COMPOUND ENZYME AND APPLICATION THEREOF IN PREPARATION OF L-ERGOTHIONEINE

Abstract

A compound enzyme, comprising L-histidine methylase, halogen methyltransferase and trimethylhistidine sulfurylase. L-histidine is catalyzed by L-histidine methylase and halogen methyltransferase to obtain trimethylhistidine, and then is catalyzed by trimethylhistidine sulfurase to obtain the L-ergothioneine. The method can realize conversion from L-histidine to L-ergothioneine only in two steps. Moreover, SAM enzyme is regenerated, such that the usage amount of the SAM enzyme is greatly reduced. Therefore, the method greatly reduces the raw material cost. Moreover, the method is high in reaction concentration (about 30 g/L), simple in process and good in industrial production prospect.

Claims

1. An enzyme composition, comprising L-histidine methyltransferase, halogen methyltransferase and L-hercynine sulfurylase.

2. The enzyme composition according to claim 1, wherein the L-histidine methyltransferase is derived from Mycobacterium smegmatismc or Chlorobium limicola; the halogen methyltransferase is derived from Chloracidobacterium thermophilum or Mycobacterium smegmatis; and the L-hercynine sulfurylase is derived from Escherichia coli or Chlorobium limicola.

3. The enzyme composition according to claim 1, wherein the L-histidine methyltransferase has an amino acid sequence set forth in SEQ ID NO: 1 or 2, the halogen methyltransferase has an amino acid sequence set forth in SEQ ID NO: 3 or 4, and the L-hercynine sulfurylase has an amino acid sequence set forth in SEQ ID NO:5 or 6.

4. (canceled)

5. A method for producing L-ergothionein by enzymatic catalysis, comprising catalyzing L-histidine by L-histidine methyltransferase and halogen methyltransferase to obtain L-hercynine; and catalyzing L-hercynine by L-hercynine sulfurylase to obtain L-ergothioneine.

6. The method according to claim 5, wherein L-hercynine is prepared by adding L-histidine, S-adenosylmethionine, L-histidine methyltransferase and halogen methyltransferase to a buffer solution, adding iodomethane dropwise, and stirring at pH7.0-9.0 for reaction to obtain L-hercynine.

7. The method according to claim 6, wherein the buffer solution is a phosphate buffer solution at 100 mmol/L with a pH of 8.0; the L-histidine has a concentration of 100 mmol/L-200 mmol/L, the S-adenosylmethionine has a concentration of 0.8 mmol/L-1.2 mmol/L, the L-histidine methyltransferase has a concentration of 500 U/L-1000 U/L, and the halogen methyltransferase has a concentration of 800 U/L-1500 U/L; and the iodomethane and S-adenosylmethionine are in a molar ratio of (360-720):1.

8. The method according to claim 5, wherein the catalyzing L-hercynine by L-hercynine sulfurylase to obtain L-ergothioneine comprises adding L-hercynine sulfurylase and potassium polysulfide for reaction in N.sub.2 atmosphere to obtain L-ergothioneine.

9. The method according to claim 8, wherein the L-hercynine sulfurylase has a concentration of 600 U/L-800 U/L, and the potassium polysulfide is added at an amount twice the equivalent of the L-hercynine.

10. The method according to claim 5, wherein the L-histidine methyltransferase is prepared by transforming a host in which S-adenosylhomocysteine nucleoside hydrolase mtnN gene is knocked out with a nucleic acid molecule encoding L-histidine methyltransferase, and performing induction and expression to obtain a bacterial solution containing the L-histidine methyltransferase; the halogen methyltransferase is prepared by transforming a host in which S-adenosylhomocysteine nucleoside hydrolase mtnN gene is knocked out with a nucleic acid molecule encoding halogen methyltransferase, and performing induction and expression to obtain a bacterial solution containing the halogen methyltransferase; and the L-hercynine sulfurylase is prepared by transforming a host in which S-adenosylhomocysteine nucleoside hydrolase mtnN gene is knocked out with a nucleic acid molecule encoding L-hercynine sulfurylase, and performing induction and expression to obtain a bacterial solution containing the L-hercynine sulfurylase.

11. A nucleic acid molecule encoding the L-histidine methyltransferase contained in the enzyme composition according to claim 1, selected from the group consisting of I) a nucleic acid molecule having a nucleotide sequence set forth in SEQ ID NO: 7 or 8; II) a nucleic acid molecule derived from I) by substitution, deletion or addition of one or more nucleotides; III) a nucleic acid molecule having at least 85% homology with I) and encoding L-histidine methyltransferase; and IV) a nucleic acid molecule partially or fully complementary to any one of I) to III).

12. A nucleic acid molecule encoding the halogen methyltransferase contained in the enzyme composition according to claim 1, selected from the group consisting of I) a nucleic acid molecule having a nucleotide sequence set forth in SEQ ID NO: 9 or 10; II) a nucleic acid molecule derived from I) by substitution, deletion or addition of one or more nucleotides; III) a nucleic acid molecule having at least 85% homology with I) and encoding halogen methyltransferase; and IV) a nucleic acid molecule partially or fully complementary to any one of I) to III).

13. A nucleic acid molecule encoding the L-hercynine sulfurylase contained in the enzyme composition according to claim 1, selected from the group consisting of I) a nucleic acid molecule having a nucleotide sequence set forth in SEQ ID NO: 11 or 12; II) a nucleic acid molecule derived from I) by substitution, deletion or addition of one or more nucleotides; III) a nucleic acid molecule having at least 85% homology with I) and encoding L-hercynine sulfurylase; and IV) a nucleic acid molecule partially or fully complementary to any one of I) to III).

14. (canceled)

15. The method according to claim 10, wherein the host is a recombinant host transformed or transfected with a recombinant vector comprising a nucleic acid molecule encoding the L-histidine methyltransferase, halogen methyltransferase or L-hercynine sulfurylase, and in the host, S-adenosyl homocysteine nucleoside hydrolase mtnN gene is knocked out.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0055] In order to more clearly illustrate the embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings needed to be used in the description of the embodiments or the prior art.

[0056] FIG. 1 shows the route of enzymatic production of L-ergothionein in the present disclosure;

[0057] FIG. 2 shows the route of production of L-ergothioneine adopted on the market;

[0058] FIG. 3 shows the SDS-PAGE gel images of L-histidine methyltransferase HisMT, halogen methyltransferase HaMT and L-hercynine sulfurylase HerS in the examples;

[0059] FIG. 4 shows the nuclear-magnetic .sup.1H-NMR data of the finally purified L-ergothioneine in 600M Varian, D.sub.2O solution.

DETAILED DESCRIPTION

[0060] The present disclosure provides an enzyme composition and use thereof in the production of L-ergothioneine. Those skilled in the art can learn from the content herein and appropriately improve the process parameters for realization. In particular, it should be noted that all similar replacements and modifications are apparent to those skilled in the art, which are all considered to be included in the present disclosure. The method and use of the present disclosure have been described through preferred embodiments, and those skilled in the art can apparently make modifications or appropriate changes and combinations to the method and use herein without departing from the content, spirit and scope of the present disclosure to realize and apply the technology of the present disclosure.

[0061] Various L-ergothioneine synthetases exist widely in nature. Organisms generally convert L-histidine into L-hercynine using L-histidine methyltransferase (EgtD, HisMT, EC 2.1.1.44). Subsequently, in most aerobic microorganisms, L-hercynine is continuously converted into L-ergothioneine by L-hercynine oxidase (EgtB, EC 1.14.99.50), glutamate hydrolase (EgtC, EC 3.5.1.118) and sulfur oxygen lyase (EgtE, EC 4.4.1.36).

[0062] The present disclosure obtains a variety of L-histidine methyltransferase HisMT and L-hercynine sulfurylase HerS through screening and selects HisMT and HerS that are overexpressed and highly active in Escherichia coli, thereby effectively realizing the scaled-up production of L-hercynine sulfuration. Moreover, a halogen methyltransferase HaMT is introduced in the production process, and the enzyme can rapidly convert S-adenosylhomocysteine (SAH) into S-adenosylmethionine (SAM) using iodomethane. Therefore, HaMT enzyme and HisMT enzyme are both used to effectively realize the cyclic regeneration of the expensive coenzyme S-adenosylmethionine.

[0063] In order to achieve the above purpose of the present disclosure, the present disclosure provides the following technical solution: regenerating S-adenosylmethionine by utilizing L-histidine methyltransferase HisMT and halogen methyltransferase HaMT to convert the raw material L-histidine into L-hercynine at once; and then converting L-hercynine into L-ergothioneine by using L-hercynine sulfurylase HerS.

[0064] In some specific embodiments of the present disclosure, L-histidine methyltransferase (HisMT, EC 2.1.1.44), halogen methyltransferase (HaMT), and L-hercynine sulfurylase HerS belong to enzyme families of PF10017, PF05724, and PF00581, respectively, which may be derived from strains such as Escherichia coli, Mycobacterium smegmatis, Chlorobium limicola, Chloracidobacterium thermophilum, and Methyllibium sp.

[0065] In the present disclosure, the amino acid sequences of enzymes are derived from bacteria strains, and the nucleic acid sequences encoding the enzymes are codon-optimized. The optimization includes: codon usage preference, elimination of secondary structures (such as hairpin structures) that are not conducive to expression, changes in GC content, CpG dinucleotide content, secondary structure of mRNA, cryptic splice sites, early polyadenylation sites, internal ribosome entry site and ribosome-binding site, negative CpG islands, RNA instability regions, repeats (direct repeats, inverted repeats, etc.) and restriction sites that may affect cloning. In the present disclosure, the nucleic acid encoding the enzyme may be DNA, RNA, cDNA or PNA. In an embodiment of the present disclosure, the nucleic acid is in the form of DNA. Such DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be either a coding strand or a non-coding strand.

[0066] The recombinant vector of the present disclosure comprises one or more regulatory sequences and coding sequences of the target protein. A regulatory sequence may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, an origin of replication, a nucleic acid restriction site, and a homologous recombination site operably linked to a nucleic acid sequence. The vector can also comprise a selective marker, such as a resistance protein marker, an amino acid screening marker or a green fluorescent protein.

[0067] The test materials used in the present disclosure are all commonly commercially available, which all can be purchased from the market, where the primer sequences used and the amino acid sequences and nucleic acid sequences of the enzymes are shown in Tables 1-3:

TABLE-US-00001 TABLE1 Primername Primersequence MsHisMT GTGAAAGGACGCCATATG forwardprimer ACGCTCTCACTG MsHisMT CACTGCTGCTCGAGCATC reverseprimer ACCGCACCGCCAG EcHerSforward CTCAAAACTACCATGGCG primer GGATTTTCGATG EcHerSreverse GACGCTGAAGTCGCTCGA primer GCGTCAGTAATTG MsHisMT: Mycobacterium smegmatis-derived L-histidine methyltransferase ClHisMT: Chlorobium limicola-derived L-histidine methyltransferase CtHaMT: Chloracidobacterium thermophilum-derived halogen methyltransferase MspHaMT: Methylibium sp.-derived halogen methyltransferase ClHerS: Chlorobium limicola-derived L-hercynine sulfurylase EcHerS: Escherichia coli-derived L-hercynine sulfurylase CkHerS: Citrobacterkoseri-derived L-hercynine sulfurylase

TABLE-US-00002 TABLE2 Abbre- Sequence viation number Aminoacidsequence MsHisMT SEQID MTLSLANYLAADSAAEALRRDVRAGLTAAP No.1 KSLPPKWFYDAVGSDLFDQITRLPEYYPTR TEAQILRTRSAEIIAAAGADTLVELGSGTS EKTRMLLDAMRDAELLRRFIPFDVDAGVLR SAGAAIGAEYPGIEIDAVCGDFEEHLGKIP HVGRRLVVFLGSTIGNLTPAPRAEFLSTLA DTLQPGDSLLLGTDLVKDTGRLVRAYDDAA GVTAAFNRNVLAVVNRELSADFDLDAFEHV AKWNSDEERIEMWLRARTAQHVRVAALDLE VDFAAGEEMLTEVSCKFRPENVVAELAEAG LRQTHWWTDPAGDFGLSLAVR C1HisMT SEQID MAYSKTNLSELPLADIDNHLTEIGFDTTIS No.2 EIITGLTANAKYIQSKYFYDKRGSALFEKI TSLSEYYPSRTEKAIISQLPPALIEDLADI DIIELGCGDHSKISLLIRRIPAESVPGLRY FPIDISQTALKQSIEDLRDLFPALKVKGIL ADYVHQMHLFPEERKRLFCFFGSTIGNLSR EETLDFMQNMGTTMHPGDMLLVGMDRVKNI ALLEKAYNDDQFITAMFNKNILRVINGLIK SDFNPDDFEHRAFYNADFNRIEMHLEATGN ISVKSAFMPELIRIKKGETIHTENSHKFEK ADILLMGQHAGLAIKNIYSDKNELFSLAHY EKK CtHaMT SEQID MLGMDADTASFWEEKYRADLTAWDRGGVSP No.3 ALEHWLAEGALKPGRILIPGCGYGHEVLAL ARRGFEVWGLDIALTPVRRLQEKLAQAGLT AHVVEGDVRTWQPEQPFDAVYEQTCLCALS PEDWPRYEAQLCRWLRPGGRLFALWMQTDR PGGPPYHCGLEAMRVLFALERWRWVEPPQR TVPHPTGFFEYAAILERLV MspHaMT SEQID MSGPDLNFWQQRFDAGQLPWDRGAPSPQLA No.4 AWLGDGSLKPGRIAVPGCGSGHEVVALARG GFSVTAIDYAPGAVRLTQGRLAAAGLAAEV VQADVLTWQPTAPLDAVYEQTCLCALHPDH WVAYFARLHAWLRPGGTLALLAMQALRESA GQGLIEGPPYHVDVNALRALLPGDRWDWPR PPYARVPHPSSTWQNWPSC C1HerS SEQID MQNKNFRAPQSEAIGILYKLIETGSKHKNM No.5 YDHTEITTDSLLALLGSEKVKIIDVRSADA YNGWRMRGEVRGGHIKGAKSLPAKWLTDPE WLNIVRFKQIRPEDAIVLYGYTPEECEQTA TRFKENGYNNVSVFHRFHPDWTGNDAFPMD RLEQYNRLVPAEWVNGLISGEEIPEYDNDT FIVCHAHYRNRDAYLSGHIPGATDMDTLAL ESPETWNRRTPEELKKALEEHGITASTTVV LYGKFMHPDNADEFPGSAAGHIGAIRLAFI MMYAGVEDVRVLNGGYQSWTDAGFAISKDD VPKTTVPEFGAPIPSRPEFAVDIDEAKEML QSEDSDLVCVRSYPEYIGEVSGYNYIKKKG RIPGAIFAECGSDAYHMENYRNHDHTTREY HEIEDIWAKSGIIPKKHLAFYCGTGWRGSE AWFNALLMGWPRVSVYDGGWFEWSNDPENP YETGVPK EcHerS SEQID MSGFSMKRVSQMTALAMALGLACASSWAAE No.6 LAKPLTLDQLQQQNGKAIDTRPSAFYNGWP QTLNGPSGHEPAALNLSASWLDKMSTEQLN EWIKQHNLKADAPVALYGNDKDVDAVKTRL QKAGFTHISILSDALSEPSRLQKLPHFEQL VYPQWLHNLQQGKEVTAKPAGDWKVIEAAW GAPKFYLISHIPGADYIDTNEVESEPLWNK VSDEQLKAMLAKHGIRHDTTVILYGRDVYA AARVAQIMLYAGVKDVRLLDGGWQTWSDAG LPVERGTPPKVKAEPDFGVKIPAQPQLMLD MEQARGLLHRQDASLVSIRSWPEFIGTTSG YSYIKPKGEIAGARWGHAGSDSTHMEDFHN PDGTMRSADDITAMWKAWNIKPDQQVSFYC GTGWRASETFMYARAMGWNNVSVYDGGWYE WSSDPKNPVATGERGPDSSK

TABLE-US-00003 TABLE3 Sequence viation number Genesequenceofenzyme MsHisMT SEQID atgacgctctcactggccaactacctggca No.1 gccgactcggccgccgaagcactgcgccgt gacgtccgcgcgggcctcaccgcggcaccg aagagtctgccgcccaagtggttctacgac gccgtcggcagtgatctgttcgaccagatc acccggctccccgagtattaccccacccgc accgaggcgcagatcctgcggacccggtcg gcggagatcatcgcggccgcgggtgccgac accctggtggaactgggcagtggtacgtcg gagaaaacccgcatgctgctcgacgccatg cgcgacgccgagttgctgcgccgcttcatc ccgttcgacgtcgacgcgggcgtgctgcgc tcggccggggcggcaatcggcgcggagtac cccggtatcgagatcgacgcggtatgtggc gatttcgaggaacatctgggcaagatcccg catgtcggacggcggctcgtggtgttcctg gggtcgaccatcggcaacctgacacccgcg ccccgcgcggagttcctcagtactctcgcg gacacgctgcagccgggcgacagcctgctg ctgggcaccgatctggtgaaggacaccggc cggttggtgcgcgcgtacgacgacgcggcc ggcgtcaccgcggcgttcaaccgcaacgtg ctggccgtggtgaaccgcgaactgtccgcc gatttcgacctcgacgcgttcgagcatgtc gcgaagtggaactccgacgaggaacgcatc gagatgtggttgcgtgcccgcaccgcacag catgtccgcgtcgcggcactggacctggag gtcgacttcgccgcgggtgaggagatgctc accgaggtgtcctgcaagttccgtcccgag aacgtcgtcgccgagctggcggaagccggt ctgcggcagacgcattggtggaccgatccg gccggggatttcgggttgtcgctggcggtg cggtga CIHisMT SEQID atggcatactcgaagaccaatctttccgaa No.2 ctgccccttgcggacatagacaaccattta actgagataggcttcgacactactatttca gagattattactgggttgactgctaatgcg aagtatattcagtccaagtacttttatgat aaacggggttctgctttatttgagaaaatt acttcattatcagagtactacccgagtaga acagagaaggctataatttcacaattaccg ccagccttaatagaagatctggctgacatc gacattatcgagttgggctgcggagatcac agcaagatctcactgcttatacgccggata cccgccgaatctgtgcctggtttgcgctat ttcccaattgatatctctcaaacggcttta aaacaatcaatcgaggacttgagagactta tttccagccctgaaggttaaaggcatcctt gcagattatgttcaccagatgcacctgttc ccagaggaacgtaaacgcttgttttgtttc tttggttctacgataggcaatctgtcacgt gaagagacactggattttatgcagaacatg ggcacaactatgcacccaggcgatatgtta ctggtcggtatggatcgtgtaaagaatatc gcattactggagaaggcatataatgacgat caattcataacggcaatgtttaacaaaaat atattgcgcgttataaacggattaataaaa tctgacttcaatcctgacgatttcgagcac cgtgctttttataatgcggactttaacaga atcgaaatgcaccttgaagctaccggtaat atctcggtgaagagcgcattcatgccagaa ctgatacggattaaaaaaggagaaactatc cacacagagaacagtcacaaatttgagaaa gctgatatattgttaatgggacagcatgca gggttagcgatcaagaacatctattccgac aagaacgaactttttagcctggcccattat gagaagaagtga CtHaMT SEQID atgctgggcatggacgctgatactgcgagc No.3 ttctgggaagagaaatatcgcgccgactta accgcatgggatcggggcggtgtatctccc gctctggaacattggttggccgagggtgcg ttgaaacccggtcgcattttgataccgggc tgcgggtatggccacgaagtcttagcttta gctcggcgcggatttgaggtttgggggctt gacatagcactgacacccgttcgtcggctt caagaaaagttggcacaggctggtcttacg gcacacgtagtcgagggcgacgtgcgtact tggcaacccgaacaaccgttcgatgcagta tatgaacaaacatgcttatgcgcccttagc cccgaagattggccacggtacgaagcccaa ttgtgccgttggcttagacctggcggcaga ttattcgcactttggatgcagacagaccgg ccaggcggaccgccataccattgtgggttg gaagcaatgcgtgttttatttgcgcttgaa agatggcgttgggtcgaaccgccccagaga acagtgccacaccctacaggtttcttcgag tatgccgcgatccttgaacggttggtctga MspHaMT SEQID atgagtggacccgacttaaatttctggcag No.4 caacgttttgacaccggccaacttccgtgg gaccgtggcgcaccttcgccacaactggcg gcttggttgggtgatgggtcattagcgcca ggtcgcattgccgtaccggggtgcggttca ggacatgaagttgttgcgttagcccgcggt gggttctcagtaacagccattgattacgcc cccggagcagtgcgcttaacccaaggccgc ttggctgcggcggggttagcggcggaagtc gtgcaggctgacgtacttacgtggcagcca acggctccgcttgacgctgtgtacgagcaa acttgtctgtgtgcattgcatcccgaccat tgggtggcatacgcagctagattacacgct tggcttagaccaggaggtactttggcattg ttggcaatgcaggcactgcgtgagggcgct ggacagggcttgatagaagggcctccttac catgttgacgttaacgctcttcgcgcgctg ctgccgggggaccgttgggactggccacgt ccaccgtatgcgcgcgttcctcacccctct tctacatgggccgagttggcaatagtcctg actcggcgttga ClHerS SEQID atgcagaataagaattttcgtgcaccccag No.5 tcagaggccataggcatcttgtataaactt attgaaactggatcgaagcacaaaaacatg tacgaccacacagagatcacaacagacagt ctgttggcacttttgggttcggaaaaggtg aagataattgacgttcgctcggctgacgcg tataacggttggcggatgcgtggtgaagta cgtggaggccacatcaaaggtgcaaagtcg ttgccagcgaagtggctgacagatccggaa tggcttaacattgttcgtttcaaacagata cgtcccgaggacgccatcgtcctttatggt tatacgcccgaggagtgcgaacaaactgcc actagattcaaagagaatggttataataat gtgagcgtatttcacagattccacccggac tggactgggaacgatgctttcccaatggat agactggagcaatataatagacttgtaccc gccgaatgggttaacggtttgataagcggg gaagagatcccagagtacgacaatgacacc ttcatcgtgtgtcatgcacattatcggaac agagatgcgtatcttagcggccacattccg ggtgctactgacatggatactttggcgtta gaatcgccagagacctggaatcgtcggact ccagaagaattaaagaaagctttagaagag catgggataacggcttcgacgactgtcgtt ctttatgggaaatttatgcatcccgataac gcagatgaatttcctggatccgcagctggc catatcggggcgattcgcttggctttcatt atgatgtacgcgggggttgaggacgttcgg gtcctgaatggcgggtatcaatcatggacc gatgcgggatttgctatttccaaagatgac gtgccaaagacgactgtcccagagttcggc gcacccattccttcccgtccggaattcgct gtagatatcgacgaagccaaggaaatgctt caatcagaggattcagatctggtgtgcgtg cgctcgtacccggagtatatcggcgaggtc agcggctataattacataaaaaaaaagggc agaatcccgggggctatatttgctgagtgc gggtcggatgcctatcacatggaaaactat agaaatcatgaccacaccacgcgcgaatat catgaaatagaagatatatgggcaaaaagc gggatcatcccgaagaaacacttggctttt tattgcggtactggatggcgtggttctgag gcttggttcaacgcgttgctgatgggttgg ccgcgcgtttccgtttatgacggcggttgg tttgagtggagcaacgaccccgaaaaccca tacgagacgggagttccgaagtga EcHerS SEQID atgtcgggattttcgatgaaacgtgtttct No.6 caaatgaccgcgctggcaatggctttaggg ctggcttgcgcttcttcgtgggccgctgaa ctggcgaagcctcttacacttgaccagctt caacaacaaaatggcaaagcgattgatact cgccccagcgcgttttataacggctggcca caaaccttaaatggcccttccggtcatgaa cctgccgccttaaacctctctgctagctgg ctcgacaaaatgagcaccgaacagctcaac gagtggatcaagcaacataacctgaaagcc gatgccccggtggcgctgtacggtaatgac aaagatgtcgacgccgtcaaaacgcgactg caaaaagcaggttttacgcatatctccatc ctgagtgacgcgctaagcgaaccttcccgt ctgcaaaaactgccgcactttgaacagctg gtatatccgcagtggctgcataacctgcaa caaggtaaagaggttacggcgaaacctgcc ggtgactggaaagtcattgaagcggcctgg ggcgctccaaaattttaccttatcagccat attcccggcgctgactacatcgacaccaac gaagtggaaagcgaaccgctgtggaacaaa gtttctgatgaacaactgaaagcgatgctg gcaaaacacggcattcgccatgacaccacg gtcattctgtacgggcgtgacgtatacgct gcagcgcgtgtggcgcaaattatgctttat gccggcgtgaaagatgtgcgcctgctggac ggaggctggcaaacctggtccgacgcgggt ctgcccgttgagcgcggaacgccaccgaaa gtgaaagcggaaccggatttcggcgtgaag atcccggcacaaccgcaactgatgcttgat atggaacaagcgcgtggactgctgcatcgc caggatgcatcgctggtgagcattcgttcg tggccagaatttatcggtacgaccagcggt tacagctatattaaaccaaaaggtgaaata gccggagcacgttggggacacgcaggtagc gactcgacgcatatggaagatttccataac ccggatggcaccatgcgcagcgccgatgat ataaccgctatgtggaaagcatggaatatc aaaccagatcagcaagtttcattctactgc ggcactggctggcgcgcgtcagaaaccttt atgtacgcacgcgcaatgggctggaataac gtctccgtttacgacggtggctggtacgaa tggagcagcgatccaaaaaatccggtagca accggtgaacgcggcccggacagcagtaaa taa CkHerS atgaccgcgctggcactggcttcaggactc gcctgcgcttcctcctgggctgctgatatg gcgcagtcgcttaccttcagccagctacag caaaaacagggcgccgcgattgatacacgc cagagcgctttttacaacggctggccgcaa acccttaatggcccttccggtcacgaacct tccgcactgaatctgtcagcctcctggctc gataaaatgagcgacgagcagctcgccgcc tggctgcaacggcatcagctcaggaaagac gccccgctggcgctgtatggcaatgacaat ggcgtacaggcggtgaaatcacgtttacag aaggcaggcttcagccagattgctacgctg agcgatgcgctgaccgatccttcccgactg caaaagctccctcacttcgagcaattagca tacccacagtggttgcacgcccttcaacaa aaccagccggttacggctaaaccggcggga gactggaaggtgattgaagcaggctggggc gcgccgaaatattatctgctcagccacatt cccggcgccggatacatcgacaccaatgag gtggaaagcgaaccgctgtggaataaagtc tctgacgcgcagttgaaagcgatgctggcg aaacacggtattcgccatgacacgacggtt attctgtatggccgcgacgtatacgctgcg gcgcgcgttgcgcagattatgctctatgcg ggcgtgaaggacgttcgtctgctcgacggc ggctggcaaacctggtccgatgcgggtctg ccagttgaacgcggcatgcccgctgacgtg aagccggaacccgactttggcgtggcgatc ccggcgcagccgcagttaatgctggatatg gaacaggcccgcgccctgctgcaccgtcag gacgcatcgctggtcagcatccgttcctgg ccggaatttatcggcaccaccagcggatac agctatatcaagccaaaaggcgaaattgcc ggcgcgcgttggggacatgcgggcagcgac tccacgcatatggaggatttccataacccg gacggcaccatgcgcagcgcagaggatatc gccgccatgtggaagcagtgggatattctg ccggatcagcacgttgccttctattgcggc accggctggcgcgcctccgaaacgtttatg tacgcgcgggcgatgggctggaaaaacgtc gccgtgtatgacggcggttggtacgaatgg agcagcgatccgaaaaatccggtgtccacc ggcgagcgcgacccggacagcagtaagtag

[0068] The present disclosure is further illustrated below in conjunction with examples:

Example 1 Preparation of MsHisMT Enzyme Solution

[0069] 1. The MsHisMT gene fragment was amplified with chromosomal DNA of Mycobacterium smegmatismc2 155 (ATCC700084) as a template by PCR using the corresponding primers in Table 1, subjected to enzyme digestion using the Nde I/Xho I purchased from NEB Company, and connected to a pET28a plasmid (purchased from Addgene) digested with the same enzyme. Then the plasmid was transformed into E. coli DH5a cells (purchased from Tsingke Biotechnology), and verified by colony PCR and gene sequencing. [0070] 2. The plasmid with verified correct sequence was transformed into E. coli KO1 strains (BL21-DE3 cells in which S-adenosyl homocysteine nucleoside hydrolase mtnN gene was knocked out, wherein the parent strain was obtained from Tsingke Biotechnology, and the gene knockout service was provided by Ubigene Biosciences), which were then cultured in a small scale in 5 ml of LB culture medium containing 50 M Kanamycin at 37 C. When the cells grew to an OD 600 value of 0.5-0.8, 0.5 mM isopropyl--D-thiogalactopyranoside (IPTG) was added to induce protein expression at 37 C. for 3 h. Finally the cells were collected, disrupted by freeze-thaw method, and centrifuged at high speed, and the collected supernatant was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the protein expression. After expression in a small scale, MsHisMT was detected to have an enzyme activity of 90-180 U/mg. [0071] 3. The strains with correct protein expression were cultured step by step in a 5 liter fermenter to be induced for expression under the condition of 1.0 mM IPTG at 37 C. for 4 h, and wet cells were collected to be 30-60 g. Then after the cells and an appropriate amount of Tris.HCl buffer solution (25 mM, pH=8.0) were mixed evenly, the cells were crushed with a high-pressure crusher at low temperature and centrifuged at high speed to remove the cell wall to obtain a MsHisMT enzyme solution, which was stored in a refrigerator at 4 C. for later use.

[0072] In the above steps, the LB culture medium was composed of 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium phosphate, 1% dipotassium phosphate and 5% glycerol.

Example 2 Preparation of ClHisMT Enzyme Solution

[0073] ClHisMT gene was synthesized by Anhui General Biology Co., Ltd. and subcloned into a pET28a plasmid. The cloning method was the same as step 1 in Example 1, and the restriction site was Nde I/Xho I. The verified correct plasmid was transfected into E. coli KO1, cultured in a small scale in 5 ml of LB culture medium to verify the protein expression, and then amplified in a fermenter for high-density fermentation. Methods for the small-scale culture and high-density fermentation were the same as steps 2 and 3 in Example 1. After expression in a small scale, ClHisMT was detected to have an enzyme activity of 30-80 U/mg. The bacterial cells after expression in a large amount were disrupted and centrifuged to remove the cell wall to obtain a ClHisMT enzyme solution.

Example 3 Preparation of CtHaMT Enzyme Solution

[0074] CtHaMT gene was synthesized by Anhui General Biology Co., Ltd. and subcloned into a pET28a plasmid. The cloning method was the same as step 1 in Example 1, and the restriction site was Nde I/Xho I. The verified correct plasmid was transfected into E. coli KO1, cultured in a small scale in 5 ml of LB culture medium to verify the protein expression, and then amplified in a fermenter for high-density fermentation. Methods for the small-scale culture and high-density fermentation were the same as steps 2 and 3 in Example 1. After expression in a small scale, CtHaMT was detected to have an enzyme activity of 20-60 U/mg. The bacterial cells after expression in a large amount were disrupted and centrifuged to remove the cell wall to obtain a CtHaMT enzyme solution.

Example 4 Preparation of MspHaMT Enzyme Solution

[0075] MspHaMT gene was synthesized by Anhui General Biology Co., Ltd. and subcloned into a pET28a plasmid. The cloning method was the same as step 1 in Example 1, and the restriction site was Nde I/Xho I. The verified correct plasmid was transfected into E. coli KO1, cultured in a small scale in 5 ml of LB culture medium to verify the protein expression, and then amplified in a 5-liter fermenter for high-density fermentation. Methods for the small-scale culture and high-density fermentation were the same as steps 2 and 3 in Example 1. After expression in a small scale, MspHaMT was detected to have an enzyme activity of 15-35 U/mg. The bacterial cells after expression in a large amount were disrupted and centrifuged to remove the cell wall to obtain an MspHaMT enzyme solution.

Example 5 Preparation of EcHerS Enzyme Solution

[0076] The EcHerS gene fragment was amplified with chromosomal DNA of Escherichia coli purchased from ATCC (Escherichia coli O6:H1, ATCC 700928) as a template by PCR using the corresponding primers in Table 1, subjected to enzyme digestion using the Nco I/Xho I purchased from NEB Company, and connected to a pET28a plasmid (purchased from Addgene) digested with the same enzyme. Then the plasmid was transformed into E. coli DHSa cells (purchased from Tsingke Biotechnology), and verified by colony PCR and gene sequencing.

[0077] The verified correct plasmid was transfected into E. coli KO1, cultured in a small scale in 5 ml of LB culture medium to verify the protein expression, and then amplified in a 5-liter fermenter for high-density fermentation. Methods for the small-scale culture and high-density fermentation were the same as steps 2 and 3 in Example 1. After expression in a small scale, EcHerS was detected to have an enzyme activity of 15-50 U/mg. The bacterial cells after expression in a large amount were disrupted and centrifuged to remove the cell wall. The resulting EcHerS enzyme solution was vacuumized with a diaphragm vacuum pump (Tianjin Jinteng Experimental Equipment Co., Ltd.) for multiple times and supplemented with N.sub.2 to remove O.sub.2 from the reaction solution, and finally sealed under N.sub.2 protection.

Example 6 Preparation of ClHerS Enzyme Solution

[0078] ClHerS gene fragment was synthesized by Anhui General Biology Co., Ltd. and subcloned into a pET28a plasmid. The cloning method was the same as step 1 in Example 1, and the restriction site was Nde I/Xho I. The verified correct plasmid was transfected into E. coli KO1, cultured in a small scale in 5 ml of LB culture medium to verify the protein expression, and then amplified in a 5-liter fermenter for high-density fermentation. Methods for the small-scale culture and high-density fermentation were the same as steps 2 and 3 in Example 1. After expression in a small scale, ClHerS was detected to have an enzyme activity of 8-25 U/mg. The bacterial cells after expression in a large amount were disrupted and centrifuged to remove the cell wall. The resulting EcHerS enzyme solution was vacuumized with a diaphragm vacuum pump (Tianjin Jinteng Experimental Equipment Co., Ltd.) for multiple times and supplemented with N.sub.2 to remove O.sub.2 from the reaction solution, and finally sealed under N.sub.2 protection.

Example 7: Preparation of L-Ergothioneine (Combination of MsHisMT, CtHaMT and EcHerS)

[0079] 1. Catalyzing L-Histidine to Obtain L-Hercynine Using MsHisMT and CtHaMT:

[0080] 31 g of L-histidine (200 mM), 0.4 g of S-adenosylmethionine (SAM) (1 mM), 1000 U of MsHisMT and 1500 U of CtHaMT were added to 1 L of 100 mM phosphate buffer solution at pH 8.0. The reaction solution was gently stirred at room temperature, and then slowly added dropwise with 102.2 g of iodomethane (720 mmol) within 4 h. After the dropwise addition was completed, the reaction solution was continuously stirred for 4 h. During the reaction, the reaction system was maintained at pH 7.0-9.0 by adding aqueous solutions of HCl and NaOH at low concentrations dropwise. After the reaction was completed, it was detected by HPLC that most of L-histidine had been converted into L-hercynine (Shimadzu LC-20AT HPLC with Phenomenex Luna 5 m SCX 100 , LC Column 1004.6 mm, liquid chromatography conversion rate 94%). The reaction solution was subjected to extraction with 250 ml of ethyl acetate three times to remove iodomethane and iodine followed by ultrafiltration (a molecular cut-off of 1000-5000 molecular weight, Hangzhou Kaijie Membrane Separation Technology Co., Ltd.) to remove enzymes (MsHisMT and CtHaMT) in the reaction system, then vacuumized with a diaphragm vacuum pump and supplemented with N.sub.2 to remove O.sub.2 from the reaction solution, and finally sealed and protected with N.sub.2 for later use. (Activity unit U represents the amount of enzyme required to convert 1 M substrate per minute at 30 C.)

[0081] 2. Converting L-Hercynine to L-Ergothioneine Using EcHerS:

[0082] The above L-hercynine reaction solution was added with 800 U of EcHerS and 12.8 g (twice the equivalent) of potassium polysulfide K.sub.2S.sub.X (Sigma-Aldrich), and gently stirred in N.sub.2 atmosphere at room temperature for 6 h. HPLC detection (same as above) showed that most of L-hercynine raw material was consumed. The reaction solution was poured into 3 L of ethanol solution for precipitation, and then centrifuged to remove EcHerS enzyme and phosphate in the reaction solution. The solution was vacuum concentrated to 400 ml, directly loaded on an anion exchange column (Amberjet4200, OH type, Sigma-Aldrich), and then eluted with pure water. The collected aqueous samples were concentrated to obtain 52 g of light gray crude product of L-ergothioneine, which was further crystallized in ethanol/water solution to obtain 33.1 g of light yellow solid, with a final total yield of 72%.

Example 8 Preparation of L-Ergothioneine (Combination of ClHisMT, CtHaMT, and ClHerS)

[0083] 1. Catalyzing L-Histidine to Obtain L-Hercynine Using ClHisMT and CtHaMT:

[0084] 15.5 g of L-histidine (100 mM), 0.2 g of S-adenosylmethionine (1 mM), 800 U of ClHisMT and 800 U of CtHaMT were added to 1 L of 100 mM phosphate buffer solution at pH 8.0. The reaction solution was gently stirred at room temperature (25 C.), and then slowly added dropwise with 51.1 g of iodomethane (360 mmol). After the dropwise addition was completed, the reaction solution was continuously stirred for 5 h. When it was detected by HPLC that most of L-histidine had been converted (liquid chromatography conversion rate of 81%), the reaction system was subjected to extraction with 250 ml of ethyl acetate to remove iodomethane and iodine in the reaction system followed by ultrafiltration to remove enzymes (ClHisMT and CtHaMT) in the solution, then deoxygenated, sealed with N.sub.2 (the method was the same as above), and stored at 4 C. for later use.

[0085] 2. Converting L-Hercynine to L-Ergothioneine Using ClHerS:

[0086] The crude solution of L-hercynine prepared above was added with 600 U of ClHerS and 6.4 g of potassium polysulfide K.sub.2S.sub.X, and gently stirred in N.sub.2 atmosphere at room temperature for 6 h. HPLC detection (same as above) showed that most of L-hercynine raw material was consumed. The reaction solution was added to 3 L of ethanol solution to terminate the reaction and precipitate ClHerS enzyme and phosphate, and centrifuged to remove the precipitate. The supernatant was vacuum concentrated to 150 ml with a rotary evaporator. The concentrated reaction solution was purified with an anion exchange column, eluted with pure water (same as above), and concentrated to obtain 41.5 g of a crude product of L-ergothioneine, which was then crystallized with ethanol/water to obtain 14.2 g of whitish solid L-ergothioneine, with a final yield of about 61%.

Example 9 Preparation of L-Ergothioneine (Combination of MsHisMT, MspHaMT and ClHerS)

[0087] 1. Catalyzing L-Histidine to Obtain L-Hercynine Using MsHisMT and MspHaMT:

[0088] Similarly, 15.5 g of L-histidine (100 mM), 0.2 g of S-adenosylmethionine (1 mM), 500 U of MsHisMT and 1000 U of MspHaMT were added to 1 L of 100 mM phosphate buffer solution at pH 8.0. The reaction system was slowly added dropwise with 51.1 g of iodomethane (360 mmole) within about 3 h, and then stirred for about 4 h for reaction. HPLC detection showed that most of L-histidine had been consumed (liquid chromatography conversion rate of 90%). The reaction solution was subjected to extraction with 250 ml of ethyl acetate to remove iodomethane and iodine remained after the reaction followed by ultrafiltration to remove enzymes (MsHisMT and MspHaMT) in the solution, then deoxygenated, sealed with N.sub.2 (the method was the same as above), and stored at 4 C. for later use.

[0089] 2. Converting L-Hercynine to L-Ergothioneine Using ClHerS:

[0090] Similarly, the above crude solution of L-hercynine was added with 600 U of ClHerS and 6.4 g of potassium polysulfide K.sub.2S.sub.X, and gently stirred in N.sub.2 atmosphere at room temperature for 6 h. HPLC detection showed that L-hercynine raw material was completely consumed. The reaction solution was poured into 3 L of ethanol solution to terminate the reaction for precipitation, and centrifuged to remove ClHerS enzyme and phosphate. The resulting solution was concentrated to about 150 ml, and then purified with an Amberjet anion exchange column. The eluate was concentrated to obtain 45 g of a crude product of L-ergothioneine, which was then purified by crystallization with ethanol/water system to obtain 17 g of L-ergothioneine as a light yellow solid, with a final yield about 74%.

Comparative Example Preparation of L-ergothioneine (combination of ClHisMT, CtHaMT and CkHerS)

[0091] The enzyme used in the second step reaction in the preparation of L-ergothioneine is important in the whole preparation system of L-ergothioneine. Most of the HerS enzymes verified in the previous experiments had poor activity and stability, for example, CsHerS derived from Clostridium scindens ATCC 35704, CkHerS derived from Citrobacter koseri ATCC BAA-895, MsHerS derived from Mannheimia succiniciproducens, and LgHerS derived from Leptotrichia goodfellowii all had poor activity and stability, resulting in a total yield of only about 10%. The reaction was carried out with CkHerS, which is described in details as follows:

[0092] 1. Preparation of CkHerS Enzyme Solution

[0093] The CkHerS enzyme solution was prepared by the same method as in Example 6. The vector insertion site was Nhe I/Xho I, and the activity of the prepared enzyme was 0.5-7 U/mg.

[0094] 2. Preparation of L-Ergothioneine

[0095] 2.1 Catalyzing L-Histidine to Obtain L-Hercynine Using ClHisMT and CtHaMT:

[0096] 15.5 g of L-histidine (100 mM), 0.2 g of S-adenosylmethionine (1 mM), 800 U of ClHisMT and 800 U of CtHaMT were added in 1 L of 100 mM phosphate buffer solution at pH 8.0. The reaction solution was stirred at room temperature (25 C.) and slowly added dropwise with 51.1 g of iodomethane (360 mmole). After the dropwise addition was completed, the reaction solution was continuously stirred for 5 h. When it was detected by HPLC that most of L-histidine had been converted (liquid chromatography conversion rate of 79%), the reaction system was subjected to extraction with 250 ml of ethyl acetate to remove iodomethane and iodine in the reaction system followed by ultrafiltration to remove enzymes (ClHisMT and CtHaMT) in the solution, then deoxygenated, sealed with N.sub.2 (the method was the same as above), and stored at 4 C. for later use.

[0097] 2.2. Converting L-Hercynine to L-Ergothioneine Using CkHerS:

[0098] The crude solution of L-hercynine prepared above was added with 600 U of CkHerS and 6.4 g of potassium polysulfide K.sub.2S.sub.X, and gently stirred in N.sub.2 atmosphere at room temperature for 10 h. HPLC detection (same as above) showed that most of L-hercynine remained. Then the reaction solution was supplemented with 600 U of CkHerS, stirred at room temperature for 20 h, added with 3 L of ethanol to terminate the reaction and precipitate ClHerS enzyme and phosphate, and centrifuged to remove the precipitate. The supernatant was vacuum concentrated to 150 ml with a rotary evaporator. The concentrated reaction solution was purified with an anion exchange column, eluted with pure water (same as above), and concentrated to obtain 43 g of a grey crude product of the reaction solution, which cannot be purified to obtain L-ergothioneine by the above ethanol/water crystallization condition. The final crude product had a HPLC purity of 5.7% and a total yield of the two-step liquid chromatography of 10.5%.

[0099] The above are only preferred embodiments of the present disclosure, and it should be noted that for those of ordinary skill in the art, several improvements and modifications can also be made without departing from the principle of the present disclosure, and these improvements and modifications should also be considered as the protection scope of the present disclosure.