5;

[0061] S-adenosylmethionine synthase as shown in SEQ ID NO.3;

[0062] Preferably, the host bacterium is Escherichia coli.

[0063] 21. A whole-cell catalyst, characterized in that it contains the genetically engineered bacteria described in item 20.

[0064] 22. A method for preparing ergothionein, comprising using the genetically engineered bacteria of item 20 or the whole-cell catalyst of item 21.

[0065] Effects of this application

[0066] (1) This application provides a key enzyme with high catalytic activity, L-histidine methyltransferase (egtD), and a highly soluble thiotransferase, solving the bottleneck problem restricting the industrial production of ergothioneine. This application first screened for key enzymes exhibiting high catalytic activity, L-histidine methyltransferase MbegtD, L-histidine methyltransferase SbegtD, and L-histidine methyltransferase eanA. Then, protein engineering was used to modify L-histidine betaine thiotransferase to obtain a highly soluble L-histidine betaine thiotransferase mutant eanB (Δ29) whose specific enzyme activity was unaffected. MbegtD or SbegtD or eanA, eanB or eanB (Δ29), and S-adenosylmethionine synthase metK were combined to form a complex enzyme, which was then used to catalyze the in vitro synthesis of ergothioneine from histidine.

[0067] (2) The key enzymes screened in this application, L-histidine methyltransferase MbegtD, L-histidine methyltransferase SbegtD, and L-histidine methyltransferase eanA, exhibit catalytic activities that are 2.1 times, 1.87 times, and 1.63 times that of egtD, which is derived from Mycobacterium smegmatis and is widely used, respectively, thus solving the problem of insufficient supply of the intermediate product histidine betaine. Furthermore, the soluble expression level of the protein-engineered sulfur-modified enzyme mutant eanB (Δ29) is increased by 19 times, and its specific enzyme activity is not affected, significantly reducing the cost of enzyme production. This application utilizes a complex enzyme composed of L-histidine methyltransferase, L-histidine betaine sulfur-modifying enzyme, and S-adenosylmethionine synthase to achieve efficient conversion of L-histidine to ergothioneine. The raw materials required for ergothioneine preparation are inexpensive L-histidine, methionine, ATP, and potassium polysulfide, reducing production costs. Finally, this application provides a cellular material for ergothioneine preparation, which enables efficient intracellular synthesis of ergothioneine and its extracellular transport, solving the problems of low stability of intermediate products and thiosulfate enzymes, and reducing the cost of downstream purification. The ergothioneine preparation method provided in this application is of great significance for achieving low-cost, high-yield production of ergothioneine. Attached Figure Description

[0068] Figure 1 This is a method route diagram for the enzyme-catalyzed production of ergothionein in this application;

[0069] Figure 2This is an SDS-PAGE image of the crude enzyme broth from the fermentation of recombinant L-histidine methyltransferase bacteria. M represents the molecular weight of the protein; the leftmost number indicates the molecular weight of the protein band in the marker lane. Lane 1 contains the crude enzyme fermentation solution of the control strain E. coil BL21(DE3) / pET28a; Lane 2 contains the crude enzyme fermentation solution of the recombinant strain E. coil BL21(DE3) / pET28a-eanA containing L-histidine methyltransferase from Chlorobium limicola; Lane 3 contains the crude enzyme fermentation solution of the recombinant strain E. coil BL21(DE3) / pET28a-SbegtD containing L-histidine methyltransferase from Streptomyces buecherae; Lane 4 contains the crude enzyme fermentation solution of the recombinant strain E. coil BL21(DE3) / pET28a-MbegtD containing L-histidine methyltransferase from Methylobacterium; Lane 5 contains the crude enzyme fermentation solution of the recombinant strain E. coil containing L-histidine methyltransferase from Mycobacterium smegmatis. Lane 6 contains the crude fermentation enzyme solution of E. coil BL21(DE3) / pET28a-MyegtD, a recombinant strain containing L-histidine methyltransferase from Schizosaccharomyces pombe; Lane 7 contains the crude fermentation enzyme solution of E. coil BL21(DE3) / pET28a-SpEgt1, a recombinant strain containing L-histidine methyltransferase from Alkalicoccus saliphilus; Lane 8 contains the crude fermentation enzyme solution of E. coil BL21(DE3) / pET28a-AsegtD, a recombinant strain containing L-histidine methyltransferase from Prosthecochloris; Lane 9 contains the crude fermentation enzyme solution of E. coil BL21(DE3) / pET28a-PsegtD, a recombinant strain containing L-histidine methyltransferase from Methanolobus. The crude enzyme broth of BL21(DE3) / pET28a-MlegtD was fermented; lane 10 contained the crude enzyme broth of E. coil BL21(DE3) / pET28a-BlegtD, a recombinant strain containing L-histidine methyltransferase derived from Bacillus lacisalsi.

[0070] Figure 3 The result is a graph of the catalytic product ERG of L-histidine methyltransferase; the relative conversion rate is calculated with the highest conversion rate as 100%.

[0071] Figure 4SDS-PAGE images of the crude enzyme broth from the recombinant L-histidine betaine sulfonase strain; Lane 1 shows the crude enzyme broth from the recombinant strain E.coil BL21(DE3) / pET28a-eanB containing the complete amino acid sequence of L-histidine betaine sulfonase; Lane 2 shows the crude enzyme broth from the recombinant strain E.coil BL21(DE3) / pET28a-eanB(Δ29) containing the L-histidine betaine sulfonase mutant.

[0072] Figure 5 The conversion rate of L-histidine under the catalysis of different concentrations of eanB enzyme solution;

[0073] Figure 6 The conversion rate of L-histidine under the catalysis of enzyme solutions of the same concentrations of eanB and eanB(Δ29);

[0074] Figure 7 The yield of ergothioneine under the catalysis of enzyme solutions of the same concentrations of eanB and eanB(Δ29);

[0075] Figure 8 The following are LC-MS results of ergothioneine prepared in this application: a) Total ion chromatogram of ergothioneine standard; b) Total ion chromatogram of reaction product; c) Characteristic mass-to-charge ratio fragment ion peaks of ergothioneine standard; d) Characteristic mass-to-charge ratio fragment ion peaks of reaction product. Detailed Implementation

[0076] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0077] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0078] In this application, eanB enzyme refers to L-histidine betaine sulfonase, which is an eanB enzyme derived from Chlorobium limicola.

[0079] In this application, L-histidine methyltransferases were screened from a variety of enzymes from different sources. For ease of recording, different terms were used to represent them. Among them, MbegtD enzyme represents MbegtD enzyme from Methylobacterium, SbegtD enzyme represents SbegtD enzyme from Streptomyces buecherae, and eanA enzyme represents eanA enzyme from Chlorobium limicola.

[0080] In this application, metK enzyme is S-adenosylmethionine synthase, specifically referring to metK enzyme derived from Escherichia coli. "Amino acid" means any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term "amino acid" includes the following 20 naturally or genetically encoded α-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). Amino acids also include non-natural amino acids, modified amino acids (e.g., those with modified side chains and / or backbones), and amino acid analogs.

[0081] For further illustration, an amino acid is typically an organic acid comprising a substituted or unsubstituted amino group, a substituted or unsubstituted carboxyl group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, for example, mercapto, selenyl, sulfonyl, alkyl, aryl, acyl, ketone, azide, hydroxyl, hydrazine, cyano, halogen, acylhydrazine, alkenyl, alkynyl, ether, borate, boronate, dioxophosphorus, phosphonyl, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids containing photosensitive crosslinking agents, metal-bound amino acids, spin-labeled amino acids, fluorescent amino acids, amino acids containing metals, amino acids containing new functional groups, amino acids that interact covalently or non-covalently with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids containing biotin or biotin analogs, glycosylated amino acids, amino acids modified with other carbohydrates, amino acids containing polyethylene glycol or polyethers, heavy atom-substituted amino acids, chemically cleavable and / or photocleavable amino acids, amino acids containing carbon-linked sugars, redox-active amino acids, amino acids containing aminothioic acids, and amino acids containing one or more toxic moieties.

[0082] The term "nucleotide" hereincludes not only naturally occurring ribonucleotide or deoxyribonucleotide monomers, but also, in this context, its related structural variants, including derivatives and analogs, which are functionally equivalent in the specific context of the use of the nucleotide, unless the context explicitly indicates otherwise. For example, "nucleotide" refers to deoxyribonucleotides or ribonucleotides. Nucleotides can be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs. Nucleotide analogs refer to nucleotides having modified purine or pyrimidine bases or modified ribose moieties. Nucleotide analogs can be naturally occurring nucleotides (e.g., inosine, pseudouridine, etc.) or non-naturally occurring nucleotides. Non-limiting examples of modifications to the sugar or base moieties of nucleotides include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxyl, methyl, phosphoryl, and thiol groups, as well as the substitution of carbon and nitrogen atoms of the base by other atoms (e.g., 7-denitropurine). Nucleotide analogs also include dideoxynucleotides, 2'-O-methylnucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino oligonucleotides.

[0083] The terms "codon-optimized," "codon-optimized," or "codon usage preference" refer to the practice of selecting codons in a way that optimizes or customizes expression as needed (i.e., codon usage) to improve protein expression in an organism by increasing the translation efficiency of the target gene. In other words, codon optimization is a method of adjusting codons to match the abundance of host tRNAs and has traditionally been used for heterologous gene expression. New strategies for optimizing heterologous expression consider global nucleotide content, such as local mRNA folding, codon pair bias, codon ramp, or codon correlation. Codon optimization is possible because codon degeneracy is inherent. Degeneracy arises because there are more codons than can encode amino acids. Therefore, the vast majority of amino acids are encoded by multiple codons, meaning there are multiple tRNAs carrying any given amino acid (with different anticodon loops). Therefore, different codons can be used without changing the encoded amino acid sequence. In other words, it is possible to mutate / alter (or synthesize de novo) a gene or segment of nucleic acid to change the codon used to encode a specific amino acid without altering the amino acid sequence of the polypeptide / protein itself. For example, rare codons can be replaced with more abundant codons while keeping the amino acid sequence unchanged.

[0084] A host cell refers to a single-celled prokaryotic or eukaryotic organism (such as bacteria, yeast, and actinomycetes) as well as a single cell derived from a higher plant or animal when grown in a cell culture. "Host cell" can be animal host cell, plant host cell, yeast host cell, fungal host cell, protozoan host cell, or prokaryotic host cell.

[0085] Expression includes any steps involved in peptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0086] A vector is a segment of DNA, usually double-stranded, into which a segment of foreign DNA may have been inserted. Vectors can be, for example, plasmid-derived. Vectors contain a "replicon" polynucleotide sequence that promotes autonomous replication of the vector within the host cell. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host cell; it may be, for example, a replication vector molecule encoding a selectable or screenable marker, or encoding a transgene. Vectors are used to transport foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of or simultaneously with the host chromosomal DNA, and several copies of the vector and its inserted DNA can be generated. Additionally, the vector may contain essential elements that allow the inserted DNA to be transcribed into mRNA molecules or otherwise cause the inserted DNA to replicate into multiple copies of RNA. Some expression vectors also contain sequence elements near the inserted DNA that increase the half-life of the expressed mRNA and / or allow the mRNA to be translated into a protein molecule. Therefore, many molecules encoding mRNA and polypeptides of the inserted DNA can be rapidly synthesized.

[0087] The expression vector comprises a linear or circular DNA molecule containing a fragment encoding the polypeptide of this application, and the fragment can be operatively linked to other fragments that enable its transcription.

[0088] The recombinant expression vector can be any vector (e.g., plasmid or virus), which can be readily processed with recombinant DNA methods and can express the nucleotide sequence. The choice of vector typically depends on its compatibility with the host cell to which it is introduced. The vector can be a linear or closed circular plasmid.

[0089] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and whose replication does not depend on the replication of chromosomes, such as plasmids, extrachromosomal elements, miniature chromosomes, or the replication of artificial chromosomes.

[0090] The vector may contain any means that ensure its own replication. Alternatively, the vector may be integrated into the genome and replicate along with the chromosome into which it is integrated upon introduction into the host cell. Alternatively, a single vector or plasmid may be used, or two or more vectors or plasmids may be used, collectively containing the entire DNA of the genome to be introduced into the host cell, or transposons may be used.

[0091] Fusion proteins are protein molecules that combine two or more proteins. They are usually obtained by expressing hybrid genes that combine two or more gene sequences, which are inserted into an expression vector in a frame-matched manner.

[0092] Enzymes

[0093] In this application, L-histidine betaine sulfonase is a sulfide transferase, which is typically used to transfer sulfur from inorganic or organic sulfides to L-histidine betaine to generate ergothioneine.

[0094] In some embodiments of this application, the L-histidine betaine sulfonase is an L-histidine betaine sulfonase derived from Chlorobium limicola.

[0095] In some embodiments of this application, the amino acid sequence of the L-histidine betaine sulfonase (eanB) is SEQ ID NO.12, that is, the amino acid sequence of the untrunculated L-histidine betaine sulfonase is SEQ ID NO.12.

[0096] Truncated L-histidine betaine sulfonase refers to L-histidine betaine sulfonase that is missing one or more amino acid residues compared to natural L-histidine betaine sulfonase.

[0097] In some embodiments of this application, the truncated L-histidine betaine thiolase is a truncated sequence based on the amino acid sequence shown in SEQ ID NO. 12, preferably an N-terminal truncated sequence of the amino acid sequence shown in SEQ ID NO. 12, and more preferably a truncated sequence of 1-50 amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO. 12.

[0098] In some embodiments of this application, structural analysis of L-histidine betaine thiosulfate enzyme (eanB protein) revealed that it has 29 disordered amino acid sequences at its N-terminus. These 29 amino acids were truncated and mutated, and are denoted as eanB (Δ29).

[0099] In one specific embodiment, the amino acid sequence of the truncated L-histidine betaine sulfonase is shown in SEQ ID NO. 4.

[0100] This application does not limit the method for obtaining truncated L-histidine betaine thiolase. It can be any molecular biology method known to those skilled in the art, as long as it can manipulate the amino acid sequence in the desired truncation manner.

[0101] In this application, L-histidine methyltransferase is a methyltransferase that can be derived from yeast, fungi, and some prokaryotes. It is typically used to transfer the methyl group of S-adenosylmethionine to histidine to generate histidine betaine, a precursor for ergothioneine synthesis.

[0102] In some embodiments of this application, the amino acid sequence of the L-histidine methyltransferase is shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5.

[0103] S-Adenosylmethionine synthase is a ligase primarily used for the ligation reaction of methionine and adenosine triphosphate (ATP) to generate a universal methyl donor, SAM (S-adenosylmethionine).

[0104] In some embodiments of this application, the amino acid sequence of the S-adenosylmethionine synthase is shown in SEQ ID NO. 3.

[0105] This application provides a combination of enzymes comprising: the above-mentioned untruncerated or truncerated L-histidine betaine sulfonase, the above-mentioned L-histidine methyltransferase; and the above-mentioned S-adenosylmethionine synthase.

[0106] The term "enzyme combination" in this application refers to the combination of L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase, whether truncated or not. In this application, "enzyme combination" signifies a combination of enzymatic functions, meaning it can be a physical mixture of the three enzyme proteins. For example, it could be a direct mixture of purchased pure enzymes, or a direct mixture of crude enzyme solutions or purified enzymes produced through gene recombination expression using molecular biology techniques. An enzyme combination can also be a fusion protein formed by fusing the three enzymes in their three-dimensional protein structures, as long as each enzyme can perform its respective function. Similarly, it can be a simple mixture of any two enzyme fusion proteins and a third enzyme.

[0107] Similarly, this application does not limit the process of producing enzymes through gene recombination expression using molecular biology techniques; any known method can be used. In this application, three enzymes or fusion proteins of three enzymes can be expressed simultaneously using the same host and a single plasmid, or one or two enzymes or fusion proteins of enzymes can be produced separately using different hosts.

[0108] In some embodiments of this application, the combination is a composition of untruncated or truncated L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase.

[0109] In some embodiments of this application, the combination is a mixture of crude fermentation enzyme broth for producing uncut or truncated L-histidine betaine sulfonase, crude fermentation enzyme broth for L-histidine methyltransferase, and crude fermentation enzyme broth for S-adenosylmethionine synthase.

[0110] In some embodiments of this application, the combination is a fusion protein formed from untruncerated or truncated L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase; or a combination of an untruncerated or truncated fusion protein of L-histidine betaine sulfonase and L-histidine methyltransferase with S-adenosylmethionine synthase; or a combination of an untruncerated or truncated fusion protein of L-histidine betaine sulfonase and S-adenosylmethionine synthase with L-histidine methyltransferase; or a combination of an L-histidine methyltransferase and S-adenosylmethionine synthase fusion protein with untruncerated or truncated L-histidine betaine sulfonase.

[0111] This application provides the use of the above-mentioned untruncated or truncated L-histidine betaine thiosulfate enzyme, the above-mentioned L-histidine methyltransferase, and the above-mentioned S-adenosylmethionine synthase in the production of ergothioneine.

[0112] This application provides a method for producing ergothioneine, comprising using the combination of the above-described enzymes.

[0113] Biomaterials

[0114] This application provides a biomaterial, which is any one of the following:

[0115] A1) The nucleic acid molecule encoding the above-mentioned L-histidine betaine sulfonase, preferably, the L-histidine betaine sulfonase nucleic acid molecule is shown in SEQ ID NO.18;

[0116] A2) An expression cassette containing the nucleic acid molecules described in A1);

[0117] A3) A recombinant vector containing the nucleic acid molecule described in A1), or a recombinant vector containing the expression cassette described in A2);

[0118] A4) Recombinant microorganisms containing the nucleic acid molecules described in A1), or recombinant microorganisms containing the expression cassette described in A2), or recombinant microorganisms containing the recombinant vector described in A3).

[0119] In some embodiments of this application, the recombinant microorganism is Escherichia coli.

[0120] This application provides the use of the above-described L-histidine betaine thiosulfate enzyme or any of the above-described biological materials in the production of ergothionein.

[0121] This application provides a biomaterial, which is any one of the following:

[0122] B1) A nucleic acid molecule encoding the above-mentioned L-histidine methyltransferase, said nucleic acid molecule being shown in SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15;

[0123] B2) An expression cassette containing the nucleic acid molecule described in B1):

[0124] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0125] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0126] In some embodiments of this application, the recombinant microorganism is Escherichia coli.

[0127] This application provides a biomaterial, which is any one of the following:

[0128] C1) The nucleic acid molecule encoding the above-mentioned S-adenosylmethionine synthase, said nucleic acid molecule is shown in SEQ ID NO.16;

[0129] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0130] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0131] C4) Recombinant microorganisms containing the nucleic acid molecules described in C1), or recombinant microorganisms containing the expression cassette described in C2), or recombinant microorganisms containing the recombinant vector described in C3).

[0132] In some embodiments of this application, the recombinant microorganism is Escherichia coli.

[0133] This application provides the application of the above-mentioned biomaterials in the production of ergothioneine.

[0134] Genetically engineered bacteria

[0135] This application provides a genetically engineered bacterium, the engineered bacterium comprising:

[0136] The L-histidine betaine sulfonase shown in SEQ ID NO.4,

[0137] L-histidine methyltransferases shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.5;

[0138] S-adenosylmethionine synthase as shown in SEQ ID NO.3.

[0139] This application provides a combination of genetically engineered bacteria, comprising:

[0140] Genetically engineered bacteria containing L-histidine betaine sulfonase as shown in SEQ ID NO.4;

[0141] Genetically engineered bacteria containing L-histidine methyltransferase as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5; and genetically engineered bacteria containing S-adenosylmethionine synthase as shown in SEQ ID NO.3.

[0142] This application provides a combination of genetically engineered bacteria, comprising:

[0143] Genetically engineered bacteria containing L-histidine betaine sulfonase as shown in SEQ ID NO.4 and L-histidine methyltransferase as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5; and genetically engineered bacteria containing S-adenosylmethionine synthase as shown in SEQ ID NO.3; or

[0144] Genetically engineered bacteria containing L-histidine betaine sulfonase as shown in SEQ ID NO.4 and S-adenosylmethionine synthase as shown in SEQ ID NO.3, and genetically engineered bacteria containing L-histidine methyltransferase as shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.5; or

[0145] Genetically engineered bacteria containing L-histidine betaine sulfonase as shown in SEQ ID NO.4; and genetically engineered bacteria containing S-adenosylmethionine synthase as shown in SEQ ID NO.3 and L-histidine methyltransferase as shown in SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.5.

[0146] This application provides a whole-cell catalyst containing the above-mentioned genetically engineered bacteria or a combination of the above-mentioned genetically engineered bacteria.

[0147] This application provides a method for preparing ergothionein, comprising using the above-mentioned genetically engineered bacteria or a combination of the above-mentioned genetically engineered bacteria or the above-mentioned whole-cell catalyst.

[0148] DNA

[0149] In some embodiments of this application, L-histidine methyltransferase is referred to as MbegtD enzyme, and the optimized sequence of the MbegtD enzyme codon is shown in SEQ ID NO.13.

[0150] In some embodiments of this application, L-histidine methyltransferase is referred to as SbegtD enzyme, and the optimized sequence of the SbegtD enzyme codon is shown in SEQ ID NO.14.

[0151] In some embodiments of this application, L-histidine methyltransferase is referred to as eanA enzyme, and the optimized sequence of the eanA enzyme codon is shown in SEQ ID NO.15.

[0152] In some embodiments of this application, S-adenosylmethionine synthase is referred to as metK enzyme, and the optimized sequence of the metK enzyme codon is shown in SEQ ID NO.16.

[0153] In some embodiments of this application, the untrunculated L-histidine betaine thiolase is denoted as eanB, and the optimized sequence of the eanB codon is shown in SEQ ID NO.17.

[0154] In some embodiments of this application, the truncated L-histidine betaine thiolase is denoted as eanB(Δ29), and the optimized sequence of the eanB(Δ29) codon is shown in SEQ ID NO.18.

[0155] Production Process

[0156] This application provides a method for the enzyme-catalyzed production of ergothioneine, comprising the following steps: obtaining ergothioneine from histidine betaine via L-histidine betaine sulfonase catalysis, wherein the L-histidine betaine sulfonase is a truncated or untruncated L-histidine betaine sulfonase.

[0157] In some embodiments of this application, L-histidine and S-adenosylmethionine are catalyzed by a truncated or untruncated L-histidine methyltransferase to obtain histidine betaine, and histidine betaine is catalyzed by L-histidine betaine sulfonase to obtain ergothioneine, wherein the L-histidine betaine sulfonase is a truncated or untruncated L-histidine betaine sulfonase.

[0158] In some embodiments of this application, in the method for enzyme-catalyzed production of ergothioneine, methionine and adenosine triphosphate are catalyzed by S-adenosylmethionine synthase to obtain S-adenosylmethionine, L-histidine and S-adenosylmethionine are catalyzed by L-histidine methyltransferase to obtain histidine betaine, and histidine betaine is catalyzed by truncated or untruncated L-histidine betaine sulfonase to obtain ergothioneine.

[0159] See Figure 1The synthetic pathway involves the reaction of methionine and adenosine triphosphate (ATP) with S-adenosylmethionine synthase to obtain S-adenosylmethionine (SAM), simultaneously generating inorganic phosphate (Pi) and inorganic pyrophosphate (PPi) as byproducts. Then, under the catalysis of L-histidine methyltransferase, S-adenosylmethionine and L-histidine transfer the methyl group of SAM to L-histidine to obtain histidine betaine. Simultaneously, S-adenosylmethionine loses its methyl group to generate S-adenosylhomocysteine ​​(SAH). Finally, histidine betaine and potassium polysulfide (K2S)... x Under the catalysis of truncated or untruncated L-histidine betaine sulfonase, K2S... x The sulfur is transferred to histidine betaine to obtain ergothioneine.

[0160] Histidine betaine (Hercynine), chemical formula C9H 15 N3O2, with a relative molecular mass of 197.23 and a melting point of 237-238℃ (dec), is produced by histidine via methyltransferase catalysis. It is commonly used as a precursor in the synthesis of ergothioneine and can be used as a food and cosmetic additive.

[0161] In some embodiments of this application, the L-histidine betaine sulfonase is an L-histidine betaine sulfonase derived from Chlorobium limicola.

[0162] In some embodiments of this application, the truncated L-histidine betaine sulfonase is a truncated sequence based on the amino acid sequence shown in SEQ ID NO. 12, preferably an N-terminal truncated sequence of the amino acid sequence shown in SEQ ID NO. 12, more preferably a truncated sequence of 1-50 amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO. 12, and more preferably the amino acid sequence of the L-histidine betaine sulfonase is as shown in SEQ ID NO. 4.

[0163] In some embodiments of this application, the amino acid sequence of the untrunculated L-histidine betaine sulfonase is shown in SEQ ID NO.12.

[0164] In some embodiments of this application, structural analysis of the eanB protein revealed that it has 29 disordered amino acid sequences at its N-terminus, and these 29 amino acids were truncated and mutated.

[0165] L-histidine is a colorless, flaky or needle-like crystal, odorless, with a slightly bitter taste. It softens at 227℃ and decomposes at 277℃. It is soluble in water. It is produced from dry flour, pig or cow blood meal, pig hair or hooves, or by fermentation of glucose.

[0166] In some embodiments of this application, the L-histidine methyltransferase is an L-histidine methyltransferase derived from Methylobacterium, Streptomyces buecherae, or Chlorobium limicola.

[0167] Methionine is a white, thin, flaky crystal or crystalline powder with a characteristic odor and a slightly sweet taste. It has a melting point of 280–281°C, and a pH of 5.6–6.1 for a 10% aqueous solution. It is optically active (possesses a chiral carbon atom), stable to heat and air, but unstable to strong acids, which can lead to demethylation. It is soluble in water (3.3 g / 100 ml, 25°C), dilute acids, and dilute alkalis.

[0168] S-Adenosylmethionine (S-Amethystine) is synthesized from adenosine triphosphate (ATP) and methionine by methionine adenosyltransferase. Transmethylation, transsulfuration, and aminopropylation all utilize the S-A-Amethystine metabolic pathway. These anabolic reactions occur throughout the body, with most S-A-Amethystine production and consumption taking place in the liver. The methyl group (CH3) attached to the sulfur atom of methionine in S-A-Amethystine is chemically active. This makes it readily transfer to the acceptor substrate in transmethylation reactions. More than 40 metabolic reactions involve the transfer of methyl groups from S-A-Amethystine to various substrates, such as nucleic acids, proteins, lipids, and secondary metabolites. In bacteria, S-A-Amethystine is linked to the SAM riboswitch, participating in the regulation of methionine and cysteine ​​synthesis. The production, consumption, and regeneration of S-adenosylmethionine (S-AMe) is known as the S-AMe cycle. The first step involves an S-AMe-dependent methyltransferase using S-A-AMe as a substrate to produce S-A-AMe homocysteine. This homocysteine ​​is further hydrolyzed by an S-AMe homocysteine ​​hydrolase into homocysteine ​​and adenosine. Homocysteine ​​is then converted back to methionine via a methyltransferase. This reaction is carried out by cobalamin-dependent or cobalamin-independent methionine synthases. Methionine can then be further converted back to S-AMe, completing the cycle.

[0169] In some embodiments of this application, ergothionein is produced by catalysis in a buffer solution.

[0170] In some embodiments of this application, the buffer solution is selected from one or more of the following: Tris-HCl buffer solution, disodium hydrogen phosphate-citric acid buffer solution, citric acid-NaOH-HCl buffer solution, citric acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution, phosphate buffer solution, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-NaOH buffer solution, sodium barbital-HCl buffer solution, NH4HCO3 buffer solution, sodium carbonate-sodium bicarbonate buffer solution, NaHCO3 buffer solution, glycine-NaOH buffer solution, boric acid-borax buffer solution, and Na2B7O4 buffer solution.

[0171] In some embodiments of this application, the buffer solution is a Tris-HCl buffer solution.

[0172] In one specific embodiment, the Tris-HCl buffer is 50 mM and pH 8.0.

[0173] In some embodiments of this application, the concentration of L-histidine in the buffer solution is 5 mmol / L to 20 mmol / L; the concentration of methionine is 5 mmol / L to 20 mmol / L; the concentration of adenosine triphosphate is 5 mmol / L to 20 mmol / L; the concentration of potassium polysulfide is 0.8 g / L to 1.2 g / L; and the sum of the concentrations of L-histidine methyltransferase, S-adenosylmethionine synthase, and L-histidine betaine sulfonase is 300 μL / mL to 600 μL / mL.

[0174] For example, in the buffer solution, the L-histidine concentration can be 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L or any range thereof;

[0175] The methionine concentration can be any range of 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L or between;

[0176] The concentration of adenosine triphosphate can be any range of 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L or between;

[0177] The concentration of potassium polysulfide can be 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L or any range thereof;

[0178] The sum of the concentrations of the L-histidine methyltransferase, the S-adenosylmethionine synthase, and the L-histidine betaine sulfhydrylase can be 300 μL / mL, 310 μL / mL, 320 μL / mL, 330 μL / mL, 340 μL / mL, 350 μL / mL, 360 μL / mL, 370 μL / mL, 380 μL / mL, 390 μL / mL, 400 μL / mL, 410 μL / mL, 420 μL / mL, 430 μL / mL, etc. The concentrations are 440 μL / mL, 450 μL / mL, 460 μL / mL, 470 μL / mL, 480 μL / mL, 490 μL / mL, 500 μL / mL, 510 μL / mL, 520 μL / mL, 530 μL / mL, 540 μL / mL, 550 μL / mL, 560 μL / mL, 570 μL / mL, 580 μL / mL, 590 μL / mL, 300 μL / mL, or any range thereof.

[0179] In some embodiments of this application, the mass ratio of the S-adenosylmethionine synthase, the L-histidine methyltransferase, and the L-histidine betaine sulfonase is 1:(0.8-2.4):(0.2-1.2).

[0180] In one specific embodiment, ergothioneine was prepared by adding 200mM L-histidine, 100mM methionine, 100mM ATP, 6.6g / L potassium polysulfide, 100g / L cell wet weight, and 0.01% Triton X-100 to 50mM Tris-HCl buffer and stirring the mixture for 48h at 25°C and pH 8.0.

[0181] This application provides a truncated L-histidine betaine thiolase, which is an N-terminal truncated sequence of the amino acid sequence shown in SEQ ID NO.12, preferably truncated by 1-50 amino acids from the N-terminus of the amino acid sequence shown in SEQ ID NO.12, and its sequence is further preferably as shown in SEQ ID NO.4.

[0182] This application provides the application of the above-mentioned truncated L-histidine betaine thiolase in the enzyme-catalyzed production of ergothionein.

[0183] This application provides a nucleotide sequence encoding an L-histidine methyltransferase, as shown in SEQ ID NO.13, SEQ ID NO.14, or SEQ ID NO.15.

[0184] This application provides the application of the L-histidine methyltransferase encoded by the above-mentioned nucleotide sequence in the enzyme-catalyzed production of ergothionein.

[0185] This application provides the use of truncated L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase in the enzyme-catalyzed production of ergothionein.

[0186] The truncated L-histidine betaine sulfonase has been described previously and will not be repeated here.

[0187] The nucleotide sequence encoding L-histidine methyltransferase has been described above and will not be repeated here.

[0188] This application provides a combination of enzymes comprising: untruncerated or truncerated L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase.

[0189] In some embodiments of this application, the combination is a composition of untruncated or truncated L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase.

[0190] In some embodiments of this application, the combination is a fusion protein formed from untruncerated or truncated L-histidine betaine sulfonase, L-histidine methyltransferase, and S-adenosylmethionine synthase; or a combination of an untruncerated or truncated fusion protein of L-histidine betaine sulfonase and L-histidine methyltransferase with S-adenosylmethionine synthase; or a combination of an untruncerated or truncated fusion protein of L-histidine betaine sulfonase and S-adenosylmethionine synthase with L-histidine methyltransferase; or a combination of an L-histidine methyltransferase and S-adenosylmethionine synthase fusion protein with untruncerated or truncated L-histidine betaine sulfonase.

[0191] The untruncerated or truncated L-histidine betaine sulfonase has been described above and will not be repeated here.

[0192] The nucleotide sequence encoding L-histidine methyltransferase has been described above and will not be repeated here.

[0193] Example

[0194] Example 1: Preparation of MbegtD enzyme solution

[0195] This application is based on the full-length gene sequence of L-histidine methyltransferase from Methylobacterium publicly available in the NCBI database. Codon optimization was performed based on the codon preference of *E. coli*. This optimization included: eliminating secondary structures unfavorable to expression (such as hairpin structures), altering GC content, CpG dinucleotide content, mRNA secondary structure, cryptic splicing sites, early polyadenylation sites, internal ribosome entry and binding sites, negative CpG islands, RNA unstable regions, repetitive sequences (direct repeats, inverted repeats, etc.), and restriction sites that may affect cloning. The optimized gene is shown in SEQ ID NO. 13.

[0196] The optimized gene was synthesized by Nanjing GenScript Biotech Co., Ltd. Using the synthesized gene as a template, primers were designed, and the gene sequence was obtained through in vitro amplification. First, the gene and plasmid pET28a were double-digested with the restriction endonucleases EcoRI / SalI. Then, the two digested DNA fragments were ligated using T4 DNA ligase to obtain the plasmid pET28a-MbegtD. DNA sequencing confirmed the recombinant sequence was correct. The recombinant expression vector was transformed into *E. coli* BL21(DE3), resulting in the recombinant strain *E. coli* BL21(DE3) / pET28a-MbegtD.

[0197] The obtained recombinant engineered E. coli BL21(DE3) / pET28a-MbegtD was cultured in shake flasks. Single colonies were picked and inoculated into 5 mL of LB resistant medium, and cultured overnight at 37°C and 220 rpm to obtain the seed culture. A 1% inoculum was then transferred to 50 mL of fermentation medium TB and cultured at 37°C and 220 rpm until OD (dose retardation). 600 =0.6-0.8, add 0.5mM IPTG, adjust the temperature to 25℃, and induce expression for 18-20h. Centrifuge at 12000rpm at 4℃ for 10min, collect the cells, resuspend the cells in 50mM Tris-HCl (pH 8.0) buffer, sonicate to disrupt the cells, centrifuge at 12000rpm at 4℃ for 20min, and collect the supernatant as the crude MbegtD enzyme solution. The LB resistant medium composition is as follows: yeast extract 5g / L, peptone 10g / L, sodium chloride 10g / L, kanamycin 50mg / L; the TB fermentation medium composition is as follows: yeast extract 24g / L, peptone 12g / L, glycerol 4g / L, K2HPO43H2O 12.64g / L, KH2PO4 2.31g / L, kanamycin 50mg / L.

[0198] Example 2: Preparation of SbegtD enzyme solution

[0199] This application is based on the full-length gene sequence of L-histidine methyltransferase from Streptomyces buecherae published in the NCBI database. Codon optimization was performed on the gene according to the codon preference of Escherichia coli. The optimized gene is shown in SEQ ID NO.14.

[0200] The optimized gene was synthesized by Nanjing GenScript Biotech Co., Ltd. First, the gene and plasmid pET28a were double-digested with the restriction endonucleases EcoRI / SalI. Then, the two digested DNA fragments were ligated using T4 DNA ligase to obtain the plasmid pET28a-SbegtD. The recombinant expression vector was transformed into *E. coli* BL21(DE3) to obtain the recombinant strain *E. coli* BL21(DE3) / pET28a-SbegtD. The construction method of the recombinant strain *E. coli* BL21(DE3) / pET28a-SbegtD was the same as in Example 1, as were the fermentation and enzyme solution acquisition methods. Crude SbegtD enzyme solution was obtained.

[0201] Example 3: Preparation of eanA enzyme solution

[0202] This application is based on the full-length gene sequence of L-histidine methyltransferase from Chlorobium limicola published in the NCBI database. Codon optimization was performed on the gene according to the codon preference of Escherichia coli. The optimized gene sequence is shown in SEQ ID NO.15.

[0203] The optimized gene was synthesized by Nanjing GenScript Biotech Co., Ltd. First, the gene and plasmid pET28a were double-digested with the restriction endonucleases EcoRI / SalI. Then, the two digested DNA fragments were ligated using T4 DNA ligase to obtain plasmid pET28a-eanA. The recombinant expression vector was transformed into *E. coli* BL21(DE3) to obtain the recombinant strain *E. coli* BL21(DE3) / pET28a-eanA. The construction method of the recombinant strain *E. coli* BL21(DE3) / pET28a-eanA was the same as in Example 1, as were the fermentation and enzyme solution acquisition methods. Crude eanA enzyme solution was obtained.

[0204] Example 4: Preparation of metK enzyme solution

[0205] This application is based on the full-length gene sequence of S-adenosylmethionine synthase from Escherichia coli published in the NCBI database. Codon optimization was performed on the gene according to the codon preference of Escherichia coli. The optimized gene sequence is shown in SEQ ID NO. 16.

[0206] The optimized gene was synthesized by Nanjing GenScript Biotech Co., Ltd. First, the gene and plasmid pET28a were double-digested with the restriction endonucleases EcoRI / SalI. Then, the two digested DNA fragments were ligated using T4 DNA ligase to obtain the plasmid pET28a-metK. The recombinant expression vector was transformed into *E. coli* BL21(DE3) to obtain the recombinant strain *E. coli* BL21(DE3) / pET28a-metK. The construction method of the recombinant strain *E. coli* BL21(DE3) / pET28a-metK was the same as in Example 1, as were the fermentation and enzyme solution acquisition methods. Crude metK enzyme solution was obtained.

[0207] Example 5: Preparation of eanB enzyme solution

[0208] This application is based on the full-length gene sequence of L-histidine betaine sulfonase from *Chlorobium limicola* publicly available in the NCBI database. Codon optimization was performed according to the codon preference of *E. coli*, and the optimized gene sequence is shown in SEQ ID NO. 17. The optimized gene was synthesized by Nanjing GenScript Biotech Co., Ltd., and the amino acid sequence is shown in SEQ ID NO. 12. Primers were designed using the synthesized gene as a template, and the gene fragment was obtained through in vitro amplification. First, the gene and plasmid pET28a were double-digested with the restriction endonucleases EcoRI / SalI. Then, the two digested DNA fragments were ligated using T4 DNA ligase to obtain plasmid pET28a-eanB. The recombinant expression vector was transformed into Escherichia coli BL21(DE3) to obtain the recombinant strain E.coli BL21(DE3) / pET28a-eanB. The construction method of the recombinant strain E.coli BL21(DE3) / pET28a-eanB is the same as in Example 1, and the fermentation and enzyme solution acquisition methods are the same as in Example 1 to obtain crude eanB enzyme solution.

[0209] Example 6: Application of the enzyme composition in the preparation of ergothioneine

[0210] 6-1

[0211] Application of a composition consisting of the MbegtD enzyme solution prepared in Example 1, the metK enzyme solution prepared in Example 4, and the eanB enzyme solution prepared in Example 5 in the preparation of ergothioneine.

[0212] The reaction system consisted of 1 mL of a mixture of 10 mM L-histidine, 15 mM methionine, 15 mM ATP, 0.83 g / L potassium polysulfide, and 400 μL of enzyme (where the mass ratio of metK enzyme: MbegtD enzyme: eanB enzyme was 1:1:0.5), which was then brought to a final volume of 1 mL with Tris-HCl (pH 8.0) buffer. The reaction was carried out at 25°C for 24 h. Immediately after the reaction, the mixture was placed in a boiling water bath for 10 min to inactivate the enzyme and terminate the reaction. The reaction products were then analyzed by liquid chromatography (LC). The LC analysis conditions were as follows:

[0213] Mobile phase: 50 mM NaH2PO4, 5 mM sodium heptanesulfonate, 15% methanol, pH adjusted to 3.9.

[0214] Liquid chromatography detection conditions: wavelength 254 nm, column temperature 35 °C, injection volume 20 μL, flow rate 1 mL / min

[0215] Chromatographic column and instrument: A CAPcell pak C18 MGⅡ column was used with an Agilent 1260 liquid chromatograph and a UV detector for gradient injection.

[0216] Preparation of standards: Weigh histidine, ATP, methionine, potassium polysulfide, and ergothioneine separately, dissolve them in water, and dilute to 10 mL to prepare a stock solution (stock solution composition is as follows: histidine 15.5 g / L, ATP 55.1 g / L, methionine 14.92 g / L, potassium polysulfide 6.67 g / L, ergothioneine 1 g / L). Take 10 mL of each stock solution and dilute to 100 mL in a volumetric flask to prepare a mixed standard (mixed standard composition is as follows: histidine 1.55 g / L, ATP 5.51 g / L, methionine 1.492 g / L, potassium polysulfide 0.667 g / L, ergothioneine 0.1 g / L).

[0217] Ergothioneine conversion calculation method: ERG standards with different concentration gradients were prepared and subjected to liquid chromatography analysis. An ergothioneine standard curve was constructed with ergothioneine concentration as the x-axis and peak area as the y-axis. The peak areas of the reaction products were obtained by liquid chromatography analysis, and the corresponding ergothioneine yield was calculated based on the standard curve. The conversion rate was calculated based on the mass of ergothioneine product and the mass of histidine substrate. 6.2

[0219] Application of a composition consisting of the SbegtD enzyme solution prepared in Example 2, the metK enzyme solution prepared in Example 4, and the eanB enzyme solution prepared in Example 5 in the preparation of ergothioneine.

[0220] The only difference from 6.1 is that the mass ratio of metK enzyme:SbegtD enzyme:eanB enzyme in the enzyme composition is 1:1:0.5, and all other conditions are the same. 6.3

[0222] Application of a composition consisting of the eanA enzyme solution prepared in Example 3, the metK enzyme solution prepared in Example 4, and the eanB enzyme solution prepared in Example 5 in the preparation of ergothioneine.

[0223] The only difference from 6.1 is that the mass ratio of metK enzyme: eanA enzyme: eanB enzyme in the enzyme composition is 1:1:0.5, and all other conditions are the same.

[0224] Example 7: eanB protein engineering modification

[0225] The crude eanB enzyme solution from Example 5 was analyzed by SDS-PAGE, and the results are as follows: Figure 4 As shown, the soluble expression level of eanB was very low, with a concentration of only 22.4 mg / L. The crude eanB enzyme solution was purified by nickel column chromatography to obtain pure enzyme. Different concentrations of the pure eanB enzyme were used for catalytic reactions, using the same reaction system as in Example 6. The conversion rate of L-histidine was calculated, and the results are shown below. Figure 5 As shown. From Figure 5 As can be seen, increasing the amount of eanB enzyme can significantly improve the substrate conversion rate. Improving the soluble expression level of eanB is the most critical issue. Therefore, this application uses protein engineering to modify the enzyme.

[0226] Analysis of the eanB protein structure revealed 29 disordered amino acid sequences at its N-terminus. These 29 amino acids were truncated and mutated using protein engineering to obtain eanB(Δ29), which does not include the first 29 amino acids at the N-terminus. Codon optimization was performed based on the codon preference of *E. coli*, and the optimized gene sequence of eanB(Δ29) is shown in SEQ ID NO.18. The optimized gene was synthesized by Nanjing GenScript Biotech Co., Ltd., and the amino acid sequence is shown in SEQ ID NO.4. Using the recombinant plasmid pET28a-eanB constructed in Example 5 as a template, primers were designed at the 30th amino acid, and the plasmid pET28a-eanB(Δ29) was obtained through in vitro amplification. The recombinant expression vector was then transformed into *E. coli* BL21(DE3), resulting in the recombinant strain *E. coli* BL21(DE3) / pET28a-eanB(Δ29). The fermentation and enzyme solution acquisition methods for the recombinant strain were the same as in Example 1. The crude enzyme solution was analyzed by SDS-PAGE, and the results are shown below. Figure 4 As shown in the results, the expression level of soluble protein in recombinant E.coil BL21(DE3) / pET28a-eanB(Δ29) is much higher than that in recombinant E.coil BL21(DE3) / pET28a-eanB. The soluble expression level of eanB(Δ29) was measured to be 448 mg / L, which is 19 times higher than that of eanB.

[0227] eanB and eanB(Δ29), after being purified by nickel column chromatography, were added to the catalytic reaction at the same concentration, and the results were as follows: Figure 6 As shown in the results, the enzyme activity of eanB(Δ29) after protein engineering was not affected, and the L-histidine conversion rate of the eanB(Δ29) catalyzed reaction was the same as that of the eanB catalyzed reaction.

[0228] The results showed that the soluble expression level of eanB(Δ29) modified by protein engineering was increased by 19 times, and the enzyme activity was not affected, which reduced the cost of enzyme production and has significance for industrial production.

[0229] Example 8

[0230] The application reaction system for ergothioneine preparation using the eanB enzyme solution prepared in Example 5 and the eanB(Δ29) enzyme solution prepared in Example 7 was as follows: 1 mL: 10 mM L-histidine, 15 mM methionine, 15 mM ATP, 0.83 g / L potassium polysulfide, 100 μL of the eanB enzyme solution prepared in Example 5, and brought to 1 mL with Tris-HCl (pH 8.0) buffer; or, 1 mL: 10 mM L-histidine, 15 mM methionine, 15 mM ATP, 0.83 g / L potassium polysulfide, 100 μL of the eanB(Δ29) enzyme solution prepared in Example 7, and brought to 1 mL with Tris-HCl (pH 8.0) buffer. The detection method for ergothioneine is shown in 6-1.

[0231] The result is as follows Figure 7 As shown, the ergothioneine yield of untreated eanB was 7.6 mg / L; the ergothioneine yield of truncated eanB (Δ29) was 215.6 mg / L.

[0232] Example 9: Preparation of cell materials containing enzyme combinations

[0233] Using the obtained recombinant plasmids pET28a-MbegtD, pET28a-metK, and pET28a-eanB(Δ29) as templates, gene fragments were amplified by PCR. The obtained MbegtD, metK, and eanB(Δ29) gene fragments were then constructed into a plasmid vector containing a dual promoter. Specifically, MbegtD and eanB(Δ29) were constructed into two cloning sites of pRSFDuet-1, respectively, to obtain the recombinant plasmid pRSFDuet-1-MbegtD-eanB(Δ29), and metK was constructed into the first cloning site of pACYCDuet-1, resulting in the recombinant plasmid pACYCDuet-1-metK. Both recombinant plasmids were then co-transformed into *E. coli* BL21(DE3) to obtain a recombinant strain containing MbegtD, metK, and eanB(Δ29).

[0234] The recombinant bacteria were fermented, and single colonies were picked and inoculated into 5 mL of LB resistant medium (LB resistant medium composition: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, kanamycin 50 mg / L), and cultured overnight at 37℃ and 220 rpm to obtain seed culture. 1% of the seed culture was transferred to 50 mL of fermentation medium TB (fermentation medium TB composition: yeast extract 24 g / L, peptone 12 g / L, glycerol 4 g / L, K₂HPO₄·3H₂O 12.64 g / L, KH₂PO₄ 2.31 g / L, kanamycin 50 mg / L), and cultured at 37℃ and 220 rpm until OD₆₀ = 0.6-0.8. 0.5 mM IPTG was added, the temperature was adjusted to 25℃, and expression was induced for 18-20 h. Centrifuge at 5000 rpm for 10 min at 4℃, collect the bacterial cells, and wash the bacterial cells three times with 50 mM Tris-HCl (pH 8.0) buffer to obtain the cell material containing the enzyme combination required for the preparation of ergothionein.

[0235] Example 10: Application of cell materials containing enzyme combinations in the production of ergothioneine.

[0236] Substrate preparation: Weigh 200mM histidine, 100mM ATP, 100mM methionine, 6.6g K2SX, and 20mM MgCl2·6H2O, and bring the volume to 1L with 50mM Tris-HCl (pH 8.0) to obtain the substrate solution.

[0237] Preparation of ergothioneine: Weigh 2g of the cell material obtained in Example 8, resuspend it in 20ml of substrate solution, add 0.01% Triton X-100, and carry out the catalytic reaction at 25℃ and 220rpm. After reacting for 48h, centrifuge to remove cells, and take the supernatant for liquid chromatography detection. The detection and analysis methods are the same as in Example 6.

[0238] Without optimized catalytic reaction conditions, the yield of ergothioneine in shake flasks can exceed 300 mg / L. The reaction products were identified by LC-MS, and the results are as follows: Figure 8 As shown in the results, ergothionein can be successfully synthesized after being catalyzed by the composite enzyme and cell material provided in this application.

[0239] Comparative Study: Application of L-histidine methyltransferases from other sources in ergothioneine synthesis

[0240] While expressing the full-length L-histidine methyltransferase gene from the aforementioned *Methylobacterium*, this application also screened and identified six other L-histidine methyltransferases from different sources using the NCBI database. These are: MyegtD amino acid sequence from *Mycobacterium smegmatis* (SEQ ID NO. 6); SpEgt1 amino acid sequence from *Schizosaccharomyces pombe* (SEQ ID NO. 7); AsegtD amino acid sequence from *Alkalicoccus saliphilus* (SEQ ID NO. 8); PsegtD amino acid sequence from *Prosthecochloris Methanolobus* (SEQ ID NO. 9); MlegtD amino acid sequence from Methanolobus (SEQ ID NO. 10); and BlegtD amino acid sequence from *Bacillus lacisalsi* (SEQ ID NO. 11).

[0241] The genes of the above-mentioned enzymes were all codon-optimized according to the codon preference of *E. coli*. The codon-optimized L-histidine methyltransferase sequence was synthesized in its entirety by Nanjing GenScript Biotech Co., Ltd., and cloned into the EcoRI and SalI restriction sites of the *E. coli* expression vector pET28a, resulting in recombinant expression vectors pET28a-MyegtD, pET28a-SpEgt1, pET28a-AsegtD, pET28a-PsegtD, pET28a-MlegtD, and pET28a-BlegtD. DNA sequencing confirmed the correct recombinant sequences. These recombinant vectors were transformed into *E. coli* BL21(DE3) to obtain recombinant bacteria. The six strains obtained were fermented in shake flasks according to the method in Example 1. The crude enzyme solutions obtained, along with the crude enzyme solutions of the control strain (containing only the pET28a empty plasmid) and the crude enzyme solutions obtained in Examples 1, 2, and 3, were analyzed by SDS-PAGE protein electrophoresis. The results are as follows: Figure 2 As shown.

[0242] The results show that the recombinant strains E. coil BL21(DE3) / pET28a-eanA in lane 2, E. coil BL21(DE3) / pET28a-SbegtD in lane 3, E. coil BL21(DE3) / pET28a-MbegtD in lane 4, E. coil BL21(DE3) / pET28a-MyegtD in lane 5, and E. coil BL21(DE3) / pET28a-SpEgt1 in lane 6 all have a protein band near their theoretical molecular weight. The protein expression levels are highest in recombinant strains E. coil BL21(DE3) / pET28a-SbegtD, E. coil BL21(DE3) / pET28a-MbegtD, and E. coil BL21(DE3) / pET28a-SpEgt1.

[0243] The crude enzyme solutions obtained were all used to prepare ergothioneine, and the reaction system was the same as in Example 6. After reacting at 25°C for 24 hours, the mixture was immediately placed in a boiling water bath for 10 minutes to inactivate the enzyme and terminate the reaction. The reaction products were detected by liquid chromatography.

[0244] The liquid chromatography detection method is the same as in Example 6. The liquid chromatography detection results are as follows: Figure 3 As shown in the results, only the crude enzyme solutions from the fermentation of recombinant strains E.coil BL21(DE3) / pET28a-eanA, E.coil BL21(DE3) / pET28a-SbegtD, E.coil BL21(DE3) / pET28a-MbegtD, and E.coil BL21(DE3) / pET28a-MyegtD can catalyze the synthesis of ergothioneine. Among them, the crude enzyme solutions from the fermentation of recombinant strains E.coil BL21(DE3) / pET28a-MbegtD, E.coil BL21(DE3) / pET28a-SbegtD, and E.coil BL21(DE3) / pET28a-eanA have the highest catalytic conversion rates, with enzyme activities that are 2.1 times, 1.87 times, and 1.63 times that of the widely used MyegtD, respectively.

[0245] In summary, the L-histidine methyltransferase genes derived from Methylobacterium, Streptomyces buecherae, or Chlorobium limicola described in this application, when ligated into the pET28a expression vector, can express L-histidine methyltransferase at a high level and efficiently catalyze the synthesis of ergothioneine from histidine. This provides the most efficient L-histidine methyltransferase currently available for the in vitro synthesis of ergothioneine, which is of great significance for the industrial production of ergothioneine.

[0246] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Anyone skilled in the art may make some modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the scope of the appended patent application.

[0247] sequence list

[0248] SEQ ID NO.1 MbegtD

[0249] MTIDPRLTAA SPAAPLSENG LFLADVWDGL GASPKVLPAK YFYDAAGSAL FEQITVLPEYYPTRTELGIL DARGPEIAAL LPEGAALVEF GSGSTAKLRR LLRHLPGLSA YLPVDVSGEF LREQAVTLRGDFPHLAVEPV VADFTRPFAL PEGFENRALA GFFPGSTIGN FEPGEAARLL DVFGRILGAG ATLVLGVDLVKDRSVLEAAY DDAAGVTAAF NLNLLHRINR ELDGEIDPDA FAHRAFFNEA ASRIEMHLVS RRAQSVRVAGRGFAFAEGES IHTENSYKYT LDGFRALAAR AGWASVEAWT DRDGLFSVHA LRRQG

[0250] SEQ ID NO.2 SbegtD

[0251] MSPFALTRTL PDDATAAAALR ADVSAGLTAE SKWLPPKWFY DARGSELFEE ITQLPEYYPTRAEREILAAR APAAAATGA RTLVELGSGS SEKTRLLLDA LPDLAAYVPV DVSGSALESA GNALLAERPGLTVHALVADF QQVLALPTTP GPRLVVFLGG TIGNLLPAER AGFLAAVRAL LAPGDALLLG TDLVKEEETLLAAYDDAAGV TAEFNKNVLS VLNRELGADF DPADFTHVAH WDREREWIEM RLRATSALTV KIPALDLAVSFAAGEELRTE VSAKFRRAGV AAELSTAGLD LRHWWTDDAE RFALSLATPV AP

[0252] SEQ ID NO.3 metK

[0253] MAKHLFTSES VSEGHPDKIA DQISDAVLDA ILEQDPKARV ACETYVKTGM VLVGGEITTSAWVDIEEITR NTVREIGYVH SDMGFDANSC AVLSAIGKQS PDINQGVDRA DPLEQGAGDQ GLMFGYATNETDVLMPAPIT YAHRLVQRQA EVRKNGTLPW LRPDAKSQVT FQYDDGKIVG IDAVVLSTQH SEEIDQKSLQEAVMEEIIKP ILPAEWLTSA TKFFINPTGR FVIGGPMGDC GLTGRKIIVD TYGGMARHGG GAFSGKDPSKVDRSAAYAAR YVAKNIVAAG LADRCEIQVS YAIGVAEPTS IMVETFGTEK VPSEQLTLLV REFFDLRPYGLIQMLDLLHP IYKETAAYGH FGREHFPWEK TDKAQLLRDA AGLK

[0254] SEQ ID NO.4 eanB(Δ29)

[0255] MYDHTEITTDSLLALLGSEKVKIIDVRSADAYNGWRMRGEVRGGHIKGAKSLPAKWLTDPEWLNIVRFKQIRPEDAIVLYGYTPEECEQTATRFKENGYNNVSVFHRFHPDWTGNDAFPMDRLEQYNRLVPAEWVNGLISGEEIPEYDNDTFIVCHAHYRNRDAYLSGHIPGATDMDTLALESPETWNRRTPEELKKALEEHGITASTTVVLYGKFMHPDNADEFPGSAAGHIGAIRLAFIMMYAGVEDVRVLNGGYQSWTDAGFAISKDDVPKTTVPEFGAPIPSRPEFAVDIDEAKEMLQSEDSDLVCVRSYPEYIGEVSGYNYIKKKGRIPGAIFAECGSDAYHMENYRNHDHTTREYHEIEDIWAKSGIIPKKHLAFYCGTGWRGSEAWFNALLMGWPRVSVYDGGWFEWSNDPENPYETGVPK

[0256] SEQ ID NO.5 eanA

[0257] MAYSKTNLSE LPLADIDNHL TEIGFDTTIS EIITGLTANA KYIQSKYFYD KRGSALFEKITSLSEYYPSR TEKAIISQLP PALIEDLADI DIIELGCGDH SKISLLIRRI PAESVPGLRY FPIDISQTALKQSIEDLRDL FPALKVKGIL ADYVHQMHLF PEERKRLFCF FGSTIGNLSR EETLDFMQNM GTTMHPGDMLLVGMDRVKNI ALLEKAYNDD QFITAMFNKN ILRVINGLIK SDFNPDDFEH RAFYNADFNR IEMHLEATGNISVKSAFMPE LIRIKKGETI HTENSHKFEK ADILLMGQHA GLAIKNIYSD KNELFSLAHY EKK

[0258] SEQ ID NO.6 MyegtD

[0259] MTLSLANYLA ADSAAEALRR DVRAGLTAAP KSLPPKWFYD AVGSDLFDQI TRLPEYYPTRTEAQILRTRS AEIIAAAGAD TLVELGSGTS EKTRMLLDAM RDAELLRRFI PFDVDAGVLR SAGAAIGAEYPGIEIDAVCG DFEEHLGKIP HVGRRLVVFL GSTIGNLTPA PRAEFLSTLA DTLQPGDSLL LGTDLVKDTGRLVRAYDDAA GVTAAFNRNV LAVVNRELSA DFDLDAFEHV AKWNSDEERI EMWLRARTAQ HVRVAALDLEVDFAAGEEML TEVSCKFRPE NVVAELAEAG LRQTHWWTDP AGDFGLSLAV R

[0260] SEQ ID NO.7 Spegt1

[0261] MTEIENIGAL EVLFSPESIE QSLKRCQLPS TLLYDEKGLR LFDEITNLKE YYLYESELDILKKFSDSIAN QLLSPDLPNT VIELGCGNMR KTKLLLDAFE KKGCDVHFYA LDLNEAELQK GLQELRQTTNYQHVKVSGIC GCFERLLQCL DRFRSEPNSR ISMLYLGASI GNFDRKSAAS FLRSFASRLN IHDNLLISFDHRNKAELVQL AYDDPYRITE KFEKNILASV NAVFGENLFD ENDWEYKSVY DEDLGVHRAY LQAKNEVTVIKGPMFFQFKP SHLILIEESW KNSDQECRQI IEKGDFKLVS KYESTIADYS TYVITKQFPA

[0262] SEQ ID NO.8 AsegtD

[0263] MRQATYEIID FHPSLTSMKE EVSFGLSQTQ KVLPPKLFYD KKGSHLFNEI TKLPEYYLTTAEKQILSRHR QDLALKMGEH CTIIDFGCGN EEKVQLLFGA LSNITAYVPV DISTAALEQT LKEMRISFPDLSIKGVRADY GVSMSFLDHF QNAKRVFTFF GSTLGNFSLK EQEDFLQKAA SHMREGDGFL LGIDLKKDKQVLEAAYNDSE GITESFNKNV LRRMNKELAM NFDLSKFEHV AFYNEEAGRI EMHLVSNTVQ QVSLDNDTFTFRKNETIHTE NSYKFSVDQG VNLLKKAGLT TDMICEDENN QFCMLYAVQ

[0264] SEQ ID NO.9 PsegtD

[0265] MDYSENYFFE CGSGTIVNCL PEIGRQTVVA EILAGLRALP KRISSKFFYD RPGSELFQKITRLDEYYPSR TEKAIFEQLP LEAVTDCRDL DIIELGSGDH SKISLLLKKV PPGRLSGIRY FPVDISRPALESSIRELGRA FPELEVQGIV MADHLYFPGFLVFCH LGSTIGNLER DEANAFVRDI GGTMEPGDAFLTGFDRVKDT AVLERAYNDA GGLTARFNRN VLNVVNGLIE SDFNPLDFEH RAFYNRARRR IEMHLEATRDIRVRTPFARE EILVGKGERI HTENSHKFDD ADIQTIAEKA GLAVRNIFSD QELKKDRVSL

[0266] SEQ ID NO.10 MlegtD

[0267] MIIEDLMPEI GESSIKEKLV ACLKSDPKTL PCMFFYDPAG SELFEKITKL EEYYPPKIEIPLLRSTARKL NSDLKDCNLV ELGSGDCSKI SVFLDEVPEE VRQTIVYPM DVSREALEKS ARILRKYPEMGIHGVNADF RHMEFLKFLKFLKFLTGFF MEFMRHLGDV MNSNDRLLLG VDMVKDITVIERAYNDSRGI TAEFNKNILK VTNNHLGTNF NPDDFEHLAF FNKELSRIEM HLKAKKDLEI TSPLLNESITFKRGETIHTE NSHKYTVDHI SKMADAGGLS LANIYADGKK WFSLAEMVKR

[0268] SEQ ID NO.11 BlegtD

[0269] MKRAMTKTDN SGMIIDLNPS LASFQKDVLT GLSRNPKVIA PKHFYDERGS RLFNTITTLSEYYPTRTEKR ILRDQSAAIA AAIGTSASLV ELGCGSEEKI ELLISAVPMV KSYTPIDISL SAVEDTVSKLKIVCPGLHIY GLCADYTSST DFLQYTASGR RVILFLGSTI GNFEELERHM FLENLRAQLS PDDGILIGIDLVKERAVLEA AYNDTSGVTA EFNLNMLERM NRELGAAFEV EKFRHTAFFN EEKSSIEMHL ESITDQIVEVAGKSFTFMAG ETVHTENSYK FHVEAFEKEA LEAGLSLKKV WKDPQGWFAL TYLEAAGK

[0270] SEQ ID NO.12 eanB

[0271] MQNKNFRAPQSEAIGILYKLIETGSKHKNMYDHTEITTDSLLALLGSEKVKIIDVRSADAYNGWRMRGEVRGGHIKGAKSLPAKWLTDPEWLNIVRFKQIRPEDAIVLYGYTPE ECEQTATRFKENGYNNVSVFHRFHPDWTGNDAFPMDRLEQYNRLVPAEWVNGLISGEEIPEYDNDTFIVCHAHYRNRDAYLSGHIPGATDMDTLALESPETWNRRTPEELKKAL EEHGITASTTVVLYGKFMHPDNADEFPGSAAGHIGAIRLAFIMMYAGVEDVRVLNGGYQSWTDAGFAISKDDVPKTTVPEFGAPIPSRPEFAVVDIDEAKEMLQSEDSDLVCVRS YPEYIGEVSGYNYIKKKGRIPGAIFAECGSDAYHMENYRNHDHTTREYHEIEDIWAKSGIIPKKHLAFYCGTGWRGSEAWFNALLMGWPRVSVYDGGWFEWSNDPENPYETGVPK

[0272] SEQ ID NO.13 MbegtD

[0273] ATGACTATAGATCCCAGGCTAACAGCAGCTTCACCGGCAGCGCCGTTAAGCGAAAACGGTTTGTTCCTGGCGGACGTTTGGGATGGTCTGGGTGCGTCCCCGAAGGTGCTGCCAGCAAAGTACTTCTATGACGCTGCGGGCTCTGCATTGTTCGAGCAAATTACCGTTCTGCCGGAGTATTACCCGACTCGTACCGAACTGGGTATTCTGGACGCGAGAGGCCCTGAGATCGCCGCCCTCCTTCCGGAGGGCGCAGCGTTGGTCGAGTTCGGCTCCGGCTCCACGGCGAAACTGCGCCGTCTGTTGCGCCACCTGCCAGGTCTGTCTGCTTATCTGCCGGTTGATGTTAGCGGTGAGTTTCTGCGCGAACAGGCAGTTACGTTGCGTGGCGATTTTCCGCATCTGGCTGTGGAGCCGGTCGTAGCCGACTTCACCCGTCCGTTTGCGCTGCCGGAGGGCTTCGAAAACCGCGCGCTCGCTGGCTTTTTTCCGGGTAGCACGATTGGTAATTTTGAACCGGGTGAAGCTGCGCGTCTGTTGGACGTGTTTGGCCGCATCCTGGGCGCAGGTGCGACCTTGGTGCTTGGTGTTGACCTGGTCAAAGATCGTAGCGTGCTGGAAGCGGCGTACGACGACGCGGCGGGCGTGACCGCAGCTTTCAACCTGAATTTACTGCATCGTATCAATCGTGAGCTGGACGGGGAGATCGATCCGGATGCGTTCGCCCACCGCGCGTTCTTTAACGAAGCGGCGAGCCGTATCGAAATGCATCTGGTTTCCCGTCGTGCCCAAAGCGTTCGTGTTGCAGGCCGTGGTTTCGCTTTTGCCGAAGGTGAGAGCATTCACACCGAGAACAGCTATAAATACACCCTGGACGGCTTCCGCGCTCTCGCGGCGCGTGCCGGTTGGGCATCGGTGGAAGCGTGGACCGATAGAGATGGTTTGTTTAGCGTGCACGCATTACGCCGTCAGGGT

[0274] SEQ ID NO.14 SbegtD

[0275] ATGTCACCCTTTGCTCTAACTAGGACATTACCAGATGACGCAACGGCGGCGGCTCTAAGAGCGGACGTGTCTGCGGGCCTCACTGCGGAGAGCAAATGGCTGCCGCCGAAATGGTTCTATGATGCGCGTGGTAGTGAACTGTTTGAAGAGATCACCCAGCTGCCGGAATATTACCCGACTCGTGCCGAGCGCGAAATCCTGGCCGCTCGTGCTCCGGCGATTGCAGCTGCGACCGGTGCACGTACCCTGGTGGAACTGGGCTCCGGTTCGAGCGAAAAGACCCGTCTGCTGCTCGACGCGTTGCCAGATTTAGCGGCGTACGTTCCGGTCGATGTGAGCGGTAGCGCGTTAGAAAGCGCGGGCAACGCATTGCTGGCGGAGCGCCCTGGTCTGACGGTTCACGCGTTGGTAGCCGATTTTCAGCAAGTTCTGGCTCTCCCGACGACGCCGGGTCCGCGTCTGGTGGTCTTTCTGGGCGGTACAATTGGTAATCTGCTTCCGGCAGAGCGCGCTGGTTTTCTGGCTGCCGTTCGTGCCCTGTTGGCGCCGGGTGATGCCTTGCTGCTGGGCACCGATCTGGTCAAAGAAGAGGAAACCCTGTTGGCGGCGTACGACGACGCTGCAGGCGTTACCGCGGAGTTCAACAAAAATGTTCTGTCCGTGCTGAACCGTGAGCTTGGCGCAGACTTCGATCCGGCAGACTTCACCCACGTGGCACATTGGGATCGTGAGCGTGAATGGATTGAAATGCGTCTGCGCGCGACCAGCGCCCTGACCGTTAAGATCCCGGCGTTAGACTTGGCGGTGAGCTTTGCAGCGGGTGAGGAGTTGCGCACCGAAGTTTCTGCCAAGTTCCGCCGTGCTGGCGTGGCGGCGGAGTTATCCACCGCAGGGCTGGACCTGCGTCATTGGTGGACCGACGACGCGGAGCGCTTCGCCCTTAGCCTGGCGACGCCGGTTGCTCCG

[0276] SEQ ID NO.15 eanA

[0277]

[0278] SEQ ID NO.16 metK

[0279]

[0280] SEQ ID NO.17 eanB

[0281]

[0282] SEQ ID NO.18 eanB(Δ29)

[0283]

Claims

1. Application of truncated L-histidine betaine sulfonase, L-histidine methyltransferase and S-adenosylmethionine synthase in the production of ergothionein; The truncated L-histidine betaine sulfonase is the L-histidine betaine sulfonase shown in SEQ ID NO. 4; The amino acid sequence of the L-histidine methyltransferase is shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein, The S-adenosylmethionine is shown in SEQ ID NO.

3.

3. The application according to claim 1, wherein, The mass ratio of the S-adenosylmethionine synthase, the L-histidine methyltransferase, and the L-histidine betaine sulfonase is (0.5–1):(0.8–1.2):(0.2–0.6).

4. A combination of enzymes, said combination of enzymes comprising: Truncated L-histidine betaine sulfonase, L-histidine methyltransferase; and S-adenosylmethionine synthase; The truncated L-histidine betaine sulfonase is the L-histidine betaine sulfonase shown in SEQ ID NO. 4; The amino acid sequence of the L-histidine methyltransferase is shown in SEQ ID NO.

1.

5. The combination according to claim 4, wherein, The amino acid sequence of the S-adenosylmethionine synthase is shown in SEQ ID NO.

3.

6. In the combination according to claim 4, the mass ratio of the S-adenosylmethionine synthase, the L-histidine methyltransferase and the L-histidine betaine sulfonase is (0.5-1):(0.8-1.2):(0.2-0.6).

7. A method for producing ergothioneine, comprising using a combination of enzymes according to any one of claims 4-6.

8. A genetically engineered bacterium, said engineered bacterium comprising: The L-histidine betaine sulfonase shown in SEQ ID NO. 4, The L-histidine methyltransferase shown in SEQ ID NO. 1; S-adenosylmethionine synthase as shown in SEQ ID NO.

3.

9. A whole-cell catalyst, wherein, It contains the genetically engineered bacteria as described in claim 8.

10. A method for preparing ergothionein, comprising using the genetically engineered bacteria of claim 8 or the whole-cell catalyst of claim 9.

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