CN115873886B - Method and carrier for biosynthesis of ergothioneine - Google Patents

Method and carrier for biosynthesis of ergothioneine Download PDF

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CN115873886B
CN115873886B CN202210457708.4A CN202210457708A CN115873886B CN 115873886 B CN115873886 B CN 115873886B CN 202210457708 A CN202210457708 A CN 202210457708A CN 115873886 B CN115873886 B CN 115873886B
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常莹莹
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Wuhan Hesheng Technology Co ltd
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Abstract

The application discloses a method and a carrier for biosynthesis of ergothioneine, wherein the method comprises the steps of encoding glutamylcysteine synthetase and Fe 2+ The nucleic acid construct of at least one of the polynucleotide sequences of the dependent oxidase, transglutaminase, histidine methyltransferase and pyridoxal phosphate dependent carbon-sulfur lyase is introduced into a host cell to obtain a nucleic acid construct capable of expressing a glutamylcysteine synthase, fe 2+ Recombinant bacteria of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyase, and the ergothioneine is biosynthesized using said recombinant bacteria. The method of the application can provide the yield of ergothioneine.

Description

Method and carrier for biosynthesis of ergothioneine
Technical Field
The application belongs to the technical field of ergothioneine biosynthesis, and particularly relates to a method and a carrier for biosynthesis of ergothioneine.
Background
Ergothioneine (EGT) is a sulfur-containing non-proteinogenic amino acid derived from histidine, exists in organs or blood such as animal livers including human beings, is considered as an important factor for maintaining cell redox and bioenergy homeostasis, is also recommended as one of vitamins for prolonging life, and has good application prospect in the fields of foods, medicines, cosmetics and the like as a natural antioxidant, and is a novel fermentation product with high added value.
EGT can be synthesized naturally by mushrooms, cyanobacteria, mycobacteria, red baker's mold, etc., but cannot be synthesized by animals and plants. In fungi, the biosynthesis of EGT requires only two enzymes, EGT1 and EGT2, whereas in mycobacterium smegmatis (Mycobacterium smegmatis), it requires five enzyme-catalyzed reactions, which are encoded by the gene cluster egthabcde. Previous studies indicated that the gene cluster consisting of egtABCDE was present only in actinomycota and that most strains belonging to the subfamily Frankinene and the subfamily Corynebacterium contained EgtE homologous proteins and that strains belonging to the subfamily Micromonospora, the subfamily Propionibacterium, the subfamily Pseudonocardia, the subfamily Streptomyces and the subfascomycetes deleted the egtE gene.
The current commercial methods for producing EGT mainly depend on extraction or chemical synthesis from mushroom except the method of extracting from animal viscera or blood, however, the low content of EGT in mushroom makes the extraction cost high, the safety of chemical synthesis of EGT cannot be ensured, and the same cost problem exists, so researchers turn the eyes to the microbial fermentation direction with low production cost and high safety.
Disclosure of Invention
The application aims to provide a method for biosynthesis of ergothioneine and a carrier required in the biosynthesis process.
The first aspect of the present application provides a nucleic acid construct comprising a nucleic acid sequence encoding a glutamylcysteine synthetase, fe 2+ At least one of the polynucleotide sequences of a dependent oxidase, a transglutaminase, a histidine methyltransferase and a pyridoxal phosphate dependent carbon-sulfur lyase, wherein the glutamylcysteine synthetase, fe 2+ The dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases are from actinoplanes.
In a second aspect, the application provides a method of biosynthesis of ergothioneine comprising employing expression of glutamylcysteine synthetase, fe 2+ Recombinant bacteria of the group consisting of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases, wherein the recombinant bacteria are obtained by introducing a nucleic acid construct provided in the first aspect of the application into a host cell.
In a third aspect, the present application provides a recombinant bacterium for the biosynthesis of ergothioneine, which is capable of expressing glutamylcysteine synthetase, fe 2+ Dependent oxidase, glutamine transferase Histidine methyltransferase and pyridoxal phosphate-dependent carbon-sulfur lyase, wherein said recombinant bacterium is obtained by introducing a nucleic acid construct provided in the first aspect of the application into a host cell. In a fourth aspect, the application provides an enzyme for ergothioneine biosynthesis having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown by Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.4, seq ID No.5 or Seq ID No. 6.
In a fifth aspect, the application provides a polynucleotide molecule comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence set forth in Seq ID No.7, seq ID No.8, seq ID No.9, seq ID No.10, seq ID No.11 or Seq ID No. 12.
According to a sixth aspect of the present application there is provided an Actinoplanes sp HS having a species deposit number of CCTCC No. M2022390, the Actinoplanes being capable of expressing glutamylcysteine synthetase, fe as shown in Seq ID No.1, seq ID No.2, seq ID No.3 and Seq ID No.4, respectively 2+ Dependent oxidases, transglutaminases and histidine methyltransferases, and pyridoxal phosphate dependent carbon-sulfur lyases with amino acid sequences as shown in Seq ID No.5 and Seq ID No. 6.
The seventh aspect of the application provides the use of a nucleic acid construct according to the first aspect of the application, a recombinant bacterium according to the third aspect of the application, an enzyme according to the fourth aspect of the application, a polynucleotide molecule according to the fifth aspect of the application or an actinoplanes according to the sixth aspect of the application in the biosynthesis of ergothioneine.
The application provides a glutamylcysteine synthetase and Fe containing coding actinoplanes 2+ Nucleic acid constructs of polynucleotide sequences of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases, the host is attenuated by introducing the nucleic acid constructs into a host cellCell recombinant expression glutamylcysteine synthetase and Fe 2+ Dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases, thereby achieving efficient synthesis of ergothioneine in the host cell. The method can obviously improve the yield of the biosynthetic ergothioneine.
And (3) strain preservation:
name: actinoplanes HS or Actinoplanes sp.HS
Preservation date: 2022, 4 and 6 days
Preservation unit: china Center for Type Culture Collection (CCTCC)
Address: chinese university of Wuhan
Preservation number: CCTCC No. M2022390
Drawings
FIG. 1A is a graph of accurate molecular weight extracted ion flow chromatograms of EGT standard and EGT product from fermentation broth of actinomycetes HS of example 1 of the present application;
FIG. 1B is a first order precise molecular weight mass spectrum of an EGT standard and an EGT product from an Actinoplanes HS fermentation broth of example 1 of the present application;
FIG. 1C is a secondary fragment mass spectrum of an EGT standard;
FIG. 1D is a mass spectrum of secondary fragments of EGT product in fermentation broth of actinoplanes HS of example 1 of the application;
FIG. 2 shows a schematic structural diagram of plasmids pCIL001A, pCIL001, pCIL003 and pCIL 008;
FIG. 3A is a precise molecular weight extraction ion flow chromatogram of intermediate HER-Cys-Sul in fermentation broths of YC300 strain and YC301 strain of example 2 of the present application;
FIG. 3B is a first order accurate molecular weight mass spectrum of intermediate HER-Cys-Sul in fermentation broths of YC300 strain and YC301 strain of example 2 of the present application;
FIG. 4 is a precise molecular weight extraction ion chromatogram of intermediate HER-Cys-Sul in fermentation broths of strains YC301, YC303 and YC308 of example 2 of the present application;
FIG. 5 is a histogram of ergothioneine production in fermentation broths of strains YC303, YC308 of example 2 of the present application;
FIGS. 6A and 6B are schematic diagrams of the structures of plasmids pCIL013 and pCIL014, respectively;
FIG. 7 is a histogram of ergothioneine production by actinoplanes HS, and recombinant strains YC313, YC314 of the application.
Detailed Description
The terms and descriptions used herein are merely used to describe particular embodiments and are not intended to limit the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.
Definition of the definition
As used herein, the terms "a" and "an" and "the" and similar referents refer to the singular and the plural, unless the context clearly dictates otherwise.
As used herein, the terms "about" and "similar to" refer to an acceptable error range for a particular value as determined by one of ordinary skill in the art, which may depend in part on the manner in which the value is measured or determined, or on the limitations of the measurement system.
The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term "nucleic acid" or "polynucleotide" also includes nucleic acids comprising known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Pat. No.8278036 to Kariko et al, which discloses mRNA molecules with uridine replaced by pseudouridine, methods of synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single Nucleotide Polymorphisms (SNPs) and complementary sequences, as well as the sequence explicitly indicated.
"construct" refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, linear or circular single-or double-stranded DNA or RNA polynucleotide molecule) that can be derived from any source, capable of integration with the genome or autonomous replication, which can be operably linked to one or more polynucleotide molecules. In the present application, constructs typically comprise a polynucleotide molecule of the application operably linked to transcriptional initiation regulatory sequences that direct the transcription of the polynucleotide molecule of the application in a host cell. Heterologous promoters or endogenous promoters may be used to direct expression of the nucleic acids of the application.
"vector" refers to any recombinant nucleic acid construct that can be used for transformation purposes (i.e., introduction of heterologous DNA into a host cell). The vector may comprise a bacterial resistance gene for growth in bacteria and a promoter for expression of the protein of interest in an organism. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., vectors having an origin of replication that is functional in the host cell). Other vectors may be introduced into a host cell and integrated into the host cell's genome and thus replicated together with the host genome. In addition, certain preferred vectors are capable of directing the expression of the foreign genes to which they are linked. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop that can be ligated into an additional DNA segment (foreign gene), and may also include linear double-stranded molecules, such as those obtained from amplification by Polymerase Chain Reaction (PCR) or treatment of circular plasmids with restriction enzymes.
The plasmid vector comprises a vector backbone (i.e., empty vector) and an expression framework.
The term "expression cassette" refers to a sequence having the potential to encode a protein.
The term "host cell" refers to a cell, such as a microorganism, capable of introducing a gene of interest and providing conditions for cloning and/or expression of the gene of interest.
The term "recombinant bacteria" refers to genetically engineered bacteria (e.g., bacteria, yeasts, actinomycetes, etc.) that have foreign gene segments introduced into them, wherein one way of modification involves a change in the genome of the bacteria after introduction of a new DNA segment, and another way involves the introduction of a artificially constructed or modified plasmid into the bacteria, thereby allowing the bacteria to gain the ability to express the gene of interest.
The first aspect of the present application provides a nucleic acid construct comprising a nucleic acid sequence encoding a glutamylcysteine synthetase, fe 2+ At least one of the polynucleotide sequences of a dependent oxidase, a transglutaminase, a histidine methyltransferase and a pyridoxal phosphate dependent carbon-sulfur lyase, wherein the glutamylcysteine synthetase, fe 2+ The dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases are from actinoplanes.
In the application, the glutamylcysteine synthetase and Fe are coded 2+ At least one of the polynucleotide sequences of the dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases is simply referred to as the gene of interest.
The inventors found in the study that glutamylcysteine synthase and Fe can be expressed in actinoplanes 2+ Dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbothiolases, wherein histidine methyltransferases transfer three methyl groups of S-adenosylmethionine (SAM) to L-histidine, catalyze the synthesis of L-histidine betaine (HER), and glutamylcysteine synthetases link glutamate with cysteine to gamma-glutamylcysteine (gamma-Glu-Cys), fe 2+ Dependent oxidases catalyze the formation of gamma-Glu-Cys and inter-HER C-S bonds, hexenyl-gamma-glutamyl cysteine sulfoxide (HER-gamma-Glu-Cys-Sul) is synthesized, after which the L-glutamic acid moiety in HER-gamma-Glu-Cys-Sul is removed by a transglutaminase to produce acetylcysteine sulfoxide (HER-Cys-Sul), which cleaves the C-S bond under the action of pyridoxal phosphate-dependent carbon-sulfur lyase, and the final product ergothioneine is obtained.The nucleic acid construct of the present application comprises a nucleic acid sequence encoding glutamylcysteine synthetase, fe 2+ At least one of polynucleotide sequences of a dependent oxidase, a transglutaminase, a histidine methyltransferase and a pyridoxal phosphate dependent carbon-sulfur lyase, which can be used to introduce polynucleotide sequences encoding these enzymes into a host cell, thereby allowing the host cell to obtain expression of glutamylcysteine synthase, fe 2+ The ability of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyase, in turn, may be used for the biosynthesis of ergothioneine.
In some embodiments, the nucleic acid construct comprises a nucleic acid encoding a glutamylcysteine synthetase, fe 2+ Polynucleotide sequences of dependent oxidases, glutamine transferases, histidine methyltransferases and pyridoxal phosphate dependent carbon sulfur lyases.
In some embodiments, the glutamylcysteine synthetase has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in Seq ID No. 1;
the Fe is 2+ The dependent oxidase has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in Seq ID No. 2;
the transglutaminase has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in Seq ID No. 3;
The histidine methyltransferase has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in Seq ID No. 4;
the pyridoxal phosphate-dependent carbon-sulfur lyase has an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in Seq ID No. 5; or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown in Seq ID No. 6.
In some embodiments, the glutamylcysteine synthetase (bc03_glimmer_04016) has an amino acid sequence as shown below:
MSSADRVLRGAAEAIEHISGICFKTGPPRHLGVELEWTTHHVDDPAVPVPAADLRDALGVHAPAALGNPQPVPLPGGGTVTAEPGGQLEISSAPADALPALHAAVTADHAALAGMLARAGLRLGDRGIDEHREPARILDTPRYAAMERSFDRAGRTMMTGTAGLQVCLDAGEAHQIAGRWAALHDFGPPLLALFANSAVHAGRDTGWASARMAAWYGIDPRRAGPAFRESGSPAEDWARYALDAPLLCVRRDDGRWDAPPGVTFADWITDGTPTVSDLEYHLSTLFPPVRPRGYLEVRYLDTQPGPDWIAPAAVLTALMADDVITAQGREIAAPVAGRWRAAARDGLRDPAVRAAAAGLAELACRHFDRTGLDDVVRKQVSDVVDARLKGNAR*(Seq ID No.1)
the Fe is 2+ The dependent oxidase (bc03_glimmer_04015) has the amino acid sequence shown below:
MINPDKNLIAAELERSRARTALLTDAVDDDDLVAQHSPLMSPLVWDLAHVGNQEELWLVRDVGGREPVRQDIDELYDAFQHARSDRPALPLLDPAEARRYIGQVRDKALDVLDRVRLDERPLLAGGFAFGMIVQHEQQHDETMLATHQLRGGSPVLSAPPPPPGDARVRGEVLIPGGPFTMGTDTDPWALDNERPAHTVEVPAFLIDATPVSNSDYLAFIDAGGYDDPRWWSARGWTHRHEAGLTAPMHWLRDGDAWLYRRFGRISPIAGDEPVVHVDYFEAEAYAAWAGKRLPTEAEWEKAARWDPATGRSRRFPWGDEPPGPEHANLGQRHLAPAPAGAYPAGASPLGVHQLIGDVWEWTSTDWHGYPGFRVFPYAEYSEVFFGGDYKVLRGGSFGTDAAACRGTFRNWDHPIRRQIFSGFRCARELV*(Seq ID No.2)
the glutamine transferase (bc03_glimmer_04014) has the amino acid sequence shown below:
MCRHLAYLGPPEPLSAWVFDPPHALSHQAWAPRDMRGGGTINADGFGVGWYPPEGGPPVRYRSAMPIWSDPTLPRLAEVTRSGAVLAAVRSATEGMPVIATATAPLQDGRWLFSHNGVVRGFPGTLADLAAALPVEDLLTLDAPTDAAALFALVRHGLRAGKTAEEALLSVVTAVLRVAPDSRLNLLLTDGDRILATTAGHALAVRATGDAVLVASEPLDDHPAWRPLPDRRLLIATPAAVELGEL*(Seq ID No.3)
the histidine methyltransferase (bc03_glimmer_04013) has the amino acid sequence shown below:
VTSLEKHLDERDLARSLRADVRDGLSADPKRLPPKWFYDARGSRLFEDITRLPEYYPTRTERAILSAAAAEIARLTDAKTLVELGSGSSEKTRLLLDAMLGRGTLGSFIPFDVSQSALAEAVDALSVTYPGLSITGVVGDFTRHLRHLPDGDSRLVAFLGGTIGNLIPAERSAFLGDLRSVLHAGEWLLLGTDLVKDPAVLVPAYDDAAGVTAEFNRNVLHVINRELRADFEPLAFEHVAAWDPDREWIEMRLRSVRAQTVRIEDLDLTVSYAAGEEMRTEISAKFRRERLAAELAAAGFALRHWWSDPQDWFGVSLAQAVTD*(Seq ID No.4)
the pyridoxal phosphate-dependent carbon-sulfur lyase has the amino acid sequence shown below:
BC03_Glimmer_04046
MDVDALRAGTPGCRHRIHLNNAGAALMSQATLDTVVAHLRLEAEIGGYEAAGAVADRVAAVYAGLAELLGGRADEIALFDNATHAWQAAFHAVPLKAGDRVLTGRNEYGSNVLGYLQAARRVGAEIVVVPNDEHGQIDTVALAGLIDERAKVIGLTHVPTAGGLVNPAAEVGRIARAAGVPYLLDATQSVGQFPVDVTEIGCDFLCGTGRKFLRGPRGTGFLWVRDGILEQLEPHVVEIQSADWDGARGFGWVPGAQRFATWELNYAAVLGLGAAVDQALNLGLGEIGKRNAELGDRMRGLLEDTPGVTVYDLGRERCAIVTAEVAGVGAEQVVARLAESGVNVTSTVPAHQQFDTEDRDPPPLVRFSPHYYNTEDEVEHAATLVSAMVTKPL*(Seq ID No.5)
Or (b)
BC03_Glimmer_04917
MSAEDPPQPIAGARLLFSLDPAVSYLNHGSFGALPITVQRAQQRLRDEMDLNPMRFFGPGLLDRIIHTRRHLAAFLGADPEGSALTSNTTTAVSLVLQSVRLKESDEVLLTDHAYGAVTMAVRRECRRTGATTRTIAVPFGASGPEVLSRVRAALRPGRTRLLIIDQVTSATATLMPVREVVAAARAQGIPVMVDGAHVPGMLPVRVEEIGADFWVGNLHKWGWAPRGTSLLAVSPDWRRRIDPLVVSWEQDQGFPLSVEFQGTIDYTPWLAAPAGIFAMRTLGPEVVREHNAALAAYGQRVVGAALGHAPADLPEPGGPGVSMRIVPLPAGVATTFPEAHALRGHIADKLGVETQINAWGGRGLLRLSAQIYNRPEEYHHLADRLPSLLHHWQW*(Seq ID No.6)。
In some embodiments, the two above carbon sulfur lyase enzymes may be introduced into the host cell alone, involved in the biosynthesis of ergothioneine, or may be introduced into the host cell together, involved in the biosynthesis of ergothioneine.
In some embodiments, the polynucleotide sequence encoding a glutamylcysteine synthetase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 7;
the code Fe 2+ The polynucleotide sequence of the dependent oxidase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence set forth in Seq ID No. 8;
the polynucleotide sequence encoding a transglutaminase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 9;
the polynucleotide sequence encoding a histidine methyltransferase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 10;
The polynucleotide sequence encoding pyridoxal phosphate-dependent carbon-sulfur lyase has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence set forth in Seq ID No.11 or Seq ID No. 11.
In some embodiments, the nucleotide sequence encoding a glutamylcysteine synthetase comprises:
BC03_Glimmer_04016
5’-atgagttcggccgatcgagtgctgcggggagccgcggaagcgatcgaacacatctccggcatctgcttcaagaccggcccgccacgccatctcggcgtcgagctggaatggaccacacaccacgtggatgacccggccgtcccggtcccggccgcggacctgcgtgacgcgttgggggtgcacgcgccggccgccctcggcaatccgcaaccggtgccgctgcccggcggcggcacggtcaccgccgaacccggcggtcagttggagatctcgtcggcgccggccgatgcgctgcccgccctgcacgcggcggtcaccgccgaccacgccgccctggccgggatgctcgcccgggccggcctgcgcctcggcgaccggggcatcgacgagcaccgggaacccgcccggatcctggacacgccgcggtacgccgcgatggaacgctccttcgaccgcgctgggcgcaccatgatgaccggcaccgcgggactgcaggtctgcctcgacgccggggaggcccaccagatcgccggccggtgggcggcgctgcacgacttcgggccgccgctgctggcgctgttcgcgaattcggcggtgcacgccgggcgggacaccggctgggcgtcggcccggatggccgcctggtacggcatcgacccccggcgcgccggcccggccttccgggagtccggctccccggccgaggactgggcccggtacgcgctcgacgcgccactgctctgcgtgcggcgcgacgacgggcggtgggacgcgccgcccggggtgaccttcgccgactggatcaccgacggcacaccgaccgtgtccgatctggagtatcacctcagcaccctgttcccgccggtgcgcccgcgcggctacctggaggtgcgctacctcgacacccagccggggccggactggatcgccccggccgcggtgctgaccgcgctgatggccgacgacgtgatcaccgctcagggccgggagatcgcggcgccggtcgcggggcggtggcgggccgcggcacgggacgggctgcgggatccggcggtccgcgcggcggcggccggactggccgagctcgcctgccggcacttcgaccggacggggctggatgacgtcgtccgaaagcaggtttccgatgttgttgacgcgcgcctgaaggggaacgctcgatga-3’(Seq ID No.7)
encoding Fe 2+ The nucleotide sequences of the dependent oxidases include:
BC03_Glimmer_04015
5’-atgatcaaccccgacaagaacctgatcgccgccgaattggagcgatcgcgggcccgcaccgccctgctgaccgacgcggtcgacgacgacgacctcgtcgcgcagcactcgccgctgatgtcgccgctggtctgggacctcgcgcacgtcggcaaccaggaggagctctggctggtccgggacgtcggcggccgcgagccggtgcgtcaggacatcgacgagttgtacgacgcgttccagcacgcccgcagcgaccgccccgccctgccgctgctcgacccggccgaggcgcgccgatacatcggccaggtgcgggacaaggcgctcgacgtgctcgaccgggtgcgcctggacgagcggccgctgctcgccggtggcttcgcgttcggcatgatcgtgcaacacgagcagcagcacgacgagaccatgctcgccacccaccagctgcgcggcggctcgccggtgctctccgccccgccgcccccgcccggggacgcccgggtgcgcggcgaggtgctgatccccggcggcccgttcaccatgggcaccgacaccgacccgtgggcgctggacaacgagcggccggcgcacaccgtcgaggtcccggcgttcctgatcgacgccaccccggtgtccaacagtgactacctcgccttcatcgacgccggcggctacgacgacccgcgctggtggtcggcgcggggctggacgcaccggcacgaggccgggctgaccgcgccgatgcactggctgcgcgacggtgacgcctggctctaccggcggttcggccggatcagcccgatcgccggcgacgagccggtggtgcacgtcgactacttcgaggccgaggcgtacgcggcctgggccgggaaacggctgcccaccgaggccgagtgggagaaggccgcccgctgggacccggcgaccggccgctcccgccgcttcccgtggggcgacgagccgcccggcccggaacacgccaacctcgggcagcggcatctcgcgccggcgccggccggggcgtatccggccggcgcgtcgccgctgggcgtgcaccagctgatcggcgatgtctgggaatggacatcgaccgactggcacggctatcccggattccgggtgttcccgtacgccgagtactccgaggtcttcttcggcggtgactacaaggtgctgcgcggcggctcgttcggcaccgacgcggccgcctgccgcggcacgttccgcaactgggaccacccgatccggcggcagatcttcagcggcttccggtgcgcgcgggagctcgtctga-3’(Seq ID No.8)
the nucleotide sequence encoding the transglutaminase comprises:
BC03_Glimmer_04014
5’-atgtgccgccacctggcctacctcgggccgcccgagccactgtcggcgtgggtgttcgacccgccgcacgcgctgtcgcaccaggcctgggcaccacgtgacatgcgcggcgggggcaccatcaacgccgacgggttcggcgtggggtggtatccgcccgagggcgggccgccggtccggtatcgcagcgcgatgccgatctggagcgatccgacactgccccggctcgcggaggtgaccaggtcgggggcggtgctcgcggcggtccggtcggcgaccgaggggatgccggtgatcgcgacggcgacggcgccgctccaggacgggcggtggcttttcagccacaacggggtggtccgcggtttccccgggaccctcgccgacctggccgccgcgcttcccgtcgaggacctgctgaccctcgacgcgccgaccgatgcggccgccctgttcgctctggtacgccatgggctgcgcgccgggaagaccgcggaggaggcactcctgtccgtggtcaccgccgtgctgcgggtcgcgccggactcccggctgaacctgctgctgaccgatggtgatcggatcctggcgaccactgccgggcacgcgctcgccgtccgggcgaccggcgacgccgtgctggtcgcctccgagcccctcgacgaccaccccgcctggcggccgctcccggaccgccggctgttgatcgcgacccccgctgcggtggaattaggagagctgtga-3’(Seq ID No.9)
the nucleotide sequence encoding histidine methyltransferase includes:
BC03_Glimmer_04013
5’-gtgacttcgctggagaagcatctcgacgagcgtgacctggcccgatccctgcgcgccgacgtgcgcgacggcctgagcgccgacccgaaacggctgccgccgaaatggttctacgacgcccgcggcagccggctcttcgaggacatcacccggcttccggagtactacccgacccgcaccgaacgcgccatcctgagcgcggccgccgcggagatcgcccggctcaccgacgcgaaaaccctcgtcgaactcggctccggttcatcggagaagacccggctgctgctcgacgcgatgctggggcggggcacgctcggctccttcatcccgttcgacgtgtcccagagtgccctcgctgaggccgtcgacgcgctcagcgtcacctaccccggcctgagcatcaccggcgtggtcggcgacttcacccggcacctgcgccacctgcccgacggcgacagccgcctggtcgccttcctcggcggcacgatcggcaacctgatcccggccgagcgctccgccttcctcggcgacctgcgctccgtgctgcacgcgggtgagtggctgctgctcggcaccgatctggtcaaggaccctgccgtgctggtgcccgcctatgacgacgcggccggcgtcaccgcggagttcaaccggaacgtgctgcatgtcatcaaccgcgagctgcgggccgactttgagccactcgccttcgagcatgtggccgcctgggacccggatcgggaatggatcgagatgcggttgcggtcggtgcgggcgcagacggtgcggatcgaggatctggacctgaccgtctcgtatgcggcgggggaggagatgcggaccgagatctcggcgaagttccggcgggagcggcttgcggcggaactggcggcggcgggcttcgccctccgccattggtggtccgacccgcaggactggttcggcgtctccctggcccaggccgtcactgattga-3’(Seq ID No.10)
the nucleotide sequence encoding pyridoxal phosphate-dependent carbon-sulfur lyase includes:
BC03_Glimmer_04046
5’-atggacgtcgacgcactccgggccggcactccgggctgccgccaccggattcatctgaacaacgcgggcgccgcgctgatgtcgcaggcgacgctggacaccgtggtcgcacacctgcggctggaggccgagatcggcgggtacgaggcggccggcgcggtcgccgaccgggtcgccgcggtctacgccggtctcgccgagctgctcggcgggcgggccgacgagatcgcgctgttcgacaatgcgacgcacgcctggcaggcggcgttccacgcggtgccgctgaaggccggcgaccgggtgctgaccgggcgcaacgagtacggcagcaacgtgctgggctacctgcaggcggcccggcgggtcggcgccgagatcgtcgtggtgccgaacgacgagcacgggcagatcgacacggtcgcgctcgccgggctgatcgacgagcgggcgaaagtgatcggcctgacccacgtgccgacggccggtggtctggtcaatccggccgccgaggtggggcggatcgcacgggccgcgggggtgccctatctgctggacgcgacgcagtcggtgggccagttcccggtcgacgtcaccgagatcggatgcgacttcctgtgcggtacggggcgcaaattcctgcgcgggccgcgcggcaccgggttcctctgggtccgcgacggcatccttgagcagctcgaaccgcacgtggtggagatccagtcggcggactgggacggcgcgcggggcttcggctgggtgcccggggcgcagcggttcgccacctgggagctgaactacgcggcggtgctcggtctgggcgcggcggtcgaccaggcgctcaatctggggctcggcgagatcggcaagcgcaacgcggagctcggcgaccggatgcgcgggctgctggaggacactcccggcgtgaccgtttatgacctcggtcgcgagcggtgcgcgatcgtcaccgcggaggtggccggggtgggcgccgagcaggtggtggcgcggctcgccgagtccggggtgaacgtgacctcgaccgtgccggcgcaccagcagttcgacaccgaggaccgcgatccgccgccgctggtccgcttctcgccgcactactacaacaccgaggacgaggtcgagcacgcggcgaccctggtcagcgccatggtcaccaagccgttgtga-3’(Seq ID No.11)
or (b)
BC03_Glimmer_04917
5’-atgagcgccgaagatccaccgcagcccattgccggggcgcggttgctgttctcgctggatccggcggtgtcctatctcaaccacgggtcgttcggcgcgttgccgatcaccgtgcagcgcgcccagcagcgcctgcgggacgagatggatctgaacccgatgcgattcttcggcccgggcctgctggaccggatcatccacacccgacggcatctggccgctttcctcggcgccgacccggagggcagcgcgctcacctcgaacaccaccaccgcggtcagcctggtgctgcagtcggtccggttgaaggaatcggacgaggtgctgctcaccgaccacgcctacggtgcggtgacgatggcggtgcgccgggagtgccggcggaccggggcgacgacccggacgatcgcggtgccgttcggcgcgagcgggccggaggtcctgtcccgggtgcgggcggcgctgcggccgggacgcacccggctgctgatcatcgaccaggtgacctcggcgacggccacgctgatgccggtgcgggaggtcgtcgccgcggcccgggcgcagggcatcccggtgatggtcgacggcgcgcacgtgccgggcatgctgccggtgcgggtggaggagatcggcgccgatttctgggtggggaatctgcacaagtggggttgggcgccgcgcggcacgtcgctgctggcggtgtcgccggactggcggcggcggatcgatccgctggtcgtctcctgggagcaggatcagggtttcccgctgtcggtcgagttccaggggacgatcgactacaccccgtggctggcggcgccggccgggatcttcgcgatgcgcacgctgggcccggaagtcgtccgggagcacaatgcggcgctcgcggcgtacggccagcgggtggtcggtgcggccctcggtcatgcgccggcggatctgccggagccgggcggacccggggtctcgatgcggatcgtgccgctgccggccggggtggccaccacctttcccgaggcgcacgcgttgcgcgggcacatcgccgacaagctcggggtggagacgcagatcaacgcgtggggcggccgggggctgctgcggctgagcgcgcagatctacaaccggccggaggagtatcaccatctggccgatcgcctacccagcctgctgcaccactggcagtggtag-3’(Seq ID No.12)。
In some embodiments, the nucleic acid construct is a plasmid vector encoding glutamylcysteine synthetase, fe 2+ Polynucleotides of dependent oxidases, glutamine transferases, histidine methyltransferases and pyridoxal phosphate dependent carbon sulphur lyase are located in the expression frame of the plasmid vector.
In some embodiments, the plasmid vector further includes a promoter for regulating the expression of the target gene, and the type, number and position of the promoter are not limited, so long as the purpose of the present application can be achieved, for example, one promoter may be disposed upstream of each target gene fragment, or one promoter may be shared by a plurality of target genes. Promoters commonly used in the art may be used, such as the ermE promoter, the kasOp promoter, the hrdB promoter, and the like.
In some embodiments, the plasmid vector is a prokaryotic expression vector. The starting plasmid of the plasmid vector can be specifically selected by those skilled in the art according to the need, for example, the kind of host cell, cleavage site, etc., and the present application is not limited thereto.
In some embodiments, the plasmid vector is an integrative plasmid capable of integrating the gene of interest of the present application into the genome of the host cell, thereby enabling the obtained recombinant bacterium to stably express the gene of interest.
In some embodiments, the starting plasmid of the plasmid vector is a pSET152 plasmid. The pSET152 plasmid is taken as a starting plasmid, and attP sites in the plasmid can be utilized to enable a target gene carried by the plasmid to be rapidly integrated into a host cell genome, so that the rapid acquisition of a recombinant strain is facilitated.
In some embodiments, the plasmid vector is structured as shown in fig. 6A.
In some embodiments, the structure of the plasmid vector is as shown in fig. 6B.
The inventors found that the plasmid vector shown in FIGS. 6A and 6B can be highly expressed in actinoplanes HS, thereby obtaining recombinant actinoplanes with high expression of ergothioneine.
In a second aspect, the application provides a method of biosynthesis of ergothioneine comprising employing expression of glutamylcysteine synthetase, fe 2+ Recombinant bacteria of the group consisting of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases, wherein the recombinant bacteria are obtained by introducing a nucleic acid construct provided in the first aspect of the application into a host cell.
The application is prepared by the steps of encoding glutamyl cysteine synthetase and Fe 2+ The nucleic acid construct of at least one of the polynucleotide sequences of the dependent oxidase, transglutaminase, histidine methyltransferase and pyridoxal phosphate dependent carbon-sulfur lyase is introduced into a host cell to obtain a nucleic acid construct capable of expressing a glutamylcysteine synthase, fe 2+ Recombinant bacteria of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyase, and the ergothioneine is biosynthesized using said recombinant bacteria.
The nucleic acid construct of the present application may comprise a nucleic acid encoding a glutamyl cysteine synthetase, fe 2+ One or more or all of the polynucleotide sequences of dependent oxidase, glutamine transferase, histidine methyltransferase and pyridoxal phosphate dependent carbon-sulfur lyase, when the nucleic acid construct comprises a nucleic acid sequence encoding a glutamylcysteine synthetase, fe 2+ Dependent oxidase, cerealWhen one or more of the polynucleotide sequences of the amino acid amide transferase, histidine methyltransferase and pyridoxal phosphate dependent carbon-sulfur lyase are present, a nucleic acid construct comprising polynucleotide sequences encoding different enzymes may be introduced into the host cell, thereby enabling simultaneous expression of glutamylcysteine synthase, fe in the host cell 2+ Dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases.
The application is prepared by the steps of encoding glutamyl cysteine synthetase and Fe 2+ Nucleic acid constructs of polynucleotide sequences of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases are introduced into host cells by well known methods to obtain recombinant bacteria. The method for introducing the nucleic acid construct into the host cell may be specifically selected by those skilled in the art according to the need, for example, the kind of host cell, the kind of nucleic acid construct, and the like, and the present application is not limited thereto. Illustratively, a calcium transfer method may be employed for E.coli, a lithium acetate transfer method may be employed for yeast, a conjugative transfer method, a protoplast transfer method, etc. may be employed for actinoplanes.
In some embodiments, the polypeptide encodes glutamylcysteine synthetase, fe 2+ The polynucleotide sequences of the dependent oxidases, glutamine transferases, histidine methyltransferases and pyridoxal phosphate dependent carbon sulfur lyases are integrated into the genome of the host cell. The target gene is integrated into the genome of the host cell, which is beneficial to the stable expression of the target gene by the recombinant bacterium.
The inventors found in the study that it encodes glutamylcysteine synthetase, fe 2+ The recombinant bacterium of the present application can be obtained by integrating polynucleotide sequences of a dependent oxidase, a transglutaminase, a histidine methyltransferase and a pyridoxal phosphate dependent carbon-sulfur lyase, each independently, into the genome of the host cell at a site which does not interfere with the normal physiological metabolism of the host cell, and, illustratively, the gene of interest of the present application can be obtained by the attP site in a nucleic acid constructThe attB site of actinoplanes was inserted, thereby obtaining the recombinant bacterium of the application.
In some embodiments, the host cell is selected from at least one of escherichia coli, saccharomyces cerevisiae, or actinoplanes.
In some embodiments, the actinoplanes is actinoplanes HS, which has a accession number of CCTCC No. M2022390. The inventors found that actinoplanes HS itself is capable of endogenous synthesis of ergothioneine and therefore has a stronger product tolerance than other strains; when the actinoplanes HS is used as a host cell, the target gene of the application under the control of the promoter is introduced, so that the copy number of the target gene is increased, the expression quantity of a gene product (enzyme) is increased, and the recombinant bacteria with higher ergothioneine yield can be obtained.
In a third aspect, the present application provides a recombinant bacterium for the biosynthesis of ergothioneine, which is capable of expressing glutamylcysteine synthetase, fe 2+ A dependent oxidase, a transglutaminase, a histidine methyltransferase and a pyridoxal phosphate dependent carbon-sulfur lyase, wherein the recombinant bacterium is obtained by introducing the nucleic acid construct provided in the first aspect of the application into a host cell. In some embodiments, the polypeptide encodes glutamylcysteine synthetase, fe 2+ The polynucleotide sequences of the dependent oxidases, glutamine transferases, histidine methyltransferases and pyridoxal phosphate dependent carbon sulfur lyases are integrated into the genome of the host cell.
In some embodiments, the host cell is selected from at least one of escherichia coli, saccharomyces cerevisiae, or actinoplanes.
In some embodiments, the host cell is actinoplanes HS, which is deposited under the accession number CCTCC No. M2022390.
In some embodiments, the recombinant bacterium is obtained by introducing the nucleic acid construct into the actinoplanes HS, wherein the nucleic acid construct is a plasmid vector, the starting plasmid of the plasmid vector is a pSET152 plasmid, and the structure of the plasmid vector is shown in fig. 6A or fig. 6B.
In a fourth aspect, the application provides an enzyme for ergothioneine biosynthesis having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence shown by Seq ID No.1, seq ID No.2, seq ID No.3, seq ID No.4, seq ID No.5 or Seq ID No. 6.
In a fifth aspect, the application provides a polynucleotide molecule encoding an enzyme provided in the fourth aspect of the application, comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence set forth in Seq ID No.7, seq ID No.8, seq ID No.9, seq ID No.10, seq ID No.11 or Seq ID No. 12.
In a sixth aspect, the present application provides an Actinoplanes (Actinoplannes sp.) HS having a seed deposit number of CCTCC No. M2022390, which actinomycetes are capable of expressing glutamylcysteine synthetase, fe as shown in Seq ID No.1, seq ID No.2, seq ID No.3, and Seq ID No.4, respectively 2+ Dependent oxidases, transglutaminases and histidine methyltransferases, and pyridoxal phosphate dependent carbon-sulfur lyases with amino acid sequences as shown in Seq ID No.5 and Seq ID No. 6. The actinoplanes HS of the application can synthesize ergothioneine naturally and efficiently.
The seventh aspect of the application provides the use of a nucleic acid construct according to the first aspect of the application, a recombinant bacterium according to the third aspect of the application, an enzyme according to the fourth aspect of the application, a polynucleotide molecule according to the fifth aspect of the application or an actinoplanes according to the sixth aspect of the application in the biosynthesis of ergothioneine.
The enzyme composition of the present application and its use are described below by way of specific examples. The following examples are only illustrative of the present application and should not be construed as limiting the scope of the application. The plasmids referred to in the examples below are all known to those skilled in the art. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: synthesis of ergothioneine by actinoplanes HS
Step 1: actinoplanes HS fermentation
The strain with high yield of ergothioneine is obtained by screening in a laboratory, and the 16s rRNA sequence is 5'-agagtttgatcctggctcaggacgaacgctggcggcgtgcttaacacatgcaagtcgagcggaaaggcccttcggggtactcgagcggcgaacgggtgagtaacacgtgagtaacctgccccagactttgggataaccctcggaaacgggggctaataccgaatatgacctagcttcgcatggagcgtggtggaaagtttttcggtttgggatggactcgcggcctatcagcttgttggtggggtaatggcctaccaaggcgacgacgggtagccggcctgagagggcgaccggccacactgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatgggcggaagcctgatgcagcgacgccgcgtgagggatgacggccttcgggttgtaaacctctttcagcagggacgaagcgcaagtgacggtacctgcagaagaagcgccggccaactacgtgccagcagccgcggtaagacgtagggcgcgagcgttgtccggatttattgggcgtaaagagctcgtaggcggcttgtcgcgtcgaatgtgaaatcccgaggctcaacttcgggcttgcattcgatacgggcaggctagagttcggtaggggagactggaattcctggtgtagcggtgaaatgcgcagatatcaggaggaacaccggtggcgaaggcgggtctctgggccgatactgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacgctgtaaacgttgggcgctaggtgtggggggcctctccggtttcctgcgccgcagctaacgcattaagcgccccgcctggggagtacggccgcaaggctaaaactcaaaggaattgacgggggcccgcacaagcggcggagcatgcggattaattcgatgcaacgcgaagaaccttacctgggtttgacatcgccggaaatctcgcagagatgcggggtccttcggggccggtgacaggtggtgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaaccctcgttcgatgttgccagcgcgtaatggcggggactcatcggagactgccggggtcaactcggaggaaggtggggatgacgtcaagtcatcatgccccttatgtccagggcttcacgcatgctacaatggccggtacaaagggctgcgataccgtaaggtggagcgaatcccaaaaagccggtctcagttcggatcggggtctgcaactcgaccccgtgaagtcggagtcgctagtaatcgcagatcagcaacgctgcggtgaatacgttcccgggccttgtacacaccgcccgtcacgtcacgaaagtcggcaacacccgaagccggtggcctaacccgtaagggagggagccgtcgaaggtggggctggcgattgggacgaagtcgtaacaaggtagccgtaccggaaggtgcggctggatcacct-3' (Seq ID No. 38) which belongs to actinoplanes, so the strain is named actinoplanes HS (preservation number CCTCC No. M2022390).
Taking glycerinum of the actinoplanes HS,separating single colony in SFM solid plate culture medium (2% soybean cake powder, 2% mannitol, 1.6% agar, pH-7.2), culturing at 28deg.C for 5-7 days, picking single colony after single colony grows out, plating on SFM solid plate culture medium, and culturing at 28deg.C for 7 days. Scraping thallus (1.5 cm×1.5 cm) from SFM solid plate culture medium grown for 7 days, inoculating 25mL seed culture medium (1% glucose, 4% soybean cake powder, 1% glycerol, 1% soluble starch, 0.2% CaCO) 3 pH 7.0), shaking culturing at 28deg.C at 250rpm for 48 hr, collecting seed solution for 48 hr, adding 2.5mL into 25mL fermentation medium (3% maltose, 3% glucose, 1% soybean cake powder, 0.3% yeast powder, 0.1% K) 2 HPO 4 ·3H 2 O，0.15％FeCl 3 ，0.25％CaCl 2 ，0.25％CaCO 3 pH 6.5), at 28℃and 250rpm, for 7 days to obtain a fermentation broth, about 40g of dry weight of cells per 1L of fermentation broth.
Step 2: identification of ergothioneine product in fermentation broth
Taking 5mL of fermentation liquor after fermenting for 7 days, adding 5mL of pure acetonitrile, uniformly mixing, performing ultrasonic crushing at 600W for 30min, centrifuging at 4000rpm for 15min, diluting the obtained supernatant 10 times, filtering by using a 0.22 micrometer filter membrane, and detecting by using a high performance liquid chromatography-high resolution mass spectrometer (HPLC-HRMS).
The HPLC-HRMS detection method comprises the following steps: the column was Thermo Scientific Hypersil GOLD aQ (150X 2.1mm,3 μm); mobile phase: aqueous solution of 0.1% formic acid for phase a and 0.1% formic acid for phase B in acetonitrile, elution gradient: 0-1 min, phase A: phase B = 99:1; 1-10 min, 99% -10% of phase A and 1% -90% of phase B; 10-15 min, phase A: phase B = 10:90; 15-15.5 min, 10-99% of phase A and 90-1% of phase B; 15.5 to 20min, phase A: phase B = 99:1; the flow rate is 0.2mL/min; the sample volume was 2. Mu.L.
The accurate molecular weight extracted ion flow chromatograms of EGT standard (aladine, L134175-10 mg) and Actinoplanes HS fermentation broth (actionopyrrole sp.hs in the figure) are shown in fig. 1A, the first-order accurate molecular weight mass chromatogram of EGT standard and Actinoplanes HS fermentation broth (actionopyrrole sp.hs in the figure) is shown in fig. 1B, and the second-order fragment mass chromatogram of EGT standard and the second-order fragment mass chromatogram of EGT of Actinoplanes HS fermentation broth (actionopyrrole sp.hs in the figure) are shown in fig. 1C and fig. 1D, respectively.
The comparison of the primary and secondary mass spectrum fragments of the fermentation liquor sample and the ergothioneine standard in the figure proves that the actinoplanes HS strain can naturally synthesize the ergothioneine.
Step 3: quantitative analysis of ergothioneine product in fermentation broth
The ergothioneine in the fermentation broth is quantitatively detected by a high performance liquid chromatography-ultraviolet visible spectrophotometry detector (HPLC-UV), and the detection method is as follows: the chromatographic column is DIONEX120 C18 (250X 4.6mm,5 μm); mobile phase: phase A is pure acetonitrile solution, phase B is pure water solution, and the elution gradient is that: 0-15 min, phase A: phase B = 3:97; 15-18 min, 3-100% of phase A and 97-0% of phase B; 18-19 min, 100% of A phase and 0 of B phase; 19-20 min, 100-0% of phase A and 0-97% of phase B; 20-30 min, phase A: phase B = 3:97; the flow rate is 0.8mL/min; the sample volume was 20. Mu.L.
The yield of the ergothioneine in the actinoplanes HS fermentation broth is 43mg/L, which is obtained through HPLC-UV detection, and indicates that the actinoplanes HS of the application can naturally and efficiently synthesize the ergothioneine.
Example 2: identification of ergothioneine synthase in actinoplanes HS
1. Gene screening
By bioinformatic predictive analysis of the zooactinomycetes HS genome, the possible genes encoding enzymes involved in ergothioneine synthesis, bc03_glimmer_04016, bc03_glimmer_04015, bc03_glimmer_04014, bc03_glimmer_04013, bc03_glimmer_04046 and bc03_glimmer_04917 were found. The sequences are shown as Seq ID No.7, seq ID No.8, seq ID No.9, seq ID No.10, seq ID No.11 and Seq ID No.12, respectively.
2. Gene functional identification
2.1 functional identification of HER-Cys-Sul Synthesis-related enzymes
The actinoplanes HS genome is used as a template for amplification, related genes are constructed on a vector pETDuet-1, and plasmids obtained by construction are transferred into an Escherichia coli strain BL21 (DE 3), and then shake flask fermentation and product determination are carried out.
The amplification primers are shown in Table 1.
TABLE 1
Step 1: the plasmid pETDuet-1 is used as a vector, ncoI/EcoRI is used for double enzyme digestion to obtain a fragmented vector, a zooactinomycete HS strain genome obtained by small-scale extraction by a phenol chloroform method is used as a template, a Primer 1/Primer 2 is used as a Primer for amplification to obtain a fragment Bc03_Glimmer_04016, a Primer 3/Primer 4 is used as a Primer for amplification to obtain a fragment Bc03_Glimmer_04015, and the fragmented vector and 2 amplified fragments are assembled by using a multi-fragment one-step rapid cloning kit (Santa Biotechnology Co., china) to obtain the plasmid pCIL 001A. The structure of the pCIL001A plasmid is schematically shown in FIG. 2 as pCIL001A, wherein 04016 and 04015 represent amplified fragments Bc03_Glimer_04016 and Bc03_Glimer_04015, respectively, and the other parts of the plasmid vector are derived from the vector backbone portion of pETDuet-1 plasmid.
The pCIL001A plasmid is used as a vector, ndeI/XhoI double enzyme digestion is used for obtaining a fragmented vector, actinoplanes HS strain genome is used as a template, primer 5/Primer 6 is used as a Primer for amplification to obtain fragment Bc03_Glimmer_04014, primer7/Primer 8 is used as a Primer for amplification to obtain fragment Bc03_Glimmer_04013, and the fragmented vector and 2 amplified fragments are assembled by using a multi-fragment one-step method rapid cloning kit (Santa Biotechnology Co., ltd., china) to obtain the pCIL001 plasmid. The structure of the pCIL001 plasmid is schematically shown in FIG. 2, wherein 04014 and 04013 represent amplified fragments Bc03_Glimer_04014 and Bc03_Glimer_04013, respectively, and the other parts of the plasmid vector are identical to the pCIL001A plasmid.
Step 2: acquisition and fermentation of heterologous expression strains in BL21 (DE 3)
Separating single colony from appropriate amount of BL21 (DE 3) glycerinum on LA (LB medium+2% agar) plate, picking single colony in 10mL LB medium, shaking and culturing overnight at 37deg.C and 220rpm, transferring to 200mL LB medium with 1% transfer amount, shaking and culturing at 37deg.C and 220rpm to OD 0.4-0.6, collecting bacterial liquid, pre-cooling on ice for 10min, centrifuging at 4deg.C and 3500rpm for 10min, removing supernatant, adding 30mLCCC1 solution (30mM KAc,80mM CaCl) 2 PH-5.8), centrifuging at 3500rpm at 4 ℃ for 10min, removing supernatant, re-suspending cells in 30mLCCC1, standing in ice for 1-2 h, centrifuging at 3500rpm at 4 ℃ for 10min, removing supernatant, re-suspending cells in 6mL CCC2 (CCC1+10% glycerol), sub-packaging 70 mu L in each 1.5mL sterile centrifuge tube, and quick freezing with liquid nitrogen to obtain BL21 (DE 3) competent cells.
After the sequence of the constructed pCIL001 plasmid is correct, pCIL001 and pETDuet-1 are respectively transformed into BL21 (DE 3) competence by a calcium transformation method to obtain strains YC301 and YC300, wherein YC301 is a heterologous expression strain, and YC300 is a control strain.
The YC300 and YC301 strains were isolated as single colonies on LA plates containing 100. Mu.g/mL of ampicillin, and the single colonies were picked up and cultured overnight at 37℃and 220rpm in 10mL of LB liquid medium containing 100. Mu.g/mL of ampicillin, and transferred to 50mL of fermentation medium (glucose 2%, yeast extract 0.2%, ammonium sulfate 1.6%, disodium hydrogen phosphate dodecahydrate 1.6%, potassium dihydrogen phosphate 0.3%, magnesium sulfate 0.06%, calcium chloride 0.001%, pH 6.8-7.0) containing 100. Mu.g/mL of ampicillin at 1% transfer rate, and cultured at 220rpm until OD was about 0.8, and then the culture was continued at 25℃under the conditions of changing the temperature to 0.2mM IPTG (isopropyl-. Beta. -D-thiogalactoside) for 120 hours.
Step 3: detection of intermediate HER-Cys-Sul in fermentation broth
And (2) fermenting YC300 strain and YC301 strain for 120h respectively, centrifuging 1mL of fermentation liquor at 10000rpm for 5min, filtering the obtained supernatant by using a 0.22 micrometer filter membrane, directly performing HPLC-HRMS detection, re-suspending the thallus by using 1mL of 50% acetonitrile, performing ultrasonic crushing for 30min at 600W, centrifuging at 10000rpm for 5min, filtering the obtained supernatant by using a 0.22 micrometer filter membrane, and performing HPLC-HRMS detection, wherein the detection method is the same as that of the step (2) of the example 1.
The accurate molecular weight extraction ion flow chromatograms of the supernatant and the thallus intermediate HER-Cys-Sul in the fermentation broths of YC300 strain and YC301 strain are shown in FIG. 3A, the HER-Cys-Sul primary accurate molecular weight mass chromatogram is shown in FIG. 3B, and the result shows that the control strain YC300 cannot synthesize the intermediate HER-Cys-Sul, and the strain YC301 which heterologously expresses Bc03_Glimer_04016, bc03_Glimer_04015, bc03_Glimer_04014 and Bc03_Glimer_04013 can produce HER-Cys-Sul, thereby confirming that the proteins (enzymes) encoded by the genes Bc03_Glimer_04016, bc03_Glimer_04014 and Bc03_Glimer_04013 in actinomycetes HS can synthesize ergothioneine precursor HER-Cys-Sul.
2.2 functional identification of carbon-sulfur lyase
The actinoplanes HS genome is used as a template for amplification, related genes are constructed on a vector pBBR1MCS-2, and plasmids obtained by construction are transferred into a strain YC301, and then shake flask fermentation and product determination are carried out.
Step 1: plasmid construction
The plasmid pBBR1MCS-2 is used as a vector, primer 9/Primer 10 is used as a Primer to amplify to obtain a vector fragment, ndeI/SacI is used to double enzyme cut to obtain a fragmented vector fragment, actinoplanes HS strain genome is used as a template, primer11/Primer 12 is used as a Primer to amplify to obtain fragment Bc03_Glimmer_04046, the fragmented vector and the amplified fragment are assembled by using a multi-fragment one-step rapid cloning kit (Santa Biotechnology Co., ltd., china) to obtain the pCIL003 plasmid, the plasmid structure diagram is shown in figure 2, wherein Bc03_Glimmer_04046 represents an amplified fragment Bc03_Glimmer_04046, and the rest part is from a vector skeleton part of the plasmid pBBR1 MCS-2.
The plasmid pBBR1MCS-2 is used as a vector, primer 9/Primer 10 is used as a Primer to amplify to obtain a vector fragment, ndeI/SacI is used for double enzyme digestion to obtain a fragmented vector fragment, actinoplanes HS strain genome is used as a template, primer13/Primer 14 is used as a Primer to amplify to obtain fragment Bc03_Glimmer_04917, and the fragmented vector and the amplified fragment are assembled by using a multi-fragment one-step rapid cloning kit (Santa biosciences, china) to obtain the pCIL008 plasmid. The schematic plasmid structure is shown in FIG. 2, wherein Bc03_Glimer_ 04917 represents the amplified fragment Bc03_Glimer_ 04917, the remainder being derived from the vector backbone portion of the pBBR1MCS-2 plasmid.
The amplification primers are shown in Table 2.
TABLE 2
Step 2: heterologous expression strain in YC301 and its fermentation
The glycerolium of the YC301 strain obtained above was isolated as a single colony on a LA plate containing 100. Mu.g/mL ampicillin, the single colony was picked up and cultured overnight in 10mL LB medium containing 100. Mu.g/mL ampicillin at 37℃and 220rpm, and transferred to 200mL LB medium containing 100. Mu.g/mL ampicillin at 1% transfer rate, and cultured until OD 0.4-0.6 was reached at 37℃and 220rpm, and the bacterial solution was collected, followed by the competent cell preparation process of the YC301 strain and BL21 (DE 3).
And after the constructed pCIL003 and pCIL008 plasmids are sequenced correctly, respectively converting the plasmids into the competence of the YC301 strain by a calcium transformation method to obtain strains YC303 and YC308.
The YC303 and YC308 strains were isolated as single colonies on LA plates containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin, and were individually picked up and cultured in 10mL LB liquid medium containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin, at 37℃and 220rpm overnight, and transferred to fermentation medium (fermentation medium of the same YC301 strain) containing 100. Mu.g/mL ampicillin and 50. Mu.g/mL kanamycin at 1% transfer rate, cultured at 37℃and 220rpm to an OD value of about 0.8, and induced by adding 0.2mM IPTG to a final concentration, the temperature was changed to 25℃and further cultured for 120 hours, and samples were taken 24 hours, 48 hours and 120 hours after induction.
Step 3: detection of ergothioneine in fermentation broths
The fermentation broth treatment and product detection method were the same as in step 3 of 2.1 of this example 2.
The accurate molecular weight extraction ion flow chromatograms of HER-Cys-Sul products in the supernatants and thalli of strains YC301, YC303 and YC308 are shown in FIG. 4, and it can be seen that intermediate HER-Cys-Sul has not been detected after the expression of Bc03_Glimer_ 04046 (YC 303), bc03_Glimer_ 04917 (YC 308) in the YC301 strain. And drawing a standard curve by using an ergothioneine standard substance, and quantifying the ergothioneine in the sample to obtain the content of the ergothioneine in the supernatant and the thalli of fermentation broths of the YC303 strain and the YC303 strain, wherein the results are shown in figure 5, and the results show that the ergothioneine in the fermentation broths of the YC303 strain and the YC308 strain is accumulated. The results indicate that the enzymes encoded by Bc03_Glimer_ 04046 and Bc03_Glimer_ 04917 have a carbon sulfur lyase function.
Example 3: acquisition of high-yield strains of ergothioneine
Step 1: construction of overexpression plasmid
The pSET152 plasmid is used as a vector, the segmented vector fragment is obtained by double enzyme digestion of XbaI/Eco32I and is marked as 152-XE, the pIB139 plasmid is used as a template, the Primer 15/Primer 16 is used as a Primer for amplification to obtain a promoter fragment ermE (1), the actinoplanes HS strain genome is used as a template, and the Primer 17/Primer 18 is used as a Primer for amplification to obtain a fragment Bc03_Glimer_04016-Bc03_Glimer_04015-Bc03_Glimer_04014-Bc03_Glimer_04013; amplifying the pIB139 plasmid as a template, primer 19/Primer 20 as a Primer to obtain a promoter fragment ermE (2), amplifying the actinoplanes HS strain genome as a template, primer 21/Primer 22 as a Primer to obtain a fragment Bc03_Glimer_ 04046, performing overlap extension PCR (OE-PCR) on the amplified fragments ermE (1) and Bc03_Glimer_04016-Bc03_Glimer_04015-Bc03_Glimer_04014-Bc03_Glimer_04013 using Primer 15/Primer 18 primers to obtain a spliced fragment ermE-Bc03_Glimer_04016-Bc03_Glimer_04015-B03_Glimer_04014-B03_Glimer_04013, denoted as K16-13, amplified fragments ermE (2) and Bc03_Glimer_ 04046 were subjected to OE-PCR using Primer 19/Primer 22 primers to obtain a spliced fragment ermE-Bc03_Glimer_ 04046, denoted E-46, and the above fragments 152-XE, K16-13 and E-46 were assembled using a multi-piece one-step rapid cloning kit (by St. Biotechnology Co., china) to obtain pCIL013 plasmids, the plasmid structures of which were shown in FIG. 6A, wherein 04016, 04015, 04014, 04013 and 04046 represent gene fragments Bc03_Glimer_04016, bc03_Glimer_04015, bc03_Glimer_04014, bc03_Glimer_04013 and Bc03_Glimer_ 04046, respectively, and the other components (e.g., attP, oriT, acc (3) IV, ori, etc.) were derived from pSET 152.
The pIB139 plasmid was used as a template, primer 19/Primer 23 was used as a Primer for amplification to obtain a promoter fragment ermE (3), the actinoplanes HS strain genome was used as a template, primer 24/Primer 25 was used as a Primer for amplification to obtain a fragment Bc03_Glimmer_04917, the amplified fragments ermE (3) and Bc03_Glimmer_04917 were subjected to OE-PCR using Primer 19/Primer25 primers to obtain a spliced fragment ermE-Bc03_Glimmer_ 04917, designated E-17, the fragments 152-XE, K16-13 and E-17 were assembled using a multi-fragment one-step rapid cloning kit (Santa biosciences, china) to obtain pCIL014 plasmids, the plasmid structure of which were shown in FIG. 6B, wherein 04016, 04015, 04014, 04013, 04917 represent gene fragments Bc03_Glimmer_16, bc03_Glimmer_15, bc03_Glimmer_14, bc03_Glimmer_03, and other plasmids such as those of 0403..
The primer sequences are shown in Table 3.
TABLE 3 Table 3
Step 2: recombinant Strain acquisition
After the correctness of the sequencing of the plasmids pCIL013 and pCIL014 was confirmed, the strains were transformed into ET12567-pUZ8002 (Chang ET al, overproduction of gentamicin B in industrial strain Micromonospora echinospora CCTCC M2018898by cloning of the missing genes genR and genS,2019), the single clone was picked up, cultured overnight at 37℃in LB liquid medium containing 50. Mu.g/mL of apramycin, 50. Mu.g/mL of kanamycin and 25. Mu.g/mL of chloramphenicol, the overnight culture broth was transferred into 4mL of LB liquid medium containing 50. Mu.g/mL of apramycin, 50. Mu.g/mL of kanamycin and 25. Mu.g/mL of chloramphenicol in a volume ratio of 1/100, cultured at 37℃to OD value of 0.6-0.8, the cells were collected by centrifugation, and the same volume of non-anti-LB liquid medium was used for washing the cells three times, and then re-suspended with 100. Mu.L of non-anti-LB liquid medium to obtain E.coli suspension as donor bacteria. Actinoplanes HS cells cultured in SFM solid medium for 7 days (28 ℃) were collected, the cells were washed twice with 30mL of LB liquid medium, and resuspended with 3mL of antibiotic-free LB liquid medium as recipient bacteria.
Mixing 100. Mu.L of donor bacteria and 100. Mu.L of acceptor bacteria suspension, and uniformly coating on a substrate containing 10mM MgCl 2 The SFM solid medium plate of (2) is cultured for 16-18H at 28 ℃, the enwrapped plate is covered with enramycin (25 mug/mL) and trimethoprim (40 mug/mL), the culture is continued for 7 days at 28 ℃, the grown-out zygote is separated from single colony on the SFM solid medium plate containing 50 mug/mL enramycin, the single colony is coated on the SFM plate containing 50 mug/mL enramycin, and the grown colony takes a proper amount of 50 mug dd H 2 In O, the solution is boiled in boiling water for 5min and then is subjected to ice bath for 5min, repeated three times, and supernatant fluid is taken after short centrifugation for PCR verification, and correct mutant strains are named YC313 and YC314.
Step 3: recombinant strain fermentation and product detection
Separating single colony from the glycerinum bacteria of YC313 and YC314 on SFM solid plate culture medium containing 50 mug/mL apramycin, culturing at 28 ℃ for 5-7 days, after single colony grows out, picking up single colony, plating on SFM solid plate culture medium containing 50 mug/mL apramycin, culturing at 28 ℃ for 7 days, scraping thalli (1.5 cm multiplied by 1.5 cm) and inoculating 25mL seed culture medium (1% glucose, 4% soybean cake powder, 1% glycerol, 1% soluble starch, 0.2% CaCO) containing 50 mug/mL apramycin 3 pH 7.0), shaking culturing at 28deg.C at 250rpm for 48 hr, collecting seed solution for 48 hr, adding 2.5mL into 25mL fermentation medium (3% maltose, 3% glucose, 1% soybean cake powder, 0.3% yeast powder, 0.1% K) 2 HPO 4 ·3H 2 O，0.15％FeCl 3 ，0.25％CaCl 2 ，0.25％CaCO 3 pH 6.5), shaking culture was carried out at 28℃and 250rpm for 7 days to obtain a fermentation broth, and the fermentation procedure of actinoplanes HS strain was the same as in step 1 of example 1.
The fermentation broths of actinoplanes HS, YC313 and YC314 were treated as in example 1, step 2, and ergothioneine in the fermentation broths of actinoplanes HS, YC313 and YC314 were quantitatively detected by HPLC-UV, and the detection method is as follows: the chromatographic column is DIONEX120 C18 (250X 4.6mm,5 μm); mobile phase: phase A is pure acetonitrile solution, phase B is pure water solution, and the elution gradient is that: 0-15 min, phase A: phase B = 3:97; 15-18 min, 3-100% of phase A and 97-0% of phase B; 18-19 min, 100% of A phase and 0 of B phase; 19-20 min, 100-0% of phase A and 0-97% of phase B; 20-30 min, phase A: phase B = 3:97; the flow rate is 0.8mL/min; the sample volume was 20. Mu.L.
The ergothioneine yield in fermentation broths of Actinoplanes HS (marked as Actinoplanes sp.HS in the figure), YC313 and YC314 strains is shown in figure 7, and the ergothioneine yields of the YC313 and YC314 strains reach 125mg/L and 108mg/L respectively, which are 2.9 times and 2.5 times of the ergothioneine yield (43 mg/L) of Actinoplanes HS of the starting strain.
Sequence listing
<110> Wuhan Syngnato science and technology Co., ltd
<120> methods and vectors for biosynthesis of ergothioneine
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Met Ser Ser Ala Asp Arg Val Leu Arg Gly Ala Ala Glu Ala Ile Glu
1 5 10 15
His Ile Ser Gly Ile Cys Phe Lys Thr Gly Pro Pro Arg His Leu Gly
20 25 30
Val Glu Leu Glu Trp Thr Thr His His Val Asp Asp Pro Ala Val Pro
35 40 45
Val Pro Ala Ala Asp Leu Arg Asp Ala Leu Gly Val His Ala Pro Ala
50 55 60
Ala Leu Gly Asn Pro Gln Pro Val Pro Leu Pro Gly Gly Gly Thr Val
65 70 75 80
Thr Ala Glu Pro Gly Gly Gln Leu Glu Ile Ser Ser Ala Pro Ala Asp
85 90 95
Ala Leu Pro Ala Leu His Ala Ala Val Thr Ala Asp His Ala Ala Leu
100 105 110
Ala Gly Met Leu Ala Arg Ala Gly Leu Arg Leu Gly Asp Arg Gly Ile
115 120 125
Asp Glu His Arg Glu Pro Ala Arg Ile Leu Asp Thr Pro Arg Tyr Ala
130 135 140
Ala Met Glu Arg Ser Phe Asp Arg Ala Gly Arg Thr Met Met Thr Gly
145 150 155 160
Thr Ala Gly Leu Gln Val Cys Leu Asp Ala Gly Glu Ala His Gln Ile
165 170 175
Ala Gly Arg Trp Ala Ala Leu His Asp Phe Gly Pro Pro Leu Leu Ala
180 185 190
Leu Phe Ala Asn Ser Ala Val His Ala Gly Arg Asp Thr Gly Trp Ala
195 200 205
Ser Ala Arg Met Ala Ala Trp Tyr Gly Ile Asp Pro Arg Arg Ala Gly
210 215 220
Pro Ala Phe Arg Glu Ser Gly Ser Pro Ala Glu Asp Trp Ala Arg Tyr
225 230 235 240
Ala Leu Asp Ala Pro Leu Leu Cys Val Arg Arg Asp Asp Gly Arg Trp
245 250 255
Asp Ala Pro Pro Gly Val Thr Phe Ala Asp Trp Ile Thr Asp Gly Thr
260 265 270
Pro Thr Val Ser Asp Leu Glu Tyr His Leu Ser Thr Leu Phe Pro Pro
275 280 285
Val Arg Pro Arg Gly Tyr Leu Glu Val Arg Tyr Leu Asp Thr Gln Pro
290 295 300
Gly Pro Asp Trp Ile Ala Pro Ala Ala Val Leu Thr Ala Leu Met Ala
305 310 315 320
Asp Asp Val Ile Thr Ala Gln Gly Arg Glu Ile Ala Ala Pro Val Ala
325 330 335
Gly Arg Trp Arg Ala Ala Ala Arg Asp Gly Leu Arg Asp Pro Ala Val
340 345 350
Arg Ala Ala Ala Ala Gly Leu Ala Glu Leu Ala Cys Arg His Phe Asp
355 360 365
Arg Thr Gly Leu Asp Asp Val Val Arg Lys Gln Val Ser Asp Val Val
370 375 380
Asp Ala Arg Leu Lys Gly Asn Ala Arg
385 390
<210> 2
<211> 430
<212> PRT
<213> Artificial Sequence
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Met Ile Asn Pro Asp Lys Asn Leu Ile Ala Ala Glu Leu Glu Arg Ser
1 5 10 15
Arg Ala Arg Thr Ala Leu Leu Thr Asp Ala Val Asp Asp Asp Asp Leu
20 25 30
Val Ala Gln His Ser Pro Leu Met Ser Pro Leu Val Trp Asp Leu Ala
35 40 45
His Val Gly Asn Gln Glu Glu Leu Trp Leu Val Arg Asp Val Gly Gly
50 55 60
Arg Glu Pro Val Arg Gln Asp Ile Asp Glu Leu Tyr Asp Ala Phe Gln
65 70 75 80
His Ala Arg Ser Asp Arg Pro Ala Leu Pro Leu Leu Asp Pro Ala Glu
85 90 95
Ala Arg Arg Tyr Ile Gly Gln Val Arg Asp Lys Ala Leu Asp Val Leu
100 105 110
Asp Arg Val Arg Leu Asp Glu Arg Pro Leu Leu Ala Gly Gly Phe Ala
115 120 125
Phe Gly Met Ile Val Gln His Glu Gln Gln His Asp Glu Thr Met Leu
130 135 140
Ala Thr His Gln Leu Arg Gly Gly Ser Pro Val Leu Ser Ala Pro Pro
145 150 155 160
Pro Pro Pro Gly Asp Ala Arg Val Arg Gly Glu Val Leu Ile Pro Gly
165 170 175
Gly Pro Phe Thr Met Gly Thr Asp Thr Asp Pro Trp Ala Leu Asp Asn
180 185 190
Glu Arg Pro Ala His Thr Val Glu Val Pro Ala Phe Leu Ile Asp Ala
195 200 205
Thr Pro Val Ser Asn Ser Asp Tyr Leu Ala Phe Ile Asp Ala Gly Gly
210 215 220
Tyr Asp Asp Pro Arg Trp Trp Ser Ala Arg Gly Trp Thr His Arg His
225 230 235 240
Glu Ala Gly Leu Thr Ala Pro Met His Trp Leu Arg Asp Gly Asp Ala
245 250 255
Trp Leu Tyr Arg Arg Phe Gly Arg Ile Ser Pro Ile Ala Gly Asp Glu
260 265 270
Pro Val Val His Val Asp Tyr Phe Glu Ala Glu Ala Tyr Ala Ala Trp
275 280 285
Ala Gly Lys Arg Leu Pro Thr Glu Ala Glu Trp Glu Lys Ala Ala Arg
290 295 300
Trp Asp Pro Ala Thr Gly Arg Ser Arg Arg Phe Pro Trp Gly Asp Glu
305 310 315 320
Pro Pro Gly Pro Glu His Ala Asn Leu Gly Gln Arg His Leu Ala Pro
325 330 335
Ala Pro Ala Gly Ala Tyr Pro Ala Gly Ala Ser Pro Leu Gly Val His
340 345 350
Gln Leu Ile Gly Asp Val Trp Glu Trp Thr Ser Thr Asp Trp His Gly
355 360 365
Tyr Pro Gly Phe Arg Val Phe Pro Tyr Ala Glu Tyr Ser Glu Val Phe
370 375 380
Phe Gly Gly Asp Tyr Lys Val Leu Arg Gly Gly Ser Phe Gly Thr Asp
385 390 395 400
Ala Ala Ala Cys Arg Gly Thr Phe Arg Asn Trp Asp His Pro Ile Arg
405 410 415
Arg Gln Ile Phe Ser Gly Phe Arg Cys Ala Arg Glu Leu Val
420 425 430
<210> 3
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<212> PRT
<213> Artificial Sequence
<400> 3
Met Cys Arg His Leu Ala Tyr Leu Gly Pro Pro Glu Pro Leu Ser Ala
1 5 10 15
Trp Val Phe Asp Pro Pro His Ala Leu Ser His Gln Ala Trp Ala Pro
20 25 30
Arg Asp Met Arg Gly Gly Gly Thr Ile Asn Ala Asp Gly Phe Gly Val
35 40 45
Gly Trp Tyr Pro Pro Glu Gly Gly Pro Pro Val Arg Tyr Arg Ser Ala
50 55 60
Met Pro Ile Trp Ser Asp Pro Thr Leu Pro Arg Leu Ala Glu Val Thr
65 70 75 80
Arg Ser Gly Ala Val Leu Ala Ala Val Arg Ser Ala Thr Glu Gly Met
85 90 95
Pro Val Ile Ala Thr Ala Thr Ala Pro Leu Gln Asp Gly Arg Trp Leu
100 105 110
Phe Ser His Asn Gly Val Val Arg Gly Phe Pro Gly Thr Leu Ala Asp
115 120 125
Leu Ala Ala Ala Leu Pro Val Glu Asp Leu Leu Thr Leu Asp Ala Pro
130 135 140
Thr Asp Ala Ala Ala Leu Phe Ala Leu Val Arg His Gly Leu Arg Ala
145 150 155 160
Gly Lys Thr Ala Glu Glu Ala Leu Leu Ser Val Val Thr Ala Val Leu
165 170 175
Arg Val Ala Pro Asp Ser Arg Leu Asn Leu Leu Leu Thr Asp Gly Asp
180 185 190
Arg Ile Leu Ala Thr Thr Ala Gly His Ala Leu Ala Val Arg Ala Thr
195 200 205
Gly Asp Ala Val Leu Val Ala Ser Glu Pro Leu Asp Asp His Pro Ala
210 215 220
Trp Arg Pro Leu Pro Asp Arg Arg Leu Leu Ile Ala Thr Pro Ala Ala
225 230 235 240
Val Glu Leu Gly Glu Leu
245
<210> 4
<211> 323
<212> PRT
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<400> 4
Val Thr Ser Leu Glu Lys His Leu Asp Glu Arg Asp Leu Ala Arg Ser
1 5 10 15
Leu Arg Ala Asp Val Arg Asp Gly Leu Ser Ala Asp Pro Lys Arg Leu
20 25 30
Pro Pro Lys Trp Phe Tyr Asp Ala Arg Gly Ser Arg Leu Phe Glu Asp
35 40 45
Ile Thr Arg Leu Pro Glu Tyr Tyr Pro Thr Arg Thr Glu Arg Ala Ile
50 55 60
Leu Ser Ala Ala Ala Ala Glu Ile Ala Arg Leu Thr Asp Ala Lys Thr
65 70 75 80
Leu Val Glu Leu Gly Ser Gly Ser Ser Glu Lys Thr Arg Leu Leu Leu
85 90 95
Asp Ala Met Leu Gly Arg Gly Thr Leu Gly Ser Phe Ile Pro Phe Asp
100 105 110
Val Ser Gln Ser Ala Leu Ala Glu Ala Val Asp Ala Leu Ser Val Thr
115 120 125
Tyr Pro Gly Leu Ser Ile Thr Gly Val Val Gly Asp Phe Thr Arg His
130 135 140
Leu Arg His Leu Pro Asp Gly Asp Ser Arg Leu Val Ala Phe Leu Gly
145 150 155 160
Gly Thr Ile Gly Asn Leu Ile Pro Ala Glu Arg Ser Ala Phe Leu Gly
165 170 175
Asp Leu Arg Ser Val Leu His Ala Gly Glu Trp Leu Leu Leu Gly Thr
180 185 190
Asp Leu Val Lys Asp Pro Ala Val Leu Val Pro Ala Tyr Asp Asp Ala
195 200 205
Ala Gly Val Thr Ala Glu Phe Asn Arg Asn Val Leu His Val Ile Asn
210 215 220
Arg Glu Leu Arg Ala Asp Phe Glu Pro Leu Ala Phe Glu His Val Ala
225 230 235 240
Ala Trp Asp Pro Asp Arg Glu Trp Ile Glu Met Arg Leu Arg Ser Val
245 250 255
Arg Ala Gln Thr Val Arg Ile Glu Asp Leu Asp Leu Thr Val Ser Tyr
260 265 270
Ala Ala Gly Glu Glu Met Arg Thr Glu Ile Ser Ala Lys Phe Arg Arg
275 280 285
Glu Arg Leu Ala Ala Glu Leu Ala Ala Ala Gly Phe Ala Leu Arg His
290 295 300
Trp Trp Ser Asp Pro Gln Asp Trp Phe Gly Val Ser Leu Ala Gln Ala
305 310 315 320
Val Thr Asp
<210> 5
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<212> PRT
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Met Asp Val Asp Ala Leu Arg Ala Gly Thr Pro Gly Cys Arg His Arg
1 5 10 15
Ile His Leu Asn Asn Ala Gly Ala Ala Leu Met Ser Gln Ala Thr Leu
20 25 30
Asp Thr Val Val Ala His Leu Arg Leu Glu Ala Glu Ile Gly Gly Tyr
35 40 45
Glu Ala Ala Gly Ala Val Ala Asp Arg Val Ala Ala Val Tyr Ala Gly
50 55 60
Leu Ala Glu Leu Leu Gly Gly Arg Ala Asp Glu Ile Ala Leu Phe Asp
65 70 75 80
Asn Ala Thr His Ala Trp Gln Ala Ala Phe His Ala Val Pro Leu Lys
85 90 95
Ala Gly Asp Arg Val Leu Thr Gly Arg Asn Glu Tyr Gly Ser Asn Val
100 105 110
Leu Gly Tyr Leu Gln Ala Ala Arg Arg Val Gly Ala Glu Ile Val Val
115 120 125
Val Pro Asn Asp Glu His Gly Gln Ile Asp Thr Val Ala Leu Ala Gly
130 135 140
Leu Ile Asp Glu Arg Ala Lys Val Ile Gly Leu Thr His Val Pro Thr
145 150 155 160
Ala Gly Gly Leu Val Asn Pro Ala Ala Glu Val Gly Arg Ile Ala Arg
165 170 175
Ala Ala Gly Val Pro Tyr Leu Leu Asp Ala Thr Gln Ser Val Gly Gln
180 185 190
Phe Pro Val Asp Val Thr Glu Ile Gly Cys Asp Phe Leu Cys Gly Thr
195 200 205
Gly Arg Lys Phe Leu Arg Gly Pro Arg Gly Thr Gly Phe Leu Trp Val
210 215 220
Arg Asp Gly Ile Leu Glu Gln Leu Glu Pro His Val Val Glu Ile Gln
225 230 235 240
Ser Ala Asp Trp Asp Gly Ala Arg Gly Phe Gly Trp Val Pro Gly Ala
245 250 255
Gln Arg Phe Ala Thr Trp Glu Leu Asn Tyr Ala Ala Val Leu Gly Leu
260 265 270
Gly Ala Ala Val Asp Gln Ala Leu Asn Leu Gly Leu Gly Glu Ile Gly
275 280 285
Lys Arg Asn Ala Glu Leu Gly Asp Arg Met Arg Gly Leu Leu Glu Asp
290 295 300
Thr Pro Gly Val Thr Val Tyr Asp Leu Gly Arg Glu Arg Cys Ala Ile
305 310 315 320
Val Thr Ala Glu Val Ala Gly Val Gly Ala Glu Gln Val Val Ala Arg
325 330 335
Leu Ala Glu Ser Gly Val Asn Val Thr Ser Thr Val Pro Ala His Gln
340 345 350
Gln Phe Asp Thr Glu Asp Arg Asp Pro Pro Pro Leu Val Arg Phe Ser
355 360 365
Pro His Tyr Tyr Asn Thr Glu Asp Glu Val Glu His Ala Ala Thr Leu
370 375 380
Val Ser Ala Met Val Thr Lys Pro Leu
385 390
<210> 6
<211> 395
<212> PRT
<213> Artificial Sequence
<400> 6
Met Ser Ala Glu Asp Pro Pro Gln Pro Ile Ala Gly Ala Arg Leu Leu
1 5 10 15
Phe Ser Leu Asp Pro Ala Val Ser Tyr Leu Asn His Gly Ser Phe Gly
20 25 30
Ala Leu Pro Ile Thr Val Gln Arg Ala Gln Gln Arg Leu Arg Asp Glu
35 40 45
Met Asp Leu Asn Pro Met Arg Phe Phe Gly Pro Gly Leu Leu Asp Arg
50 55 60
Ile Ile His Thr Arg Arg His Leu Ala Ala Phe Leu Gly Ala Asp Pro
65 70 75 80
Glu Gly Ser Ala Leu Thr Ser Asn Thr Thr Thr Ala Val Ser Leu Val
85 90 95
Leu Gln Ser Val Arg Leu Lys Glu Ser Asp Glu Val Leu Leu Thr Asp
100 105 110
His Ala Tyr Gly Ala Val Thr Met Ala Val Arg Arg Glu Cys Arg Arg
115 120 125
Thr Gly Ala Thr Thr Arg Thr Ile Ala Val Pro Phe Gly Ala Ser Gly
130 135 140
Pro Glu Val Leu Ser Arg Val Arg Ala Ala Leu Arg Pro Gly Arg Thr
145 150 155 160
Arg Leu Leu Ile Ile Asp Gln Val Thr Ser Ala Thr Ala Thr Leu Met
165 170 175
Pro Val Arg Glu Val Val Ala Ala Ala Arg Ala Gln Gly Ile Pro Val
180 185 190
Met Val Asp Gly Ala His Val Pro Gly Met Leu Pro Val Arg Val Glu
195 200 205
Glu Ile Gly Ala Asp Phe Trp Val Gly Asn Leu His Lys Trp Gly Trp
210 215 220
Ala Pro Arg Gly Thr Ser Leu Leu Ala Val Ser Pro Asp Trp Arg Arg
225 230 235 240
Arg Ile Asp Pro Leu Val Val Ser Trp Glu Gln Asp Gln Gly Phe Pro
245 250 255
Leu Ser Val Glu Phe Gln Gly Thr Ile Asp Tyr Thr Pro Trp Leu Ala
260 265 270
Ala Pro Ala Gly Ile Phe Ala Met Arg Thr Leu Gly Pro Glu Val Val
275 280 285
Arg Glu His Asn Ala Ala Leu Ala Ala Tyr Gly Gln Arg Val Val Gly
290 295 300
Ala Ala Leu Gly His Ala Pro Ala Asp Leu Pro Glu Pro Gly Gly Pro
305 310 315 320
Gly Val Ser Met Arg Ile Val Pro Leu Pro Ala Gly Val Ala Thr Thr
325 330 335
Phe Pro Glu Ala His Ala Leu Arg Gly His Ile Ala Asp Lys Leu Gly
340 345 350
Val Glu Thr Gln Ile Asn Ala Trp Gly Gly Arg Gly Leu Leu Arg Leu
355 360 365
Ser Ala Gln Ile Tyr Asn Arg Pro Glu Glu Tyr His His Leu Ala Asp
370 375 380
Arg Leu Pro Ser Leu Leu His His Trp Gln Trp
385 390 395
<210> 7
<211> 1182
<212> DNA
<213> Artificial Sequence
<400> 7
atgagttcgg ccgatcgagt gctgcgggga gccgcggaag cgatcgaaca catctccggc 60
atctgcttca agaccggccc gccacgccat ctcggcgtcg agctggaatg gaccacacac 120
cacgtggatg acccggccgt cccggtcccg gccgcggacc tgcgtgacgc gttgggggtg 180
cacgcgccgg ccgccctcgg caatccgcaa ccggtgccgc tgcccggcgg cggcacggtc 240
accgccgaac ccggcggtca gttggagatc tcgtcggcgc cggccgatgc gctgcccgcc 300
ctgcacgcgg cggtcaccgc cgaccacgcc gccctggccg ggatgctcgc ccgggccggc 360
ctgcgcctcg gcgaccgggg catcgacgag caccgggaac ccgcccggat cctggacacg 420
ccgcggtacg ccgcgatgga acgctccttc gaccgcgctg ggcgcaccat gatgaccggc 480
accgcgggac tgcaggtctg cctcgacgcc ggggaggccc accagatcgc cggccggtgg 540
gcggcgctgc acgacttcgg gccgccgctg ctggcgctgt tcgcgaattc ggcggtgcac 600
gccgggcggg acaccggctg ggcgtcggcc cggatggccg cctggtacgg catcgacccc 660
cggcgcgccg gcccggcctt ccgggagtcc ggctccccgg ccgaggactg ggcccggtac 720
gcgctcgacg cgccactgct ctgcgtgcgg cgcgacgacg ggcggtggga cgcgccgccc 780
ggggtgacct tcgccgactg gatcaccgac ggcacaccga ccgtgtccga tctggagtat 840
cacctcagca ccctgttccc gccggtgcgc ccgcgcggct acctggaggt gcgctacctc 900
gacacccagc cggggccgga ctggatcgcc ccggccgcgg tgctgaccgc gctgatggcc 960
gacgacgtga tcaccgctca gggccgggag atcgcggcgc cggtcgcggg gcggtggcgg 1020
gccgcggcac gggacgggct gcgggatccg gcggtccgcg cggcggcggc cggactggcc 1080
gagctcgcct gccggcactt cgaccggacg gggctggatg acgtcgtccg aaagcaggtt 1140
tccgatgttg ttgacgcgcg cctgaagggg aacgctcgat ga 1182
<210> 8
<211> 1293
<212> DNA
<213> Artificial Sequence
<400> 8
atgatcaacc ccgacaagaa cctgatcgcc gccgaattgg agcgatcgcg ggcccgcacc 60
gccctgctga ccgacgcggt cgacgacgac gacctcgtcg cgcagcactc gccgctgatg 120
tcgccgctgg tctgggacct cgcgcacgtc ggcaaccagg aggagctctg gctggtccgg 180
gacgtcggcg gccgcgagcc ggtgcgtcag gacatcgacg agttgtacga cgcgttccag 240
cacgcccgca gcgaccgccc cgccctgccg ctgctcgacc cggccgaggc gcgccgatac 300
atcggccagg tgcgggacaa ggcgctcgac gtgctcgacc gggtgcgcct ggacgagcgg 360
ccgctgctcg ccggtggctt cgcgttcggc atgatcgtgc aacacgagca gcagcacgac 420
gagaccatgc tcgccaccca ccagctgcgc ggcggctcgc cggtgctctc cgccccgccg 480
cccccgcccg gggacgcccg ggtgcgcggc gaggtgctga tccccggcgg cccgttcacc 540
atgggcaccg acaccgaccc gtgggcgctg gacaacgagc ggccggcgca caccgtcgag 600
gtcccggcgt tcctgatcga cgccaccccg gtgtccaaca gtgactacct cgccttcatc 660
gacgccggcg gctacgacga cccgcgctgg tggtcggcgc ggggctggac gcaccggcac 720
gaggccgggc tgaccgcgcc gatgcactgg ctgcgcgacg gtgacgcctg gctctaccgg 780
cggttcggcc ggatcagccc gatcgccggc gacgagccgg tggtgcacgt cgactacttc 840
gaggccgagg cgtacgcggc ctgggccggg aaacggctgc ccaccgaggc cgagtgggag 900
aaggccgccc gctgggaccc ggcgaccggc cgctcccgcc gcttcccgtg gggcgacgag 960
ccgcccggcc cggaacacgc caacctcggg cagcggcatc tcgcgccggc gccggccggg 1020
gcgtatccgg ccggcgcgtc gccgctgggc gtgcaccagc tgatcggcga tgtctgggaa 1080
tggacatcga ccgactggca cggctatccc ggattccggg tgttcccgta cgccgagtac 1140
tccgaggtct tcttcggcgg tgactacaag gtgctgcgcg gcggctcgtt cggcaccgac 1200
gcggccgcct gccgcggcac gttccgcaac tgggaccacc cgatccggcg gcagatcttc 1260
agcggcttcc ggtgcgcgcg ggagctcgtc tga 1293
<210> 9
<211> 741
<212> DNA
<213> Artificial Sequence
<400> 9
atgtgccgcc acctggccta cctcgggccg cccgagccac tgtcggcgtg ggtgttcgac 60
ccgccgcacg cgctgtcgca ccaggcctgg gcaccacgtg acatgcgcgg cgggggcacc 120
atcaacgccg acgggttcgg cgtggggtgg tatccgcccg agggcgggcc gccggtccgg 180
tatcgcagcg cgatgccgat ctggagcgat ccgacactgc cccggctcgc ggaggtgacc 240
aggtcggggg cggtgctcgc ggcggtccgg tcggcgaccg aggggatgcc ggtgatcgcg 300
acggcgacgg cgccgctcca ggacgggcgg tggcttttca gccacaacgg ggtggtccgc 360
ggtttccccg ggaccctcgc cgacctggcc gccgcgcttc ccgtcgagga cctgctgacc 420
ctcgacgcgc cgaccgatgc ggccgccctg ttcgctctgg tacgccatgg gctgcgcgcc 480
gggaagaccg cggaggaggc actcctgtcc gtggtcaccg ccgtgctgcg ggtcgcgccg 540
gactcccggc tgaacctgct gctgaccgat ggtgatcgga tcctggcgac cactgccggg 600
cacgcgctcg ccgtccgggc gaccggcgac gccgtgctgg tcgcctccga gcccctcgac 660
gaccaccccg cctggcggcc gctcccggac cgccggctgt tgatcgcgac ccccgctgcg 720
gtggaattag gagagctgtg a 741
<210> 10
<211> 972
<212> DNA
<213> Artificial Sequence
<400> 10
gtgacttcgc tggagaagca tctcgacgag cgtgacctgg cccgatccct gcgcgccgac 60
gtgcgcgacg gcctgagcgc cgacccgaaa cggctgccgc cgaaatggtt ctacgacgcc 120
cgcggcagcc ggctcttcga ggacatcacc cggcttccgg agtactaccc gacccgcacc 180
gaacgcgcca tcctgagcgc ggccgccgcg gagatcgccc ggctcaccga cgcgaaaacc 240
ctcgtcgaac tcggctccgg ttcatcggag aagacccggc tgctgctcga cgcgatgctg 300
gggcggggca cgctcggctc cttcatcccg ttcgacgtgt cccagagtgc cctcgctgag 360
gccgtcgacg cgctcagcgt cacctacccc ggcctgagca tcaccggcgt ggtcggcgac 420
ttcacccggc acctgcgcca cctgcccgac ggcgacagcc gcctggtcgc cttcctcggc 480
ggcacgatcg gcaacctgat cccggccgag cgctccgcct tcctcggcga cctgcgctcc 540
gtgctgcacg cgggtgagtg gctgctgctc ggcaccgatc tggtcaagga ccctgccgtg 600
ctggtgcccg cctatgacga cgcggccggc gtcaccgcgg agttcaaccg gaacgtgctg 660
catgtcatca accgcgagct gcgggccgac tttgagccac tcgccttcga gcatgtggcc 720
gcctgggacc cggatcggga atggatcgag atgcggttgc ggtcggtgcg ggcgcagacg 780
gtgcggatcg aggatctgga cctgaccgtc tcgtatgcgg cgggggagga gatgcggacc 840
gagatctcgg cgaagttccg gcgggagcgg cttgcggcgg aactggcggc ggcgggcttc 900
gccctccgcc attggtggtc cgacccgcag gactggttcg gcgtctccct ggcccaggcc 960
gtcactgatt ga 972
<210> 11
<211> 1182
<212> DNA
<213> Artificial Sequence
<400> 11
atggacgtcg acgcactccg ggccggcact ccgggctgcc gccaccggat tcatctgaac 60
aacgcgggcg ccgcgctgat gtcgcaggcg acgctggaca ccgtggtcgc acacctgcgg 120
ctggaggccg agatcggcgg gtacgaggcg gccggcgcgg tcgccgaccg ggtcgccgcg 180
gtctacgccg gtctcgccga gctgctcggc gggcgggccg acgagatcgc gctgttcgac 240
aatgcgacgc acgcctggca ggcggcgttc cacgcggtgc cgctgaaggc cggcgaccgg 300
gtgctgaccg ggcgcaacga gtacggcagc aacgtgctgg gctacctgca ggcggcccgg 360
cgggtcggcg ccgagatcgt cgtggtgccg aacgacgagc acgggcagat cgacacggtc 420
gcgctcgccg ggctgatcga cgagcgggcg aaagtgatcg gcctgaccca cgtgccgacg 480
gccggtggtc tggtcaatcc ggccgccgag gtggggcgga tcgcacgggc cgcgggggtg 540
ccctatctgc tggacgcgac gcagtcggtg ggccagttcc cggtcgacgt caccgagatc 600
ggatgcgact tcctgtgcgg tacggggcgc aaattcctgc gcgggccgcg cggcaccggg 660
ttcctctggg tccgcgacgg catccttgag cagctcgaac cgcacgtggt ggagatccag 720
tcggcggact gggacggcgc gcggggcttc ggctgggtgc ccggggcgca gcggttcgcc 780
acctgggagc tgaactacgc ggcggtgctc ggtctgggcg cggcggtcga ccaggcgctc 840
aatctggggc tcggcgagat cggcaagcgc aacgcggagc tcggcgaccg gatgcgcggg 900
ctgctggagg acactcccgg cgtgaccgtt tatgacctcg gtcgcgagcg gtgcgcgatc 960
gtcaccgcgg aggtggccgg ggtgggcgcc gagcaggtgg tggcgcggct cgccgagtcc 1020
ggggtgaacg tgacctcgac cgtgccggcg caccagcagt tcgacaccga ggaccgcgat 1080
ccgccgccgc tggtccgctt ctcgccgcac tactacaaca ccgaggacga ggtcgagcac 1140
gcggcgaccc tggtcagcgc catggtcacc aagccgttgt ga 1182
<210> 12
<211> 1188
<212> DNA
<213> Artificial Sequence
<400> 12
atgagcgccg aagatccacc gcagcccatt gccggggcgc ggttgctgtt ctcgctggat 60
ccggcggtgt cctatctcaa ccacgggtcg ttcggcgcgt tgccgatcac cgtgcagcgc 120
gcccagcagc gcctgcggga cgagatggat ctgaacccga tgcgattctt cggcccgggc 180
ctgctggacc ggatcatcca cacccgacgg catctggccg ctttcctcgg cgccgacccg 240
gagggcagcg cgctcacctc gaacaccacc accgcggtca gcctggtgct gcagtcggtc 300
cggttgaagg aatcggacga ggtgctgctc accgaccacg cctacggtgc ggtgacgatg 360
gcggtgcgcc gggagtgccg gcggaccggg gcgacgaccc ggacgatcgc ggtgccgttc 420
ggcgcgagcg ggccggaggt cctgtcccgg gtgcgggcgg cgctgcggcc gggacgcacc 480
cggctgctga tcatcgacca ggtgacctcg gcgacggcca cgctgatgcc ggtgcgggag 540
gtcgtcgccg cggcccgggc gcagggcatc ccggtgatgg tcgacggcgc gcacgtgccg 600
ggcatgctgc cggtgcgggt ggaggagatc ggcgccgatt tctgggtggg gaatctgcac 660
aagtggggtt gggcgccgcg cggcacgtcg ctgctggcgg tgtcgccgga ctggcggcgg 720
cggatcgatc cgctggtcgt ctcctgggag caggatcagg gtttcccgct gtcggtcgag 780
ttccagggga cgatcgacta caccccgtgg ctggcggcgc cggccgggat cttcgcgatg 840
cgcacgctgg gcccggaagt cgtccgggag cacaatgcgg cgctcgcggc gtacggccag 900
cgggtggtcg gtgcggccct cggtcatgcg ccggcggatc tgccggagcc gggcggaccc 960
ggggtctcga tgcggatcgt gccgctgccg gccggggtgg ccaccacctt tcccgaggcg 1020
cacgcgttgc gcgggcacat cgccgacaag ctcggggtgg agacgcagat caacgcgtgg 1080
ggcggccggg ggctgctgcg gctgagcgcg cagatctaca accggccgga ggagtatcac 1140
catctggccg atcgcctacc cagcctgctg caccactggc agtggtag 1188
<210> 13
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 13
tttaacttta agaaggagat ataccatggg cagcagcatg agttcggccg atcgagtgc 59
<210> 14
<211> 36
<212> DNA
<213> Artificial Sequence
<400> 14
catggttaat tcctccttca tcgagcgttc cccttc 36
<210> 15
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 15
tgaaggagga attaaccatg atcaaccccg acaagaacct g 41
<210> 16
<211> 56
<212> DNA
<213> Artificial Sequence
<400> 16
cctgcaggcg cgccgagctc gaattcggat cctcagacga gctcccgcgc gcaccg 56
<210> 17
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 17
aagtataaga aggagatata catatgtgcc gccacctggc ctacc 45
<210> 18
<211> 41
<212> DNA
<213> Artificial Sequence
<400> 18
tcatagtgta atcctccttc acagctctcc taattccacc g 41
<210> 19
<211> 39
<212> DNA
<213> Artificial Sequence
<400> 19
tgaaggagga ttacactatg acttcgctgg agaagcatc 39
<210> 20
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 20
gcagcggttt ctttaccaga ctcgagtcaa tcagtgacgg cctgggccag ggag 54
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 21
cgccatatgg agctccaatt cgccctatag 30
<210> 22
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 22
cgccatatgg gttaattcct cctactgcag g 31
<210> 23
<211> 54
<212> DNA
<213> Artificial Sequence
<400> 23
actcactata gggcgaattg gagctctcac aacggcttgg tgaccatggc gctg 54
<210> 24
<211> 55
<212> DNA
<213> Artificial Sequence
<400> 24
tgcagtagga ggaattaacc catatgatgg acgtcgacgc actccgggcc ggcac 55
<210> 25
<211> 48
<212> DNA
<213> Artificial Sequence
<400> 25
actcactata gggcgaattg gagctcctac cactgccagt ggtgcagc 48
<210> 26
<211> 46
<212> DNA
<213> Artificial Sequence
<400> 26
tgcagtagga ggaattaacc catatgatga gcgccgaaga tccacc 46
<210> 27
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 27
cggccagtgc caagcttggg ctgcaggtcg actctagagc gagtgtccgt tcgagtggc 59
<210> 28
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 28
cggctccccg cagcactcga tcggccgaac tcattggatc ctaccaaccg gcacgattg 59
<210> 29
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 29
ctgttgtggg cacaatcgtg ccggttggta ggatccaatg agttcggccg atcgagtgc 59
<210> 30
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 30
tcgggcgcaa gccgccactc gaacggacac tcgctcaatc agtgacggcc tgggccagg 59
<210> 31
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 31
ggcccaggcc gtcactgatt gagcgagtgt ccgttcgagt ggcggcttgc gcccgatgc 59
<210> 32
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 32
cagcccggag tgccggcccg gagtgcgtcg acgtccattg gatcctacca accggcacg 59
<210> 33
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 33
gggcacaatc gtgccggttg gtaggatcca atggacgtcg acgcactccg ggccggcac 59
<210> 34
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 34
acagctatga catgattacg aattcgatat ctcacaacgg cttggtgacc atggcgctg 59
<210> 35
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 35
cggcaatggg ctgcggtgga tcttcggcgc tcattggatc ctaccaaccg gcacgattg 59
<210> 36
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 36
ttgtgggcac aatcgtgccg gttggtagga tccaatgagc gccgaagatc caccgcagc 59
<210> 37
<211> 59
<212> DNA
<213> Artificial Sequence
<400> 37
caggaaacag ctatgacatg attacgaatt cgatatccta ccactgccag tggtgcagc 59
<210> 38
<211> 1504
<212> DNA
<213> Artificial Sequence
<400> 38
agagtttgat cctggctcag gacgaacgct ggcggcgtgc ttaacacatg caagtcgagc 60
ggaaaggccc ttcggggtac tcgagcggcg aacgggtgag taacacgtga gtaacctgcc 120
ccagactttg ggataaccct cggaaacggg ggctaatacc gaatatgacc tagcttcgca 180
tggagcgtgg tggaaagttt ttcggtttgg gatggactcg cggcctatca gcttgttggt 240
ggggtaatgg cctaccaagg cgacgacggg tagccggcct gagagggcga ccggccacac 300
tgggactgag acacggccca gactcctacg ggaggcagca gtggggaata ttgcacaatg 360
ggcggaagcc tgatgcagcg acgccgcgtg agggatgacg gccttcgggt tgtaaacctc 420
tttcagcagg gacgaagcgc aagtgacggt acctgcagaa gaagcgccgg ccaactacgt 480
gccagcagcc gcggtaagac gtagggcgcg agcgttgtcc ggatttattg ggcgtaaaga 540
gctcgtaggc ggcttgtcgc gtcgaatgtg aaatcccgag gctcaacttc gggcttgcat 600
tcgatacggg caggctagag ttcggtaggg gagactggaa ttcctggtgt agcggtgaaa 660
tgcgcagata tcaggaggaa caccggtggc gaaggcgggt ctctgggccg atactgacgc 720
tgaggagcga aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgctgtaaa 780
cgttgggcgc taggtgtggg gggcctctcc ggtttcctgc gccgcagcta acgcattaag 840
cgccccgcct ggggagtacg gccgcaaggc taaaactcaa aggaattgac gggggcccgc 900
acaagcggcg gagcatgcgg attaattcga tgcaacgcga agaaccttac ctgggtttga 960
catcgccgga aatctcgcag agatgcgggg tccttcgggg ccggtgacag gtggtgcatg 1020
gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccctcg 1080
ttcgatgttg ccagcgcgta atggcgggga ctcatcggag actgccgggg tcaactcgga 1140
ggaaggtggg gatgacgtca agtcatcatg ccccttatgt ccagggcttc acgcatgcta 1200
caatggccgg tacaaagggc tgcgataccg taaggtggag cgaatcccaa aaagccggtc 1260
tcagttcgga tcggggtctg caactcgacc ccgtgaagtc ggagtcgcta gtaatcgcag 1320
atcagcaacg ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacgtcacg 1380
aaagtcggca acacccgaag ccggtggcct aacccgtaag ggagggagcc gtcgaaggtg 1440
gggctggcga ttgggacgaa gtcgtaacaa ggtagccgta ccggaaggtg cggctggatc 1500
acct 1504

Claims (8)

1. A method for biosynthesis of ergothioneine comprises expressing glutamylcysteine synthetase, fe 2+ A recombinant bacterium of a dependent oxidase, a transglutaminase, a histidine methyltransferase and a pyridoxal phosphate dependent carbon-sulfur lyase for the synthesis of ergothioneine, wherein the recombinant bacterium is obtained by introducing a nucleic acid construct into a host cell, which is Actinoplanes sp HS with a accession number cctcccno: M2022390;
the nucleic acid construct comprises a nucleic acid sequence encoding a glutamylcysteine synthetase, fe 2+ Polynucleotide sequences of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases, wherein the glutamylcysteine synthetases, fe 2+ Dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases are from Actinoplanes sp.);
the glutamylcysteine synthetase has an amino acid sequence shown as a Seq ID No. 1;
The Fe is 2+ The dependent oxidase is the amino acid sequence shown in Seq ID No. 2;
the transglutaminase is an amino acid sequence shown in Seq ID No. 3;
the histidine methyltransferase is an amino acid sequence shown in Seq ID No. 4;
the pyridoxal phosphate dependent carbon-sulfur lyase is an amino acid sequence shown in Seq ID No. 5; or the amino acid sequence shown in Seq ID No. 6.
2. The method of claim 1, wherein,
the polynucleotide sequence encoding a glutamylcysteine synthetase has at least 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 7;
the code Fe 2+ The polynucleotide sequence of the dependent oxidase has at least 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence set forth in Seq ID No. 8;
the polynucleotide sequence encoding a transglutaminase has at least 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 9;
the polynucleotide sequence encoding histidine methyltransferase has at least 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 10;
the polynucleotide sequence encoding pyridoxal phosphate-dependent carbon-sulfur lyase has at least 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown as Seq ID No.11 or Seq ID No. 12.
3. The method according to claim 1 or 2, wherein the glutamylcysteine synthetase, fe, is encoded 2+ The polynucleotide sequences of the dependent oxidases, glutamine transferases, histidine methyltransferases and pyridoxal phosphate dependent carbon sulfur lyases are integrated into the genome of the host cell.
4. An actinomycete is Actinoplanes sp HS with a preservation number of CCTCC No. M2022390.
5. A first partRecombinant bacteria for biosynthesis of ergothioneine capable of expressing glutamylcysteine synthetase, fe 2+ Dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulphur lyase, wherein the recombinant bacterium is prepared by a method wherein the recombinant bacterium encodes a glutamylcysteine synthase, fe 2+ Integration of polynucleotide sequences of dependent oxidases, transglutaminases, histidine methyltransferases and pyridoxal phosphate dependent carbon-sulfur lyases into the genome of said host cell,
the glutamylcysteine synthetase has an amino acid sequence shown as a Seq ID No. 1;
the Fe is 2+ The dependent oxidase is the amino acid sequence shown in Seq ID No. 2;
The transglutaminase is an amino acid sequence shown in Seq ID No. 3;
the histidine methyltransferase is an amino acid sequence shown in Seq ID No. 4;
the pyridoxal phosphate dependent carbon-sulfur lyase is an amino acid sequence shown in Seq ID No. 5; or the amino acid sequence shown in Seq ID No. 6;
the host cell is Actinoplanes sp HS, the strain preservation number of which is CCTCC No. M2022390, and the Actinoplanes sp can express the glutamylcysteine synthetase and the Fe 2+ A dependent oxidase, said glutamine transferase, said histidine methyltransferase, and said pyridoxal phosphate dependent carbon sulfur lyase.
6. The recombinant bacterium according to claim 5, wherein,
the polynucleotide sequence encoding a glutamylcysteine synthetase has at least 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 7;
the code Fe 2+ The polynucleotide sequence of the dependent oxidase has at least 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence set forth in Seq ID No. 8;
the polynucleotide sequence encoding a transglutaminase has at least 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 9;
The polynucleotide sequence encoding histidine methyltransferase has at least 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence shown in Seq ID No. 10;
the polynucleotide sequence encoding pyridoxal phosphate-dependent carbon-sulfur lyase has at least 97%, 98%, 99% or 100% sequence identity with the nucleotide sequence shown as Seq ID No.11 or Seq ID No. 12.
7. Use of actinoplanes as claimed in claim 4 for the biosynthesis of ergothioneine.
8. Use of the recombinant bacterium of claim 5 or 6 in the biosynthesis of ergothioneine.
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