CN110551697B

CN110551697B - Application of ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus in synthesis of ergothioneine

Info

Publication number: CN110551697B
Application number: CN201910789954.8A
Authority: CN
Inventors: 林俊芳; 林金德; 郭丽琼; 陈春金; 梁思敏
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-08-26
Filing date: 2019-08-26
Publication date: 2021-02-19
Anticipated expiration: 2039-08-26
Also published as: CN110551697A

Abstract

The invention discloses the application of ergothioneine synthase PEGT1 and PEGT2 of Pleurotus edible fungus in synthesizing ergothioneine. The present invention starts from Pleurotus oyster mushroom, Bailing mushroom and Pleurotus eryngii, cloned and functionally identified the three kinds of Pleurotus edible mushroom ergothioneine synthesis-encoding related genes, and their nucleotide sequences are respectively as SEQ ID NO: As shown in 2, 4, 6, 8, 10, and 12, it was respectively connected with the pET-32a(+) expression vector and realized heterologous expression in Escherichia coli, and realized in vitro synthesis of ergothioneine. The present invention clarifies the biosynthetic pathway of ergothioneine of Pleurotus edible fungus for the first time, indicating that it has a more concise synthesis pathway, namely two genes Pegt1 and Pegt2 are involved in the synthesis of ergothioneine, and at the same time, the genetic engineering cell constructed by the present invention is safe It is stable and has a short production cycle, showing its great value in the application development of ergothioneine.

Description

Application of ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus in synthesis of ergothioneine

Technical Field

The invention belongs to the field of synthetic biology, and particularly relates to application of pleurotus edible fungus ergothioneine synthetases PEGT1 and PEGT2 in synthesis of ergothioneine.

Background

Ergothioneine (EGT for short) is a rare natural chiral amino acid strong antioxidant, is mainly synthesized in microorganisms such as bacteria and fungi, has unique biological function and pharmacological action, and has great application prospect in industries such as food, medicine and the like.

However, the following disadvantages exist in the background art:

(1) the biosynthesis yield is generally low and the synthesis process is complicated, and after the reported five genes EgtA, B, C, D and E from the Mycobacterium smegmatis and participating in the ergothioneine synthesis are expressed in a heterogeneous mode, the yield of the synthesized ergothioneine reaches 24mg/L, but the Mycobacterium smegmatis has certain biological hazard, and the ergothioneine biosynthesis process is long in period.

(2) The direct extraction with organisms as raw materials has high cost, low yield and limited sources.

(3) Because of the structural characteristics of ergothioneine, the chemical synthesis difficulty is high, and the synthesis raw materials are expensive.

(4) The ergothioneine obtained by directly utilizing the submerged fermentation of edible fungus mycelium is a main means for producing the ergothioneine in recent years, but the large-scale production of the ergothioneine is seriously limited by the problems of long production period, high production cost, low yield and the like.

(5) The literature reports show that the first EGT biosynthetic pathway in the fungus is analyzed in the crude Neurospora crassa EGT biosynthetic pathway in 2014, which provides a better research basis for the discovery of more fungus EGT biosynthesis related enzyme genes and the analysis of the EGT biosynthetic pathway. The edible fungi has a simpler ergothioneine biosynthesis pathway, and is a good source of an ergothioneine synthase gene as a good medicinal and edible fungi, and at present, no document report exists on an encoding gene of the ergothioneine synthase of pleurotus edible fungi.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of the ergothioneine synthetases PEGT1 and PEGT2 of pleurotus edible fungi in the synthesis of ergothioneine.

Another object of the present invention is to provide ergothioneine synthase PEGT1 and PEGT2 of Pleurotus ostreatus.

Another object of the present invention is to provide genes Pegt1 and Pegt2 encoding ergothioneine synthase PEGT1 and PEGT2 of the Pleurotus ostreatus.

The purpose of the invention is realized by the following technical scheme:

the invention provides application of ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus in synthesis of ergothioneine.

Preferably, the ergothioneine synthetase PEGT1 and PEGT2 of the Pleurotus ostreatus are applied to the in vitro combined biosynthesis of the ergothioneine.

Preferably, the pleurotus edible fungi is pleurotus ostreatus, pleurotus nebrodensis or pleurotus eryngii.

The amino acid sequence of the oyster mushroom ergothioneine synthase PEGT1(PBEGT1) is shown as SEQ ID NO: 1, and the nucleotide sequence of the coding gene (PBegt1) is shown as SEQ ID NO: 2 is shown in the specification;

the amino acid sequence of the oyster mushroom ergothioneine synthase PEGT2(PBEGT2) is shown as SEQ ID NO: 3, and the nucleotide sequence of the coding gene (PBegt2) is shown as SEQ ID NO: 4 is shown in the specification;

the amino acid sequence of the pleurotus nebrodensis ergothioneine synthase PEGT1(PtEGT1) is shown as SEQ ID NO: 5, the nucleotide sequence of the coding gene (Ptegt1) is shown as SEQ ID NO: 6 is shown in the specification;

the amino acid sequence of the pleurotus nebrodensis ergothioneine synthase PEGT2(PtEGT2) is shown as SEQ ID NO: 7, and the nucleotide sequence of the coding gene (Ptegt2) is shown as SEQ ID NO: 8 is shown in the specification;

the amino acid sequence of the pleurotus eryngii ergothioneine synthase PEGT1(PeEGT1) is shown as SEQ ID NO: 9, and the nucleotide sequence of the coding gene (Peegt1) is shown as SEQ ID NO: 10 is shown in the figure;

the amino acid sequence of the pleurotus eryngii ergothioneine synthase PEGT2(PeEGT2) is shown as SEQ ID NO: 11, and the nucleotide sequence of the coding gene (Peegt2) is shown as SEQ ID NO: 12 is shown in the specification;

SEQ ID NO: 1. 3, 5, 7, 9 and 11, and/or one or more amino acid residues are substituted and/or deleted and/or added, and the derived proteins with the same functions also belong to the protection scope of the invention.

SEQ ID NO: 2. 4, 6, 8, 10 and 12, and the nucleotide sequence of which one or more nucleotides are substituted and/or deleted and/or added and which expresses the same functional protein also belongs to the protection scope of the invention.

The invention also provides ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus, which are specifically (1) or (2):

(1) SEQ ID NO: 1. 3, 5, 7, 9 and 11;

(2) SEQ ID NO: 1. 3, 5, 7, 9 and 11, and/or one or more amino acid residues are substituted and/or deleted and/or added, and the functions of the derived proteins are the same.

The invention also provides genes encoding ergothioneine synthase PEGT1 and PEGT2 of Pleurotus ostreatus, which are (1) or (2):

(1) SEQ ID NO: 2. 4, 6, 8, 10 and 12;

(2) SEQ ID NO: 2. 4, 6, 8, 10 and 12, and expressing the same functional protein by substituting and/or deleting and/or adding one or more nucleotides;

the recombinant vector, the expression cassette and the recombinant bacterium containing the coding gene also belong to the protection scope of the invention.

The edible fungus ergothioneine synthase PEGT1 and PEGT2 of Pleurotus ostreatus or the coding gene thereof are applied to the preparation of recombinant vectors, expression cassettes and recombinant bacteria containing the edible fungus ergothioneine synthase coding gene of Pleurotus ostreatus.

The application of the ergothioneine synthetase PEGT1 and PEGT2 of the pleurotus edible fungi or the coding gene thereof in preparing the fermentation liquor containing the ergothioneine adopts the following steps: constructing a recombinant expression vector containing the coding gene, transforming the recombinant expression vector into escherichia coli BL21(DE3), carrying out induced expression on the obtained genetically engineered bacteria, washing and collecting thalli subjected to induced expression by PBS, finally mixing the engineering bacteria containing PEGT1 with the engineering bacteria containing PEGT2, dissolving and uniformly mixing the engineering bacteria with PBS, adding a reaction substrate, crushing the mixture, and reacting for 2 hours to obtain a fermentation liquid containing ergothioneine.

The invention also provides a specific primer pair for obtaining the cDNA sequences of the ergothioneine synthetase PEGT1 and PEGT2 encoding genes of the pleurotus edible fungi, and the primers are synthesized by Tianyihui gene technology company Limited in Guangzhou city.

The ergothioneine synthetase genes PEGT1 and PEGT2 of the pleurotus edible fungi provided by the invention are ergothioneine synthetase genes cloned from pleurotus edible fungi oyster mushrooms, pleurotus nebrodensis and pleurotus eryngii, and can be cloned in S-adenosylmethionine (SAM) and Fe²⁺Specifically catalyzing histidine (His) and cysteine (Cys) to form Ergothioneine (EGT) in the presence of pyridoxal phosphate (PLP), and particularly showing that the edible fungus ergothioneine synthetase encoding gene PEGT1 catalyzes histidine (His) and cysteine (Cys) to form histidine betaine cysteine sulfoxide (HerSul), and the edible fungus ergothioneine synthetase encoding gene PEGT2 converts the histidine betaine cysteine sulfoxide (HerSul) into Ergothioneine (EGT).

The discovery of the ergothioneine synthetase of the pleurotus edible fungi enriches the diversity of the ergothioneine synthetase, and can solve the problems of the source and the resource of the compound to a certain extent.

Compared with the prior art, the invention has the following advantages and effects:

the invention starts from pleurotus edible fungi oyster mushroom, pleurotus nebrodensis and pleurotus eryngii, clones and functionally identifies three pleurotus edible fungi ergothioneine synthesis coding related genes, and nucleotide sequences of the genes are respectively shown as SEQ ID NO: 2. 4, 6, 8, 10 and 12, respectively connected with pET-32a (+) expression vector and realizing heterologous expression in Escherichia coli, and realizing in vitro synthesis of ergothioneine. The ergothioneine has important application value, the invention firstly clarifies the ergothioneine biosynthesis route of pleurotus edible fungi, and shows that the ergothioneine biosynthesis route has a simpler synthesis route, namely, two genes Pegt1 and Pegt2 participate in the synthesis of the ergothioneine, and meanwhile, the gene engineering cell constructed by the invention is safe and stable, has short production period and shows the important value of the ergothioneine in the application and development of the ergothioneine.

Drawings

FIG. 1 is a PCR agarose gel electrophoresis of genes encoding ergothioneine synthase PEGT1 and PEGT2 of Pleurotus ostreatus, wherein a is an RNA electrophoresis of Pleurotus ostreatus, and lanes 1 and 2: oyster mushroom, lanes 3, 4: pleurotus nebrodensis, lanes 5, 6: pleurotus eryngii; b is an electrophoresis picture of the edible fungus ergothioneine synthetase encoding gene cDNA obtained by RT-PCR amplification, wherein a lane M: DNAMarker3, lane 1: PBegt1, lane 2: PBegt2, lane 3: ptegt1, lane 4: ptegt2, lane 5: peegt1, lane 6: peegt 2.

FIG. 2 is a schematic structural diagram of Escherichia coli expression plasmids pET-32a-PBegt1 and pET-32a-PBegt2 for expressing coding genes of Pleurotus ostreatus ergothioneine synthetase PBEGT1 and PBEGT 2.

FIG. 3 is the structure schematic diagram of the Escherichia coli expression plasmids pET-32a-Ptegt1 and pET-32a-Ptegt2 for expressing the encoding genes of the ergothioneine synthase PtEGT1 and PtEGT2 of Pleurotus nebrodensis.

FIG. 4 is a schematic structural diagram of Escherichia coli expression plasmids pET-32a-Peegt1 and pET-32a-Peegt2 for expressing coding genes of Pleurotus eryngii ergothioneine synthetase PeEGT1 and PeEGT 2.

FIG. 5 is SDS-PAGE of expression vectors of genes encoding ergothioneine synthase PEGT1 and PEGT2 of Pleurotus ostreatus, wherein lane 1: PBEGT1, lane 2: PBEGT2, lane 3: PtEGT1, lane 4: PtEGT2, lane 5: PeEGT1, lane 6: PeEGT 2.

FIG. 6 is an HPLC identification chart of a biosynthetic ergothioneine reaction product after the induction expression of an encoding gene PeEGT of the pleurotus eryngii ergothioneine synthetase.

FIG. 7 is HPLC identification chart of biosynthetic ergothioneine reaction products after the induction expression of coding genes of Pleurotus ostreatus, Pleurotus nebrodensis ergothioneine synthetase PBEGT and PtEGT.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

Example 1

Obtaining the cDNA sequences of the ergothioneine synthetase PEGT1 and PEGT2 coding genes of the pleurotus edible fungi and analyzing the biological informatics:

the reported amino acid sequences of Neurospora crassa ergothioneine synthetase Nc-EGT1(Protein ID: XP-956324.3) and Nc-EGT2(Protein ID: XP-001728131.1) were used (biological and biological transformations of C-S bond formation and cleavage enzymes in the rare Neurospora crassa ergothioneine pathway [ J ] biological pathway]Organic Letters,2014,16(20):5382-5385.) put in NCBI to perform homology search with oyster mushroom by using BLASTP, find two corresponding proteins similar to the oyster mushroom, thereby finding the cDNA sequence of the corresponding protein in the oyster mushroom, and respectively performing analysis and comparison with the genomes of Pleurotus nebrodensis and Pleurotus eryngii by using the two cDNA sequences of the oyster mushroom and BLASTN, thereby finding the corresponding encoding genes of Pleurotus nebrodensis, Erythroculture synthesis enzymes PEGT1 and PEGT2 of the Pleurotus ostreatus. PDB culture medium (potato is peeled by 200g/L, glucose is peeled by 20g/L, MgSO is used₄·7H₂O g/L，KH₂PO₄g/L, natural pH) is shaken to collect mycelium of edible fungi of pleurotus for RNA extraction (see figure 1a), RNA of the edible fungi of pleurotus with good quality is taken as a template, a cDNA chain is synthesized by adopting a method of TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit of the whole gold company, encoding genes of ergothioneine synthase PEGT1 and PEGT2 of the edible fungi of pleurotus are taken as primer design templates, and primer premier5 is used for designing primers to respectively amplify cDNA sequences of the encoding genes of the ergothioneine synthase PEGT1 and PEGT2 of the edible fungi of pleurotus (such as SEQ ID NO: 2. 4, 6, 8, 10, 12), the specific amplification primers are as shown in table 1 below (the sequencing results show that the amplified individual sequences have individual base mismatch with the primers designed by the original genome as the template, and the primers have been optimized according to the actually obtained sequencing sequences).

TABLE 1 primers for the cDNA amplification of the ergothioneine synthase PEGT1 and PEGT2 genes of Pleurotus ostreatus

Amino acid sequence alignment, protein three-dimensional structure homology simulation and structure rationality online analysis are carried out on coding gene cDNA of the ergothioneine synthetase PEGT1 and PEGT2 of the edible fungi of pleurotus which is obtained by amplification, and the ergothioneine synthetase PEGT1 of the edible fungi of pleurotus has the activity similar to sulfoxide synthase EgtB and histidine specificity methyltransferase EgtD. The ergothioneine synthetase PEGT2 of Pleurotus ostreatus has cysteine sulfoxide lyase activity similar to Nc-EGT2, and according to the analysis result, it can be preliminarily deduced that the ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus can synthesize ergothioneine.

Example 2

Constructing the expression vector of the ergothioneine synthetase PEGT1 and PEGT2 coding gene cDNA sequence of the pleurotus edible fungi:

(1) strains and plasmids

(2) The method comprises the following steps of collecting mycelium of pleurotus edible fungi by shaking the PDB culture medium to extract RNA, wherein the specific operation process of RNA extraction is as follows:

1) freshly collected fungal mycelia were placed in a liquid nitrogen precooled mortar without rnase, an appropriate amount of liquid nitrogen was added, the mycelia were rapidly ground to a powder with a rnase-free grinding pestle, and approximately 80mg of the powder was rapidly taken in a rnase-free 1.5mL centrifuge tube.

2) mu.L of extraction Buffer (200. mu.L of 10XSTE Buffer was pipetted (3.025 g Tris, 0.146g EDTA, 2.9g NaCl in a 50mL beaker, about 30mL ddH was added first) was immediately added₂Dissolving O, adjusting pH to 8.0 with hydrochloric acid, diluting to 50mL, autoclaving), and 100 μ L10% SDS (5g SDS dissolved in 50mL ddH)₂O) and 700 μ L sterile deionized water in a 2mL centrifuge tube, mixed well) and 600 μ L PCI (PCI is made from phenol: chloroform: isoamyl alcohol is mixed in a ratio of 25:24:1And) the tube was set in an oscillator and oscillated for 30 seconds.

3) Centrifuging at 14000rpm for 5min at 4 deg.C, sucking the supernatant with a pipette into another centrifuge tube, adding an equal volume of PCI, and shaking in a shaker for 20 s.

4) Centrifuge at 14000rpm for 5min at 4 deg.C, carefully pipette the supernatant into another centrifuge tube using a pipette gun, add 1/10 volumes of 3M NaAc (pH5.2) (24.6g anhydrous NaAc in a beaker, add about 40mL deionized water to stir and dissolve, adjust pH to 5.2 with glacial acetic acid, bring volume to 100mL with deionized water), and shake well.

5) Adding 2-2.5 times volume of anhydrous ethanol, placing in an oscillator, oscillating, mixing, and precipitating at-20 deg.C for about 40 min.

6) Centrifuging at 14000rpm for 15min at 4 deg.C, and discarding the supernatant. Adding 70% ethanol, washing the precipitate, centrifuging at 14000rpm at 4 deg.C for 5min, and removing ethanol.

7) The droplets were removed as much as possible with a pipette to remove the washing ethanol and dried in a vacuum desiccator for 5min or in a clean bench for 5 min.

8) With 10. mu.L of RNase-free ddH₂Dissolving the precipitate with O to obtain total RNA solution, and storing at-80 deg.C.

(3) The extracted high-quality edible fungus RNA of the pleurotus is used as a template, and a cDNA chain is synthesized by adopting a method of a TransScript One-Step gDNA Removal and cDNA Synthesis Supermix reverse transcription kit of the general formula gold company, and the specific operation is described in the specification.

(4) Taking the synthesized cDNA chain as a template, utilizing designed cDNA amplification primers (shown in table 1) of encoding genes of ergothioneine synthetase PEGT1 and PEGT2 of edible fungi of Pleurotus ostreatus, and adopting high fidelity enzyme KOD-Fx to carry out PCR amplification, wherein the amplification system is 25 mu L, and the reaction program is as follows: 2xPCR Buffer 12.5. mu.L, 2mM dNTPs 5. mu.L, Primer F0.75. mu.L, Primer R0.75. mu.L, Template 0.5. mu.L, KOD-Fx 0.5. mu.L, ddH₂Make up to 25. mu.L of O. The PCR procedure was: pre-denaturation at 94 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing of the Pegt1 primer at 57 ℃, extension at 30s and 68 ℃ for 2min34s and 30 cycles, annealing of the Pegt2 primer at 60 ℃, extension at 30s and 68 ℃ for 1min14s and 30 cycles, and 1% agarose after the end of the procedureThe size of the gel electrophoresis detection strip is shown in figure 1b, wherein M represents DNA Marker3, 1, 2, 3, 4, 5 and 6 represent PBegt1, PBegt2, Ptegt1, Ptegt2, Peegt1 and Peegt2 electrophoresis strips respectively, and the amplified strips are consistent with the expected strip sizes (about 2562bp and 1245bp of coding gene cDNA of the ergothioneine synthetase PEGT1 and PEGT2 of the edible fungi of the pleurotus) so as to preliminarily judge that the coding gene cDNA of the ergothioneine synthetase PEGT1 and the PEGT2 of the edible fungi of the pleurotus is successfully amplified.

(5) Construction of clone vector of ergothioneine synthase PEGT1 and PEGT2 coding gene pCloneEZ-TOPO of Pleurotus ostreatus

Respectively connecting the target bands amplified by the PCR with pCloneEZ-TOPO vectors, wherein the specific operation is described in the specification, the connecting products are transformed into escherichia coli DH5a, the escherichia coli DH5 is coated on an LB solid plate added with ampicillin (Amp:100 mu g/mL) for screening, the escherichia coli DH5 is cultured in a constant-temperature incubator at 37 ℃ overnight until a single colony grows out, the single colony is selected for colony PCR verification, and positive clones are selected for DNA sequencing verification.

(6) The pleurotus edible fungi PEGT1 and PEGT2 coding genes pCloneEZ-TOPO cloning vector plasmid and pET-32a (+) plasmid which are successfully constructed by shake fungus extraction are selected, EcoRI is selected as a restriction enzyme cutting site, and EcoRI restriction enzyme is adopted to carry out single enzyme cutting linearization on pET-32a (+) (reaction system: pET-32a (+)<5 μ g, 5 μ L of 10xFastDigest Buffer, 3 μ L of FastDigestenzyme, supplemented with ddH₂O to 50 μ L), reaction at 37 ℃ for 25 minutes and reaction at 85 ℃ for 5 minutes to inactivate the enzyme and recover and purify the product. CE Design V1.04 software is adopted to Design the edible fungus ergothioneine synthetase PEGT1 and PEGT2 coding gene cDNA amplification primers of Pleurotus with pET-32a (+) linearization homologous arm, and the specific primer sequences are shown in the following table 2. Taking the ergothioneine synthetase PEGT1 and PEGT2 encoding gene pCloneEZ-TOPO cloning vector plasmids of the edible fungi of the pleurotus as a template, adopting high fidelity enzyme PrimerSTAR Max Premix (2x) to carry out PCR amplification, wherein the PCR system is 25 mu L, and the reaction program is as follows: PrimerSTAR Max Premix (2X) 12.5. mu.L, Primer F0.75. mu.L, Primer R0.75. mu.L, Template 0.2. mu.L, ddH₂Make up to 25. mu.L of O. The PCR procedure was: denaturation at 98 deg.C for 10s, adding Pegt1 primer for annealing at 60 deg.C, 30s, extension at 68 deg.C for 2min35s,30 cycles, adding a homology arm Pegt2 primer for annealing at 60 ℃, 30s, extending at 68 ℃ for 1min for 15s, 30 cycles, detecting the size of a strip by using 1% agarose gel electrophoresis after the program is finished, and recovering and purifying the product. Adopting a Clonexpress II One Step Cloning Kit homologous recombination Kit to connect coding gene cDNA of ergothioneine synthetase PEGT1 and PEGT2 of pleurotus edible fungi with homologous arms to an EcoRI single-enzyme digestion linearized pET-32a (+) vector (the structural schematic diagram is shown in the figures 2, 3 and 4), and the specific operation is detailed in the specification; the ligation product was transformed into E.coli BL21(DE3), applied to LB (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, pH7.4, agar powder 20g/L (solids)) solid plates supplemented with ampicillin (Amp: 100. mu.g/mL) for screening, incubated overnight in a 37 ℃ incubator until single colonies grew, and single clones were picked for PCR-based assay, and positive clones were selected for DNA sequencing.

TABLE 2 edible fungus ergothioneine synthetase PEGT1 and PEGT2 encoding gene cDNA amplification primers with homologous brachial ears

Example 3

Protein induction expression analysis of ergothioneine synthase PEGT1 and PEGT2 of Pleurotus ostreatus:

the successfully constructed engineering bacteria liquid is inoculated into 50mL LB liquid culture medium according to the inoculation amount of 1% (v/v), cultured at 37 ℃ and 220rpm until the OD600nm is about 0.6-0.8, added with IPTG with the final concentration of 0.6mM, and induced to express for 16h at 20 ℃. PBS (8 g NaCl, 0.2g KCl, 1.42g Na weighed) at pH7.4 was used₂HPO₄、0.27gKH₂PO₄In a beaker, add about 800mL of deionized water to dissolve thoroughly, adjust pH to 7.4 with hydrochloric acid and add ddH₂O constant volume to 1L high pressure steam sterilization), washing the engineering strain after induction expression, taking a proper amount of thallus, dissolving the thallus in PBS, mixing with 2x protein sample Buffer (adding 0.5mL of 1M Tris-HCl with pH of 6.8 (weighing 121.1g of Tris, placing in a 1L beaker, adding about 800mL of deionized water, fully stirring and dissolving, adjusting pH to 6.8 with concentrated hydrochloric acid, constant volume to 1L, and high pressure steamSterilized, stored at room temperature), 0.2g SDS (sodium dodecyl sulfate), 10mg BPB (bromophenol blue), 1mL glycerol in a 10mL centrifuge tube, adding deionized water to a constant volume of 5mL, dispensing 500 μ L aliquots into the centrifuge tube, adding 10 μ L β -mercaptoethanol per aliquot before use) according to a ratio of 1: 1 proportion, mixing uniformly, cooling to room temperature after boiling water bath for 10 minutes, utilizing 7.5% SDS-PAGE (preparation of separation gel: 1.25mL of separation gel stock solution, 0.625mL of Tris-HCl buffer solution (pH 8.8), 0.05mL of 10% SDS, 0.025mL of 10% ammonium persulfate, 0.5mL of 1% TEMED and 2.55mL of deionized water, mixing uniformly, adding a 1mL gun head between two glass plates, the height of which is about 1cm from the lower edge of a sample mold comb, removing bubbles on the surface of the gel, adding 800 μ L of distilled water continuously to flatten the surface of the gel, isolating air, after 45 minutes, pouring off distilled water after the gel blocks are solidified, and sucking off excessive water by using absorbent paper. preparation of concentrated gel, taking 0.6mL of concentrated gel stock solution, 0.25mL of Tris-HCl buffer solution (pH 6.8), 0.02mL of 10% SDS, 0.01mL of 10% ammonium persulfate, 0.4mL of 1% TEMED, 0.92mL of deionized water, adding a 1mL gun head to the upper layer of the separation gel between the two glass plates, slightly inserting the sample die comb teeth, pulling out the comb teeth after the gel is solidified), and detecting whether the recombinant protein is correctly expressed. The results are shown in FIG. 5, wherein M represents PageRuler Prestained Protein Ladder, and 1, 2, 3, 4, 5, 6 represent PBEGT1, PBEGT2, PtEGT1, PtEGT2, PeEGT1, PeEGT2 recombinant Protein SDS-PAGE electrophoresis bands, and it can be seen from the figure that the expressed recombinant proteins are consistent with the expected band sizes (the Cladosporium Clavipitanum synthetase PEGT1, the Cladosporium 2 recombinant proteins are respectively about 114.7kDa and 64.1kDa), which indicates that the constructed expression vector can effectively express Cladosporium Clavipitanum synthetase PEGT1 and PEGT2 in the form of recombinant proteins.

Example 4

In-vitro enzyme activity test analysis of ergothioneine synthetase PEGT1 and PEGT2 of pleurotus edible fungi:

1) the successfully constructed engineering bacteria liquid and a control strain (containing no target gene and plasmid pET-32a (+)) are inoculated into 50mL LB liquid culture medium according to the inoculation amount of 1% (v/v), cultured at 37 ℃ and 220rpm until OD600nm is about 0.6-0.8, added with IPTG with the final concentration of 0.6mM, and induced to express for 16h at 20 ℃.

2) Adding 30 mu L IPTG, controlling the final concentration of IPTG at 0.6mM, and carrying out induced expression for 16h at 20 ℃;

3) the collected cells were washed separately with PBS ph7.4, and finally PEGT1 and PEGT2 engineered strains derived from the same species were mixed and dissolved in 10mL PBS, and reaction substrates were added to control the final concentrations: 20mM His, 31 mg; 20mM Cys, 24.3 mg; 5mM SAM, 20 mg; 10mM Fe²⁺，20mg；5mM PLP，12.4mg；

4) Mixing, ultrasonically crushing bacteria, wherein the crushing condition is as follows: 33% power, about 250W; the total ultrasonic time is 6min, the work time is 3s, the interval is 3s, and the alarm temperature is 40 ℃;

5) after the ultrasonic treatment is finished, the reaction system is placed at 36 ℃ for reaction for 2 hours, and the reaction system is uniformly mixed every 20 minutes;

6) the reaction product is filtered by a 0.22 mu m water system filter membrane and then used for HPLC detection, wherein the liquid phase detection conditions adopted by the in vitro enzyme activity test of the oyster mushroom and pleurotus nebrodensis ergothioneine synthetase PBEGT and PtEGT are as follows: the detection wavelength is 257nm, the flow rate is 1mL/min, the mobile phase (acetonitrile: 20mM, pH6.0 ammonium acetate: 85:15) and the single sample analysis time is 60min, and the liquid phase detection conditions are adopted in the in vitro enzyme activity test of the pleurotus eryngii ergothioneine synthase PeEGT: detection wavelength 257nm, flow rate 1mL/min, mobile phase (acetonitrile: water 80:20), single sample analysis time 60 min.

The results are shown in FIGS. 6 and 7, where in FIG. 6 the PeEGT1 and PeEGT2 enzymatic reaction products peaked at 41.700min, which is essentially identical to the peak time of 41.614min for the EGT standard. In FIG. 7, the peak times of the enzymatic reaction products of PBEGT1 and PBEGT2 and the enzymatic reaction products of PtEGT1 and PtEGT2 were 46.482min and 46.459min, respectively, and also substantially coincided with the peak time 46.863 of the EGT standard. In both FIG. 6 and FIG. 7, no product was detected in the blank control group at the peak time of the EGT standard, and it can be seen that the clonally expressed Pleurotus eryngii thionin synthetases PEGT1 and PEGT2 are capable of co-biosynthesizing ergothioneine in vitro.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> application of ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus in synthesis of ergothioneine

<160> 36

<170> SIPOSequenceListing 1.0

<211> 853

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Ile Gln Ile Val Asp Val Gln Thr Leu Asn Gln Ser Arg His

1 5 10 15

Leu Asp Leu Gln Ser Asp Leu Ser Gln Gln Leu Val Asp Gly Leu Thr

20 25 30

Arg Pro His Gly Glu Lys Glu Ile Pro Thr Val Leu Leu Tyr Asp Glu

35 40 45

Arg Gly Leu Arg Leu Tyr Asp Ala Ile Thr Thr Glu Val Ser Glu Tyr

50 55 60

Tyr Leu Phe Gly Ala Glu Glu Glu Ile Leu Lys Asn Lys Ala Asp Glu

65 70 75 80

Ile Val Arg Thr Met His Ser Gly Leu Gly Asp Gly Asp Pro Asp Ser

85 90 95

Glu Val Val Leu Glu Leu Gly Ala Gly Ser Leu Arg Lys Thr Ser His

100 105 110

Ile Leu Ala Gly Leu Ser Arg Ile Val Pro Ser Gln Cys Asp Thr Ala

115 120 125

Pro Ile Thr Tyr Tyr Ala Leu Asp Leu Glu Glu Arg Glu Leu Gln Arg

130 135 140

Val Leu Asp Ser Ile Ser Asn Ser Ser Val Gly Glu Met Leu Arg Gly

145 150 155 160

Lys Val Glu Thr Lys Gly Leu Trp Gly Thr Tyr Glu Asp Gly Leu Lys

165 170 175

Phe Val Glu Asp Glu Gly Ser Pro Ile Ser Ser His Ser Asn Ser Pro

180 185 190

Thr Ser Gln Ser Ser Arg Arg Glu Leu Gly Pro Pro Ser Pro Thr Pro

195 200 205

Arg Ser Gly Ser Ser Ser Pro Leu His Ile Leu Phe Leu Gly Ser Ser

210 215 220

Leu Gly Asn Phe Asp Arg Lys Asp Ser Val Lys Phe Leu Gln Ser Met

225 230 235 240

Pro Leu Arg Pro Gly Ser Gly Asp Thr Leu Leu Ile Gly Leu Asp His

245 250 255

Asp Asn Asp Lys Thr Leu Ile Glu Glu Ala Tyr Asn Asp Arg Lys Gly

260 265 270

Tyr Thr Lys Thr Phe Ile Met Asn Gly Leu Arg Ala Ala Gly Arg Ala

275 280 285

Leu Gly Asn Glu Asp Met Phe Glu Glu Asp Lys Trp Glu Tyr Val Asn

290 295 300

Arg Tyr Asn Glu Ala Glu Arg Arg His Glu Ala Tyr Tyr Lys Ser Lys

305 310 315 320

Cys Pro Gln Lys Leu Glu Asp Pro Lys Ala Glu His Gly Tyr Glu Phe

325 330 335

Leu Glu Asp Glu Leu Val Lys Ile Glu Val Ser Tyr Lys Tyr Ser Glu

340 345 350

Thr Asp Ala Tyr Ser Leu Phe Thr Asp Ser Gly Leu Arg Pro Ile Gln

355 360 365

Arg Trp Thr Asp Ser Ala Thr Gln Tyr Ser Leu Trp Leu Leu Glu Arg

370 375 380

Pro Pro Phe Met Phe Pro Leu Leu Lys Ser Pro Ile Ala Phe Asn Gly

385 390 395 400

Leu Gly Glu Ile Val Lys Asn Phe Pro Leu Ser Asn Thr Pro Phe Gly

405 410 415

Val Pro Ser Pro Gln Glu Trp Ser Asn Leu Trp Ala Ala Trp Asp Phe

420 425 430

Val Thr Leu Lys Met Ile Pro Pro Ser Met Leu His Val Lys Pro Ile

435 440 445

His Leu Arg His Ile Cys Leu Phe Tyr Leu Gly His Ile Pro Thr Phe

450 455 460

Leu Asp Ile His Leu Ser Arg Leu Leu Lys Glu Pro His Thr Glu Pro

465 470 475 480

Glu Ser Phe Lys Tyr Ile Phe Glu Arg Gly Ile Asp Pro Asn Val Asp

485 490 495

Asn Pro Ala Glu Cys His Pro His Ser Glu Val Pro Gln Asn Glu Gly

500 505 510

Asp Trp Pro Ser Leu Glu Ala Ile Leu Ser Phe Gln Ala Gln Val Arg

515 520 525

Ser Arg Leu Leu His Leu Tyr Asp Asp Val Leu Thr Gly Lys Lys Val

530 535 540

Leu Thr Arg Lys Val Cys Arg Ile Leu Ser Met Thr Phe Glu His Glu

545 550 555 560

Ala Phe His Ile Glu Thr Leu Leu Tyr Met Leu Leu Gln Ala Ala Gly

565 570 575

Asn Gly Thr Thr Pro Pro Pro Gly Phe Thr Pro Pro Pro Trp Ser Ser

580 585 590

Leu Ser Ala Asn Trp Asn Ala Leu Pro Ala Leu Glu Asn Thr Thr Val

595 600 605

Thr Leu Gly Pro Glu Ile Val Ala Leu Gly His Asp Asp Asp Glu Arg

610 615 620

Glu Asp Glu Asp Pro Ala Phe Ile Thr Asp Val Lys Asp His Glu Phe

625 630 635 640

Ala Trp Asp Asn Glu Asn Pro Lys Arg Gln Val Glu Val Lys Gln Phe

645 650 655

Lys Ile Glu Trp Arg Pro Val Thr Asn Gly Gln Phe Tyr Glu Phe Tyr

660 665 670

Lys Glu Tyr Lys Asp Asp Met Lys Leu Gln Leu Pro Ala Ser Trp Leu

675 680 685

Glu Thr Asp Asp Lys Met Met Val Arg Thr Leu Tyr Gly Pro Val Pro

690 695 700

Met Lys Val Ala Gln Asn Trp Pro Val Val Ile Ser Tyr Asp Gly Leu

705 710 715 720

Ser Ala Tyr Ala Ile Val Lys Gly Gly Arg Leu Pro Thr Glu Pro Glu

725 730 735

Leu Arg Leu Phe Leu Asp Lys Phe Glu Ser Gly Tyr Glu Gly Gly Ala

740 745 750

Asn Val Gly Phe Arg Asn Trp His Pro Val Pro Ala Thr Thr Gly Gly

755 760 765

Lys Lys Tyr Gly Gly Lys Gly His Asn Gly Gly Val Trp Glu Trp Thr

770 775 780

Ser Thr Thr Leu Thr Lys His Asp Gly Phe Glu Pro Ser Lys His Tyr

785 790 795 800

Pro Gly Phe Thr Ala Asp Phe Phe Asp Gly Cys His Asn Val Ile Leu

805 810 815

Gly Gly Ser Tyr Ala Phe Ile Pro Arg Met Ala Glu Arg Arg Ser Leu

820 825 830

Arg Asn Trp Tyr Gln Arg Asn Tyr Pro Tyr Ala Trp Thr Gly Gly Arg

835 840 845

Ile Val Tyr Asp Ile

850

<211> 2562

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggccattc aaatcgtcga cgtccagacg ctcaatcagt ccagacattt ggatctgcag 60

agcgacctct cccagcagct tgttgacggc ttaactcggc cgcatggcga aaaggaaatc 120

cccaccgtgc tcttatacga cgaacgaggg ttaagactct acgacgccat cacgaccgaa 180

gtctcggaat actacctctt tggcgccgag gaggaaatcc tcaagaataa agccgatgag 240

attgttcgga cgatgcacag tggattgggg gacggggatc cagactcgga ggtcgtcctc 300

gaattaggcg ccggttctct aaggaagacc tcccacattc tcgcagggct ttcacgcatc 360

gttcctagcc agtgcgatac agcgccgatc acctactacg ccctcgatct tgaagaacgc 420

gaacttcagc gagtactaga ctccatatca aactcaagcg ttggagagat gttgcgagga 480

aaagtagaaa cgaagggcct ctggggcact tacgaagacg ggctgaagtt tgtagaagac 540

gagggttctc ccatcagcag ccattcaaat tcccctacta gccaatcttc gagaagagag 600

ttggggccgc catcaccgac ccccagatcg ggatcgagtt ccccactgca tattctattc 660

ttaggttcgt cgttggggaa ctttgaccgc aaggatagcg tcaagttctt gcagtccatg 720

ccccttcgcc ctggctcagg cgatacgcta ctaattggtc tcgatcacga taacgataag 780

acgttgatcg aagaggcgta caatgaccgc aaaggatata cgaagacgtt catcatgaac 840

gggttgagag cggctggacg tgcgctagga aacgaggata tgtttgaaga agacaaatgg 900

gaatatgtta atagatataa tgaggcggag cgtcggcatg aggcatatta caagtccaag 960

tgtccccaga aactcgaaga tccgaaggcg gaacatggct acgagttcct tgaagatgaa 1020

ctagtcaaaa tcgaggtctc ctataagtac tcggaaaccg atgcctatag cctattcact 1080

gactcgggcc tccgccctat ccaacgttgg acagacagtg ccacccagta ctccctttgg 1140

ctactcgagc gtcccccctt tatgttcccc cttttgaagt ccccaatcgc cttcaatggc 1200

ctcggcgaga tcgtcaaaaa ctttcccctc tccaacacgc ccttcggcgt tccttcaccc 1260

caagaatggt caaacctgtg ggccgcttgg gacttcgtga ccttgaagat gattccaccc 1320

tcgatgctcc acgtgaagcc tatccacctg aggcacatat gcctatttta tctgggccat 1380

atcccgacgt tcttggacat ccatttgagt cgcttgctga aagaaccgca cacggagcct 1440

gaaagtttca agtatatctt tgagcgaggg attgatccta atgtggacaa cccggccgaa 1500

tgtcatcccc attcagaggt tccgcagaat gaaggcgatt ggccttcgct tgaagctata 1560

ttatcctttc aagcccaagt ccgctcaagg cttttgcatc tctacgacga tgtcttgaca 1620

ggaaagaagg tgttgacgcg aaaggtctgt cgcatattgt caatgacttt cgaacacgag 1680

gctttccaca ttgagacgtt gctctacatg cttttgcaag ccgccggaaa cgggactacc 1740

cctcccccag gattcacacc tccaccctgg agctcgcttt ccgcaaactg gaacgctctc 1800

ccagcgttgg aaaacacgac agttactctg ggtccggaaa tcgtagcatt aggccacgac 1860

gacgatgaaa gggaggatga agaccctgcg ttcatcactg acgtgaaaga tcacgaattt 1920

gcatgggata atgagaatcc caagcgtcag gtcgaagtca agcagttcaa gattgagtgg 1980

aggccggtca cgaatggcca gttctacgaa ttctacaaag agtataagga cgacatgaag 2040

ctccaattac cggccagttg gttggagaca gatgataaga tgatggttcg caccctgtac 2100

ggccctgtac ctatgaaagt ggcgcagaac tggccggtcg tcatttcgta cgatggcttg 2160

tctgcctatg cgatcgttaa aggcggaagg cttccaaccg agcctgagct tcgtctcttc 2220

ctagataaat ttgagagcgg ctacgaaggc ggcgcaaatg ttgggttccg taactggcat 2280

cccgttcccg caacgaccgg cgggaagaag tacggtggaa aaggtcacaa cggcggcgtc 2340

tgggaatgga cttcaactac gctcacgaaa cacgatggat tcgaaccatc taagcactac 2400

ccaggattca cggcggattt ctttgacgga tgtcacaacg tgattttggg cgggtcatac 2460

gcattcatcc ctcgcatggc tgaaaggcgc agtttgcgca actggtacca gaggaactac 2520

ccgtatgctt ggacgggagg gcgaattgtt tacgatattt ga 2562

<211> 414

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Leu Lys Tyr Phe Ser Phe Glu Pro Gly Tyr Ile Asn Leu Asn Asn

1 5 10 15

Gly Ser Phe Gly Ser Leu Pro Lys Pro Val Ala Glu Phe Cys Val Gln

20 25 30

His Ala Ala Arg Val Glu Ala Asn Pro Asp Arg Tyr His Arg Leu Glu

35 40 45

Phe Ala Glu Leu Leu Thr Ala Ala Arg Ala Arg Val Ala Arg Leu Ile

50 55 60

Gly Ala Asp Thr Asp Glu Cys Val Phe Val Pro Ser Thr Thr Ala Gly

65 70 75 80

Ile Asn Thr Ile Leu Arg Asn Ile Glu Trp Ser Ser Gly Asp Val Ile

85 90 95

Ile Arg Phe Ser Thr Thr Tyr Gln Gly Val Glu Arg Val Ala Arg Tyr

100 105 110

Ile Ala Asp Thr His Ser Glu Val Thr Leu Ser Thr Val Glu Leu Thr

115 120 125

Phe Pro Ile Ser His Gln Glu Ile Val Lys Arg Phe Arg Glu His Val

130 135 140

Val Gly Val Lys Ala Ser Cys His Ser Ser Ser Arg Val Tyr Ala Ile

145 150 155 160

Ile Asp Ser Val Val Ser His Pro Gly Val Ala Leu Pro Trp Lys Gln

165 170 175

Met Thr Gln Ile Cys Arg Glu Glu Gly Val Cys Ser Ile Ile Asp Ala

180 185 190

Ala His Ser Ile Gly Gln Glu Met Asn Ile Asp Leu Ala Ser Ala Gln

195 200 205

Pro Asp Phe Trp Val Ser Asn Cys His Lys Trp Leu Phe Ala Lys Arg

210 215 220

Gly Ser Thr Val Leu Tyr Val Pro Lys Arg Asn Gln His Ile Ile Lys

225 230 235 240

Ser Ala Leu Val Thr Ser Trp Asp Tyr Pro Ser Pro Lys Glu Ser Ala

245 250 255

Pro Ala Ala Tyr Asp Thr Gly Phe Val Leu Gln His Glu Trp Pro Gly

260 265 270

Ala Ile Asp Phe Ser Pro Tyr Leu Ser Val Cys Ala Ala Met Asp Phe

275 280 285

Arg Glu Lys Ile Gly Gly Glu Gln Ala Ile Asn Asp Tyr Cys His Lys

290 295 300

Leu Ala Leu Glu Gly Gly Arg Lys Leu Ala Ser Ala Leu Gly Gly Cys

305 310 315 320

Val Leu Asp Glu Ser Pro Ser Ala Glu Leu Thr Leu Asn Met Val Asn

325 330 335

Val Gln Leu Pro Leu Pro Pro Thr Thr Ser Lys Glu Leu Gln Gly Lys

340 345 350

Ile Asp Ser Phe Leu Glu Tyr Glu Leu Leu His Lys His Asn Val Ser

355 360 365

Ser Val His Phe Tyr His Gly Gly Ser Trp Trp Val Arg Cys Ser Ala

370 375 380

Gln Ile Trp Asn Glu Val Ser Asp Phe Glu Tyr Leu Gly Lys Ala Leu

385 390 395 400

Ser Asn Ala Cys Gln Glu Ala Arg Ser Lys Phe Phe Ala Asp

405 410

<211> 1245

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgctaaagt acttttcatt tgagcccgga tacatcaacc tcaacaatgg ttcatttggg 60

tccctcccca agccggtggc tgagttttgt gtgcaacatg ctgccagggt tgaagcgaat 120

ccggacagat atcatcgact cgagtttgcg gagctcctca cagctgcgcg cgctcgtgtt 180

gccagactga tcggagcaga cacggacgaa tgcgtgtttg ttccgagcac cactgcaggc 240

atcaacacaa tattacggaa cattgaatgg agttcaggcg atgtaatcat tagattctca 300

acaacttatc aaggggtgga acgggttgct agatacatag ccgacactca ttccgaagtg 360

acgctatcaa ccgtggagtt gacatttccc atctctcatc aagagattgt aaaacgcttc 420

cgagaacacg ttgttggggt caaggcaagc tgtcacagct catcgagggt ctacgccata 480

atcgactcgg tcgtatcgca cccgggtgtt gctttgccct ggaaacagat gacccaaatc 540

tgtcgcgaag agggcgtctg cagcattatt gacgcagccc actccattgg ccaggagatg 600

aacatagatc tggcatctgc gcagccagac ttttgggtat cgaactgcca caaatggctt 660

ttcgccaaac gtggttctac cgttttatat gtgccgaagc ggaaccaaca catcatcaag 720

tctgcgctgg taacatcctg ggactatccc tcccctaagg agtctgctcc tgctgcgtac 780

gatactggtt tcgttctcca acatgaatgg ccgggggcga tcgacttttc gccttacctg 840

agtgtctgtg ctgcgatgga cttccgcgag aagatcggtg gcgaacaggc gatcaacgac 900

tattgccaca agctggcact ggagggcggc cggaaattag cttctgctct tggaggttgt 960

gtcctcgacg agtcaccttc tgcggaattg acactcaaca tggtgaatgt acaactaccg 1020

cttcctccga caacatcgaa agagctccag ggaaagatcg atagctttct ggaatacgag 1080

ctgttacata aacacaacgt atcctccgtg cacttctacc acggtggttc gtggtgggtg 1140

cgctgcagtg ctcaaatttg gaacgaggtt tctgacttcg agtaccttgg caaagctctg 1200

agcaacgcgt gtcaagaggc tagatcgaag ttcttcgcgg actag 1245

<211> 855

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Ala Ile Gln Ile Val Asp Ala Gln Thr Leu Asn Gln Ser Arg His

1 5 10 15

Leu Asp Leu Gln Ser Asp Leu Ser Gln Gln Leu Val Asp Gly Leu Thr

20 25 30

Arg Pro His Gly Glu Lys Glu Ile Pro Thr Val Leu Leu Tyr Asp Glu

35 40 45

Pro Gly Leu Arg Leu Tyr Asp Ala Ile Thr Thr Glu Val Ser Glu Tyr

50 55 60

Tyr Leu Phe Gly Ala Glu Glu Glu Ile Leu Lys Asn Lys Ala Asp Glu

65 70 75 80

Ile Val Arg Thr Met His Ser His Asn Gly Leu Gly Asp Gly Val Leu

85 90 95

Asp Ser Glu Val Val Leu Glu Leu Gly Ala Gly Ser Leu Arg Lys Thr

100 105 110

Ser His Ile Leu Ala Gly Leu Ser Arg Ile Val Pro Ser Glu Cys Asp

115 120 125

Thr Ala Pro Ile Thr Tyr Tyr Ala Leu Asp Leu Glu Glu Arg Glu Leu

130 135 140

Gln Arg Val Leu Asp Ser Ile Ser Asn Ser Ser Val Gly Glu Met Leu

145 150 155 160

Arg Gly Lys Val Glu Thr Lys Gly Leu Trp Gly Thr Tyr Glu Asp Gly

165 170 175

Leu Lys Phe Val Glu Asp Glu Gly Ser Pro Leu Ser Ser His Ser Asn

180 185 190

Ser Pro Thr Ser Gln Ser Ser Gly Arg Glu Leu Thr Pro Pro Ser Pro

195 200 205

Thr Pro Arg Ser Gly Ser Ser Ser Pro Leu His Ile Leu Phe Leu Gly

210 215 220

Ser Ser Leu Gly Asn Phe Gly Arg Lys Asp Ser Val Lys Phe Leu Gln

225 230 235 240

Ser Met Pro Leu Arg Pro Gly Ser Gly Asp Thr Leu Leu Ile Gly Leu

245 250 255

Asp His Asp Asn Asp Lys Thr Leu Ile Glu Glu Ala Tyr Asn Asp Arg

260 265 270

Lys Gly Tyr Thr Lys Thr Phe Ile Met Asn Gly Leu Arg Ala Ala Gly

275 280 285

Arg Ala Leu Gly Asn Glu Asp Met Phe Glu Glu Asp Lys Trp Glu Tyr

290 295 300

Val Asn Arg Tyr Asn Glu Ala Glu Arg Arg His Glu Ala Tyr Tyr Lys

305 310 315 320

Ser Lys Cys Pro Gln Lys Leu Glu Asp Pro Lys Ala Glu His Gly Tyr

325 330 335

Glu Phe Leu Glu Asp Glu Leu Val Lys Ile Glu Val Ser Tyr Lys Tyr

340 345 350

Ser Glu Thr Asp Ala Tyr Ser Leu Phe Thr Asp Ser Gly Leu Arg Pro

355 360 365

Ile Gln Arg Trp Thr Asp Ser Ala Thr Gln Tyr Ser Leu Trp Leu Leu

370 375 380

Glu Arg Pro Pro Phe Met Phe Pro Leu Leu Lys Ser Pro Ile Ala Phe

385 390 395 400

Asn Gly Leu Gly Glu Ile Val Lys Asn Ser Pro Leu Ser Asn Ser Pro

405 410 415

Phe Gly Val Pro Ser Pro Gln Glu Trp Ser Asn Leu Trp Ala Ala Trp

420 425 430

Asp Phe Val Thr Leu Lys Met Ile Pro Pro Ser Met Leu His Val Lys

435 440 445

Pro Ile His Leu Arg His Ile Cys Leu Phe Tyr Leu Gly His Ile Pro

450 455 460

Thr Phe Leu Asp Ile His Leu Ser Arg Leu Leu Lys Glu Pro His Thr

465 470 475 480

Glu Pro Glu Ser Phe Lys Tyr Ile Phe Glu Arg Gly Ile Asp Pro Asn

485 490 495

Val Asp Asn Pro Ala Glu Cys His Pro His Ser Glu Val Pro Gln Asn

500 505 510

Glu Gly Asp Trp Pro Ser Leu Glu Gly Ile Leu Ser Phe Gln Ala Arg

515 520 525

Val Arg Ser Arg Leu Leu Thr Leu Tyr Asp Asp Val Leu Thr Gly Lys

530 535 540

Lys Val Leu Thr Arg Lys Val Cys Arg Ile Leu Ser Met Thr Phe Glu

545 550 555 560

His Glu Ala Phe His Ile Glu Thr Leu Leu Tyr Met Leu Met Gln Val

565 570 575

Ala Gly Asn Gly Thr Ile Pro Pro Pro Gly Phe Thr Pro Pro Pro Trp

580 585 590

Ser Ser Leu Ser Ala Asn Trp Asn Ala Leu Pro Ala Leu Glu Asn Thr

595 600 605

Thr Val Thr Leu Gly Pro Glu Ile Val Ser Val Gly His Asp Asp Asp

610 615 620

Glu Arg Glu Asp Glu Asp Pro Ala Phe Ala Thr Asp Val Lys Asp Tyr

625 630 635 640

Glu Phe Ala Trp Asp Asn Glu Asn Pro Glu Arg Gln Val Glu Val Lys

645 650 655

Gln Phe Arg Ile Glu Trp Arg Pro Val Thr Asn Gly Gln Phe Tyr Glu

660 665 670

Phe Tyr Lys Ala Tyr Lys Asp Asp Leu Lys Leu Gln Leu Pro Ala Ser

675 680 685

Trp Leu Glu Thr Asp Asp Thr Met Met Val Arg Thr Leu His Gly Pro

690 695 700

Val Pro Met Lys Val Ala Gln Asn Trp Pro Val Val Ile Ser Tyr Asp

705 710 715 720

Gly Leu Ser Ala Tyr Ala Ile Val Lys Gly Gly Arg Leu Pro Thr Glu

725 730 735

Pro Glu Leu Arg Leu Phe Leu Asp Lys Phe Glu Ser Gly Tyr Glu Gly

740 745 750

Gly Ala Asn Val Gly Phe Arg Asn Trp His Pro Val Pro Ala Thr Thr

755 760 765

Gly Gly Lys Lys Tyr Gly Gly Lys Gly His Asn Gly Gly Val Trp Glu

770 775 780

Trp Thr Ser Thr Thr Leu Thr Lys His Asp Gly Phe Glu Pro Ser Lys

785 790 795 800

His Tyr Pro Gly Phe Thr Ala Asp Phe Phe Asp Gly Cys His Asn Val

805 810 815

Ile Leu Gly Gly Ser Tyr Ala Phe Ile Pro Arg Met Ala Glu Arg Arg

820 825 830

Ser Leu Arg Asn Trp Tyr Gln Arg Asn Tyr Pro Tyr Ala Trp Thr Gly

835 840 845

Gly Arg Ile Val Tyr Asp Val

850 855

<211> 2568

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggcaattc aaatcgtcga cgcccagacg ctcaatcagt ccaggcattt ggatctccag 60

agcgacctct cccagcaact tgttgacggc ttaactcggc cgcatggcga aaaggaaatc 120

cctaccgtgc tcctatatga cgaaccaggg ttaagattgt acgacgccat cacgactgag 180

gtctcagaat actatctttt tggcgccgag gaggaaatcc tcaagaataa agccgatgaa 240

attgttcgga cgatgcacag tcacaatgga ttgggggacg gggttctaga ctcagaagtc 300

gtcctcgaat taggcgccgg ttccctgagg aagacgtccc acatcctcgc agggctttca 360

cgcatcgttc ctagcgagtg cgatacagcg ccgatcacct attacgccct cgatctcgaa 420

gaacgcgaac ttcagcgagt gctggactcc atatcaaact caagcgtcgg agagatgttg 480

cgaggaaaag tcgaaacgaa gggcctgtgg ggaacctacg aagatgggct gaagttcgta 540

gaagacgagg gttctcctct cagcagccat tcgaattccc ctactagtca atcatcggga 600

agagagttga cgccgccatc accgaccccg agatcaggat cgagttcccc actgcacatt 660

ctattcttag gatcgtcgtt ggggaacttt ggccgcaagg acagcgtcaa gttcttgcag 720

tctatgcccc ttcgccccgg ctcaggcgat acgctgttaa ttggtctcga tcacgataac 780

gataaaacgc tgatcgaaga ggcgtacaat gaccgcaaag gatatacgaa gacgttcatc 840

atgaacgggt tgagagcggc tggacgcgca ttaggaaacg aggatatgtt tgaagaagac 900

aaatgggaat atgtcaacag atataatgag gcggagcgtc gacatgaggc gtactacaag 960

tccaagtgtc cgcaaaaact cgaagatccg aaggcggaac atggctacga gttccttgaa 1020

gatgaactag tcaaaatcga ggtctcctat aagtactcgg aaaccgatgc ctatagccta 1080

ttcaccgact cgggcctccg ccctatccaa cgttggacag acagtgccac ccagtactcc 1140

ctttggctac tcgagcgtcc cccctttatg ttcccccttc tgaagtcccc aatcgccttc 1200

aatggtctcg gcgagatcgt caaaaactcg ccactctcca actcgccctt cggcgttcct 1260

tcgccccaag aatggtcaaa tctgtgggcc gcttgggact tcgttacctt gaagatgatc 1320

ccaccctcga tgctccacgt gaagcctatc cacctgaggc acatatgcct gttttatttg 1380

ggccatattc cgacgttctt ggacatccat ttgagtcgct tgctgaaaga accgcacacc 1440

gaacccgaaa gtttcaagta tatcttcgag cgagggattg atcccaatgt ggacaacccg 1500

gccgaatgcc atcctcattc agaggttccg cagaatgaag gcgattggcc ttcgcttgaa 1560

ggcatattat ctttccaagc ccgagtccgc tcaaggcttt tgactctcta tgacgatgtc 1620

ttgacaggaa agaaggtgtt gacgcgaaag gtctgtcgca tcttatcaat gactttcgaa 1680

cacgaggctt tccacattga gacgctgctc tacatgctta tgcaagtcgc cggaaatggg 1740

accatccctc cgccaggatt cacacctcca ccatggagct cactttccgc aaactggaac 1800

gctcttccag cgttggaaaa tacgacagtt actctcggtc cggaaatcgt atcagtgggc 1860

cacgacgacg atgaaaggga ggatgaagac cctgcgtttg ccactgacgt gaaagattac 1920

gaatttgcat gggacaatga gaatcccgag cggcaggtcg aagttaagca gttcaggatt 1980

gagtggaggc cggtcacgaa tggccagttc tacgaattct acaaggcgta taaggacgac 2040

ctgaagctcc aattaccagc cagctggttg gagacagatg atacaatgat ggttcgcacc 2100

ctgcacggcc ctgtacctat gaaagtggct cagaactggc ccgtcgtcat ttcgtacgat 2160

ggcttgtctg cctatgcgat cgtcaaaggc ggaaggcttc cgacggagcc tgagcttcgt 2220

ctcttcctag acaagtttga gagcggctac gaaggaggcg caaatgttgg attccgtaac 2280

tggcatcccg ttcccgcaac gaccggcggg aagaagtacg gtggcaaagg tcacaacggc 2340

ggcgtttggg aatggacttc gactacactc acgaaacacg atggattcga accatccaag 2400

cactaccctg gattcacggc cgatttcttc gacggatgtc acaatgtgat cttgggaggg 2460

tcatatgctt tcatccctcg catggctgaa agacgcagtt tgcgcaactg gtaccagagg 2520

aactacccgt atgcttggac aggaggaaga attgtttacg acgtttga 2568

<211> 414

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Leu Lys His Phe Ser Phe Glu Pro Gly Tyr Ile Asn Leu Asn Asn

1 5 10 15

Gly Ser Phe Gly Ser Leu Pro Lys Pro Val Ala Glu Phe Cys Val Gln

20 25 30

His Ala Ala Arg Val Glu Ala Asn Pro Asp Arg Tyr His Arg Leu Glu

35 40 45

Phe Ala Glu Leu Leu Thr Ala Ala Arg Ala Arg Val Ala Arg Leu Ile

50 55 60

Gly Ala Asp Thr Asp Glu Cys Val Phe Val Pro Ser Thr Thr Ala Gly

65 70 75 80

Ile Asn Thr Ile Leu Arg Asn Ile Glu Trp Ser Ser Gly Asp Val Ile

85 90 95

Ile Lys Phe Ser Thr Thr Tyr Gln Gly Val Glu Arg Val Val Arg Tyr

100 105 110

Ile Ala Asp Thr His Ser Glu Val Thr Leu Ser Thr Val Glu Leu Thr

115 120 125

Phe Pro Ile Ser His Gln Glu Ile Val Lys Arg Phe Arg Glu His Val

130 135 140

Val Gly Val Lys Ala Ser Cys His Ser Ser Ser Arg Val Tyr Ala Ile

145 150 155 160

Ile Asp Ser Val Val Ser His Pro Gly Val Ala Leu Pro Trp Lys Gln

165 170 175

Met Thr Gln Ile Cys Arg Glu Glu Gly Val Cys Ser Ile Ile Asp Ala

180 185 190

Ala His Ser Ile Gly Gln Glu Met Asn Ile Asp Leu Ala Ser Ala Gln

195 200 205

Pro Asp Phe Trp Val Ser Asn Cys His Ile Trp Leu Phe Ala Lys Arg

210 215 220

Gly Ser Thr Val Leu Tyr Val Pro Lys Arg Asn Gln His Ile Ile Lys

225 230 235 240

Ser Ala Leu Val Thr Ser Trp Asp Tyr Pro Ser Pro Lys Glu Ser Ala

245 250 255

Pro Ala Ala Tyr Asp Thr Gly Phe Val Leu Gln His Glu Trp Pro Gly

260 265 270

Ala Ile Asp Phe Ser Pro Tyr Leu Ser Val Cys Ala Ala Met Asp Phe

275 280 285

Arg Glu Lys Ile Gly Gly Glu Gln Ala Ile Asn Asp Tyr Cys His Lys

290 295 300

Leu Ala Leu Glu Gly Gly Arg Lys Leu Ala Ser Ala Leu Gly Gly Cys

305 310 315 320

Val Leu Asp Glu Ser Pro Ser Ala Asp Leu Thr Leu Asn Met Val Asn

325 330 335

Val Gln Leu Pro Leu Pro Pro Thr Thr Ser Lys Glu Leu Gln Gly Lys

340 345 350

Ile Asp Ser Phe Leu Glu Tyr Glu Leu Leu His Lys His Asn Val Ser

355 360 365

Ser Val His Phe Cys His Gly Gly Ser Trp Trp Val Arg Cys Ser Ala

370 375 380

Gln Ile Trp Asn Glu Val Ser Asp Phe Glu Tyr Leu Gly Lys Ala Leu

385 390 395 400

Asn Asn Ala Cys Gln Glu Ala Arg Ser Lys Phe Phe Ala Asp

405 410

<211> 1245

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgctaaagc acttttcatt tgagcccgga tacatcaacc tcaacaatgg ttcatttggg 60

tccctcccca agccggtggc tgagttttgt gtgcaacatg ctgccagggt tgaagcgaat 120

ccggacagat atcatcgtct cgagtttgcg gagctcctca cagctgcgcg cgctcgtgtt 180

gccagactga tcggagcaga cacggacgaa tgtgtgtttg ttccgagcac cactgcaggc 240

atcaacacaa tcctacggaa cattgaatgg agttcaggcg atgtaatcat taaattctca 300

acaacttatc aaggggtgga acgggttgtt agatacatag ccgacactca ttccgaagtg 360

acgctatcaa ccgtggagtt gacatttccc atctctcatc aagagattgt aaaacgcttc 420

cgagaacacg ttgttggggt caaggcaagc tgtcacagct catcgagggt ctacgccata 480

atcgactcgg tcgtatcgca cccgggtgtt gctttgccct ggaaacagat gacccaaatc 540

tgtcgcgaag agggcgtctg cagcattatt gacgcagccc actccattgg ccaggagatg 600

aacatagatc tggcatctgc gcagccagac ttttgggtat cgaactgcca catatggctt 660

ttcgccaaac gtggttctac cgttttatat gtgccgaagc ggaaccaaca catcatcaag 720

tctgcgctgg taacatcctg ggactacccc tcccctaagg agtctgctcc tgctgcgtac 780

gatactggtt tcgttctcca acatgaatgg ccgggtgcga tcgacttttc gccttacctg 840

agtgtctgtg ctgcgatgga cttccgcgag aagatcggtg gcgaacaggc gatcaacgac 900

tattgccaca agctggcact ggagggcggc cggaaattag cttctgctct tggaggttgt 960

gtcctcgacg agtcaccttc tgcggatttg acactcaaca tggtgaatgt acaactaccg 1020

cttcctccga caacatcgaa agagctccag ggaaagatcg atagctttct ggaatacgag 1080

ctgttacata aacacaacgt atcctccgtg cacttctgcc acggtggttc gtggtgggtg 1140

cgctgcagtg ctcaaatttg gaacgaggtt tctgacttcg agtaccttgg caaagctctg 1200

aacaacgcgt gtcaagaggc tagatcgaag ttcttcgcgg actag 1245

<211> 854

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Ala Ile Gln Ile Val Asp Ala Gln Thr Leu Asn Gln Ser Arg His

1 5 10 15

Leu Asp Leu Gln Ser Asp Leu Ser Gln Gln Leu Val Asp Gly Leu Thr

20 25 30

Arg Pro His Gly Glu Lys Glu Ile Pro Thr Val Leu Leu Tyr Asp Glu

35 40 45

Ser Gly Leu Arg Leu Tyr Asp Ala Ile Thr Thr Glu Val Ser Glu Tyr

50 55 60

Tyr Leu Phe Gly Ala Glu Glu Glu Ile Leu Lys Asn Lys Ala Asp Glu

65 70 75 80

Ile Val Arg Thr Met His Ser His Asn Gly Leu Gly Asp Gly Val Leu

85 90 95

Asp Ser Glu Val Ile Leu Glu Leu Gly Ala Gly Ser Leu Arg Lys Thr

100 105 110

Ser His Ile Leu Ala Gly Leu Ser Arg Ile Val Pro Ser Glu Cys Asp

115 120 125

Thr Ala Pro Ile Thr Tyr Tyr Ala Leu Asp Leu Glu Glu Arg Glu Leu

130 135 140

Gln Arg Val Leu Asp Ser Ile Ser Asn Ser Ser Val Gly Glu Met Leu

145 150 155 160

Arg Gly Lys Val Glu Thr Lys Gly Leu Trp Gly Thr Tyr Glu Asp Gly

165 170 175

Leu Lys Phe Val Glu Asp Glu Gly Ser Pro Leu Ser Ser His Ser Asn

180 185 190

Ser Pro Thr Ser Gln Ser Ser Arg Gly Glu Leu Ala Pro Pro Ser Pro

195 200 205

Thr Pro Arg Ser Gly Leu Ser Ser Pro Leu His Ile Leu Phe Leu Gly

210 215 220

Ser Ser Leu Gly Asn Phe Asp Arg Lys Asp Ser Val Lys Phe Leu Gln

225 230 235 240

Ser Met Pro Leu Arg Pro Gly Ser Gly Asp Thr Leu Leu Ile Gly Leu

245 250 255

Asp His Asp Asn Asp Lys Thr Leu Ile Glu Glu Ala Tyr Asn Asp Arg

260 265 270

Lys Gly Tyr Thr Lys Thr Phe Ile Met Asn Gly Leu Arg Ala Ala Gly

275 280 285

Arg Val Leu Gly Asn Glu Asp Met Phe Glu Glu Asp Lys Trp Glu Tyr

290 295 300

Val Asn Arg Tyr Asn Glu Ala Glu Arg Arg His Glu Ala Tyr Tyr Lys

305 310 315 320

Ser Lys Cys Pro Gln Lys Leu Glu Asp Pro Lys Ala Glu His Gly Tyr

325 330 335

Glu Phe Leu Glu Asp Glu Leu Val Lys Ile Glu Val Ser Tyr Lys Tyr

340 345 350

Ser Glu Thr Asp Ala Tyr Ser Leu Phe Thr Asp Ser Gly Leu Arg Pro

355 360 365

Ile Gln Arg Trp Thr Asp Ser Ala Thr Gln Tyr Ser Leu Trp Leu Leu

370 375 380

Glu Arg Pro Pro Phe Met Phe Pro Leu Leu Lys Ser Pro Ile Ala Phe

385 390 395 400

Asn Ser Leu Gly Glu Ile Val Glu Asn Ser Pro Leu Ser Asn Ser Pro

405 410 415

Phe Gly Val Pro Ser Pro Gln Glu Trp Ser Asn Leu Trp Ala Ala Trp

420 425 430

Asp Phe Val Thr Leu Lys Met Ile Pro Pro Ser Met Leu His Val Lys

435 440 445

Pro Ile His Leu Arg His Ile Cys Leu Phe Tyr Leu Gly His Ile Pro

450 455 460

Thr Phe Leu Asp Ile His Leu Ser Arg Leu Leu Lys Glu Ser His Thr

465 470 475 480

Glu Pro Glu Ser Phe Lys Tyr Ile Phe Glu Arg Gly Ile Asp Pro Asn

485 490 495

Val Asp Asn Pro Ala Glu Cys His Pro His Ser Glu Val Pro Gln Asn

500 505 510

Glu Gly Asp Trp Pro Ser Leu Glu Ala Ile Leu Ser Phe Gln Ala Arg

515 520 525

Val Arg Ser Arg Leu Leu Thr Leu Tyr Asp Asn Val Leu Ile Gly Lys

530 535 540

Val Leu Thr Arg Lys Val Cys Arg Ile Leu Ser Met Thr Phe Glu His

545 550 555 560

Glu Ala Phe His Leu Glu Thr Leu Leu Tyr Met Leu Leu Gln Val Ala

565 570 575

Gly Asn Gly Thr Ile Pro Pro Pro Gly Phe Thr Pro Pro Ser Trp Thr

580 585 590

Ser Leu Ser Ala Asn Trp Asn Val Leu Pro Ala Leu Glu Asn Ala Thr

595 600 605

Val Thr Leu Gly Pro Glu Ile Val Thr Leu Gly His Asp Asp Asp Glu

610 615 620

Arg Glu Asp Glu Asp Pro Ala Phe Ala Thr Asp Val Lys Asp His Glu

625 630 635 640

Phe Ala Trp Asp Asn Glu Asn Pro Lys Arg Gln Val Glu Val Lys Gln

645 650 655

Phe Arg Ile Glu Leu Arg Pro Val Thr Asn Gly Gln Phe Tyr Glu Phe

660 665 670

Tyr Lys Ala Tyr Lys Asp Asp Met Lys Leu Gln Leu Pro Ala Ser Trp

675 680 685

Leu Glu Thr Asp Asp Lys Met Met Val Arg Thr Leu Tyr Gly Pro Val

690 695 700

Pro Met Lys Val Ala Gln Asn Trp Pro Val Val Ile Ser Tyr Asp Gly

705 710 715 720

Leu Ser Ala Tyr Ala Ile Val Lys Gly Gly Arg Leu Pro Thr Glu Pro

725 730 735

Glu Leu Arg Leu Phe Leu Asp Lys Phe Glu Ser Gly Tyr Glu Gly Gly

740 745 750

Ala Asn Val Gly Phe Arg Asn Trp His Pro Ile Pro Ala Thr Thr Gly

755 760 765

Gly Lys Lys Tyr Gly Gly Lys Gly His Asn Gly Gly Val Trp Glu Trp

770 775 780

Thr Ser Thr Thr Leu Thr Lys His Asp Gly Phe Glu Pro Ser Lys His

785 790 795 800

Tyr Pro Gly Phe Thr Ala Asp Phe Phe Asp Gly Cys His Asn Val Ile

805 810 815

Leu Gly Gly Ser Tyr Ala Phe Ile Pro Arg Met Ala Glu Arg Arg Ser

820 825 830

Leu Arg Asn Trp Tyr Gln Arg Asn Tyr Pro Tyr Ala Trp Thr Gly Gly

835 840 845

Arg Ile Val Tyr Asp Val

850

<211> 2565

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggcaattc aaatcgtcga cgcccagacg cttaatcagt ccaggcattt ggatcttcag 60

agcgacctct cccagcaact tgttgacggc ttaactcggc cgcatggcga aaaggaaatc 120

cctaccgtgc tcctatacga cgaatcaggg ttaagattgt acgacgccat cacgaccgag 180

gtctcagaat actatctttt tggcgccgag gaggaaatcc tcaagaataa agctgatgaa 240

attgttcgga cgatgcacag tcacaacgga ttgggggacg gggttctaga ctcggaagtc 300

atcctcgaat taggcgccgg ttccctgagg aagacgtccc acatcctcgc agggctttca 360

cgcatcgttc ctagcgagtg cgatacagcg ccgatcacct attacgccct cgatcttgaa 420

gaacgcgaac ttcagcgagt actagactcc atatcaaact cgagcgtcgg agagatgtta 480

agaggaaaag tcgaaacgaa ggggctgtgg ggaacctacg aagacgggct gaagttcgta 540

gaagacgagg gttctcctct cagcagccat tcgaattccc ccactagtca atcttcgaga 600

ggagagttgg cgccgccatc accgaccccg agatcaggat tgagttcccc actgcacatt 660

ctattcttag gatcgtcgtt ggggaacttt gaccgcaagg acagcgtcaa gttcttgcag 720

tccatgcccc ttcgccccgg ctcaggcgat acgctgttaa tcggtctcga tcacgataac 780

gataaaacgc tgatcgaaga ggcgtacaat gaccgcaaag gttatacgaa gacattcatc 840

atgaacgggt tgagagcggc tggacgtgtg ttaggaaacg aggacatgtt tgaagaagac 900

aaatgggaat atgttaacag atataatgag gccgagcgtc gacatgaggc gtactacaag 960

tccaagtgtc cgcaaaaact cgaagatccg aaggcggaac atggctacga gttccttgaa 1020

gatgaactag tcaaaatcga ggtctcctat aagtactcgg aaaccgatgc ctatagccta 1080

ttcaccgact cgggcctccg ccctatccaa cgctggacag acagtgctac ccagtactcc 1140

ctttggctac tcgagcgtcc cccctttatg ttcccccttt tgaagtcccc aatcgccttc 1200

aatagtctcg gcgagatcgt cgaaaactct ccactctcca actcgccctt cggcgttcct 1260

tcgccccaag aatggtcaaa tctgtgggcc gcatgggact tcgttacctt gaagatgatc 1320

cccccctcaa tgctccacgt gaagcctatc cacctgaggc acatatgcct gttttatttg 1380

ggccatattc cgacgttctt ggacatccat ttgagtcgct tgctgaaaga atcgcacacg 1440

gaacctgaaa gttttaagta tatctttgag cgagggattg atcccaatgt ggacaacccg 1500

gccgaatgtc atccccattc ggaggttccg cagaatgaag gcgattggcc ttcgcttgaa 1560

gctatcttat ctttccaagc tcgagttcgc tcaagacttt tgactctcta cgacaatgtc 1620

ttgataggaa aggtgttgac gcgaaaggtc tgtcgcatct tgtcaatgac tttcgaacac 1680

gaggcttttc accttgagac gctgctctac atgcttttgc aagttgccgg aaatgggacc 1740

atccctccgc caggatttac acctccatca tggacctcac tttctgctaa ctggaacgtt 1800

cttccagcgt tggaaaatgc gacagttact ctcggcccgg aaatcgtaac attgggccac 1860

gacgacgatg aaagggagga tgaagaccct gcgtttgcca ctgacgtgaa agatcacgaa 1920

tttgcatggg acaatgagaa tcccaagcgg caggtcgaag ttaagcagtt caggattgag 1980

ttgaggccgg tcacgaatgg ccagttttac gaattctaca aggcgtataa ggacgacatg 2040

aagctccaat taccagccag ctggttggag acagatgata agatgatggt tcgcaccctg 2100

tacggccctg tgcctatgaa agtggctcag aactggcccg tcgtcatttc gtacgatggc 2160

ctgtctgcct atgcgatcgt caaaggcgga aggcttccga cggagcctga gcttcgtctc 2220

ttcctagaca agtttgagag cggctacgaa ggtggcgcaa atgttggatt tcgtaactgg 2280

catcccattc ccgcaacgac cggcgggaag aagtacggtg gaaagggtca caacggcggc 2340

gtttgggaat ggacttcaac tacactcacg aaacacgatg gattcgaacc atccaagcac 2400

tacccaggat tcacggcgga tttcttcgac ggatgtcaca atgtgatctt gggagggtca 2460

tatgccttca tccctcgcat ggctgaaaga cgcagtttgc gcaactggta ccagaggaac 2520

tacccgtatg cttggacagg gggaagaatt gtttacgacg tttga 2565

<211> 414

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Leu Lys Tyr Phe Ser Phe Glu Pro Gly Tyr Ile Asn Leu Asn Asn

1 5 10 15

Gly Ser Phe Gly Ser Leu Pro Lys Pro Val Ala Glu Phe Cys Val Gln

20 25 30

His Ala Ala Arg Val Glu Ala Asn Pro Asp Arg Tyr His Arg Leu Glu

35 40 45

Phe Ala Glu Leu Leu Thr Ala Ala Arg Ala Arg Val Ala Arg Leu Ile

50 55 60

Gly Ala Asp Thr Asp Glu Cys Val Phe Val Pro Ser Thr Thr Ala Gly

65 70 75 80

Ile Asn Thr Ile Leu Arg Asn Ile Glu Trp Ser Ser Gly Asp Val Ile

85 90 95

Ile Arg Phe Ser Thr Thr Tyr Gln Gly Val Glu Arg Val Ala Arg Tyr

100 105 110

Ile Ala Asp Thr His Ser Glu Val Thr Leu Ser Thr Val Glu Leu Thr

115 120 125

Phe Pro Ile Ser His Gln Glu Ile Val Lys Arg Phe Arg Glu His Val

130 135 140

Val Gly Val Lys Ala Ser Cys His Ser Ser Ser Arg Val Tyr Ala Ile

145 150 155 160

Ile Asp Ser Val Val Ser His Pro Gly Val Ala Leu Pro Trp Lys Gln

165 170 175

Met Thr Gln Ile Cys Arg Glu Glu Gly Val Cys Ser Ile Ile Asp Ala

180 185 190

Ala His Ser Ile Gly Gln Glu Met Asn Ile Asp Leu Ala Ser Ala Gln

195 200 205

Pro Asp Phe Trp Val Ser Asn Cys His Lys Trp Leu Phe Ala Lys Arg

210 215 220

Gly Ser Thr Val Leu Tyr Val Pro Lys Arg Asn Gln His Ile Ile Lys

225 230 235 240

Ser Ala Leu Val Thr Ser Trp Asp Tyr Pro Ser Pro Lys Glu Ser Ala

245 250 255

Pro Ala Ala Tyr Asp Thr Gly Phe Val Leu Gln His Glu Trp Pro Gly

260 265 270

Ala Ile Asp Phe Ser Pro Tyr Leu Ser Val Cys Ala Ala Met Asp Phe

275 280 285

Arg Glu Lys Ile Gly Gly Glu Gln Ala Ile Asn Asp Tyr Cys His Lys

290 295 300

Leu Ala Leu Glu Gly Gly Arg Lys Leu Ala Ser Ala Leu Gly Gly Cys

305 310 315 320

Val Leu Asp Glu Ser Pro Ser Ala Glu Leu Thr Leu Asn Met Val Asn

325 330 335

Val Gln Leu Pro Leu Pro Pro Thr Thr Ser Lys Glu Leu Gln Gly Lys

340 345 350

Ile Asp Ser Phe Leu Glu Tyr Glu Leu Leu His Lys His Asn Val Ser

355 360 365

Ser Val His Phe Tyr His Gly Gly Ser Trp Trp Val Arg Cys Ser Ala

370 375 380

Gln Ile Trp Asn Glu Val Ser Asp Phe Glu Tyr Leu Gly Lys Ala Leu

385 390 395 400

Ser Asn Ala Cys Gln Glu Ala Arg Ser Lys Phe Phe Ala Asp

405 410

<211> 1245

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

atgctaaagt acttttcatt tgagcccgga tacatcaacc tcaacaatgg ttcatttggg 60

tccctcccca agccggtggc tgagttttgt gtgcaacatg ctgccagggt tgaagcgaat 120

ccggacagat atcatcgtct cgagtttgcg gagctcctca cagctgcgcg cgctcgtgtt 180

gccagactga tcggagcaga cacggacgaa tgcgtgtttg ttccgagcac cactgcaggc 240

atcaacacaa tcctacggaa cattgaatgg agttcaggcg atgtaatcat tagattctca 300

acaacttatc aaggggtgga acgggttgct agatacatag ccgacactca ttccgaagtg 360

acgctatcaa ccgtggagtt gacatttccc atctctcatc aagagattgt aaaacgcttc 420

cgagaacacg ttgttggggt caaggcaagc tgtcacagct catcgagggt ctacgccata 480

atcgactcgg tcgtatcgca cccgggtgtt gctttgccct ggaaacagat gacccaaatc 540

tgtcgcgaag agggcgtctg cagcattatt gacgcagccc actccattgg ccaggagatg 600

aacatagatc tggcatctgc gcagccagac ttttgggtat cgaactgcca caaatggctt 660

ttcgccaaac gtggttctac cgttttatat gtgccgaagc ggaaccaaca catcatcaag 720

tctgcgctgg taacatcctg ggactatccc tcccctaagg agtctgctcc tgctgcgtac 780

gatactggtt tcgttctcca acatgaatgg ccgggggcga tcgacttttc gccttacctg 840

agtgtctgtg ctgcgatgga cttccgcgag aagatcggtg gcgaacaggc gatcaacgac 900

tattgccaca agctggcact ggagggcggc cggaaattag cttctgctct tggaggttgt 960

gtcctcgacg agtcaccttc tgcggaattg acactcaaca tggtgaatgt acaactaccg 1020

cttcctccga caacatcgaa agagctccag ggaaagatcg atagctttct ggaatacgag 1080

ctgttacata aacacaacgt atcctccgtg cacttctacc acggtggttc gtggtgggtg 1140

cgctgcagtg ctcaaatttg gaacgaggtt tctgacttcg agtaccttgg caaagctctg 1200

agcaacgcgt gtcaagaggc tagatcgaag ttcttcgcgg actag 1245

<210> 13

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggccattc aaatcgtcga c 21

<210> 14

tcaaatatcg taaacaattc gccctcc 27

<210> 15

atgctaaagt acttttcatt tgagcccgg 29

<210> 16

ctagtccgcg aagaacttcg atctagc 27

<210> 17

atggcaattc aaatcgtcga cg 22

<210> 18

tcaaacgtcg taaacaattc ttcctcc 27

<210> 19

atgctaaagc acttttcatt tgagcccg 28

<210> 20

ctagtccgcg aagaacttcg atc 23

<210> 21

atggcaattc aaatcgtcg 19

<210> 22

tcaaacgtcg taaacaattc 20

<210> 23

atgctaaagt acttttcatt tgagcccg 28

<210> 24

ctagtccgcg aagaacttcg atc 23

<210> 25

gctgatatcg gatccgaatt catggccatt caaatcgtcg a 41

<210> 26

ttgtcgacgg agctcgaatt ctcaaatatc gtaaacaatt cgccc 45

<210> 27

gctgatatcg gatccgaatt catgctaaag tacttttcat ttgagcc 47

<210> 28

ttgtcgacgg agctcgaatt cctagtccgc gaagaacttc gat 43

<210> 29

gctgatatcg gatccgaatt catggcaatt caaatcgtcg a 41

<210> 30

ttgtcgacgg agctcgaatt ctcaaacgtc gtaaacaatt cttcc 45

<210> 31

gctgatatcg gatccgaatt catgctaaag cacttttcat ttgagcc 47

<210> 32

ttgtcgacgg agctcgaatt cctagtccgc gaagaacttc gat 43

<210> 33

gctgatatcg gatccgaatt catggcaatt caaatcgtcg ac 42

<210> 34

ttgtcgacgg agctcgaatt ctcaaacgtc gtaaacaatt cttcc 45

<210> 35

gctgatatcg gatccgaatt catgctaaag tacttttcat ttgagcc 47

<210> 36

ttgtcgacgg agctcgaatt cctagtccgc gaagaacttc gat 43

Claims

1. the application of Pleurotus edible fungus ergothioneine synthase PEGT1 and PEGT2 in synthesizing ergothioneine, it is characterized in that:

The Pleurotus edible mushrooms are oyster mushroom, Bailing mushroom or King oyster mushroom;

The amino acid sequence of described oyster mushroom ergothioneine synthase PEGT1 is shown in SEQ ID NO: 1;

The amino acid sequence of described oyster mushroom ergothioneine synthase PEGT2 is shown in SEQ ID NO: 3;

The amino acid sequence of the described Bailing mushroom ergothioneine synthase PEGT1 is shown in SEQ ID NO: 5;

The amino acid sequence of the described Bailing mushroom ergothioneine synthase PEGT2 is shown in SEQ ID NO: 7;

The amino acid sequence of the described Pleurotus eryngii ergothioneine synthase PEGT1 is shown in SEQ ID NO: 9;

The amino acid sequence of the ergothioneine synthase PEGT2 of Pleurotus eryngii is shown in SEQ ID NO: 11.

2. application according to claim 1, is characterized in that:

The application of the ergothioneine synthase PEGT1 and PEGT2 of the Pleurotus edible fungus in the combined biosynthesis of ergothioneine in vitro.

3. application according to claim 1 or 2, is characterized in that:

The nucleotide sequence of the ergothioneine synthase PEGT1 encoding gene of Oyster mushroom is shown in SEQ ID NO: 2;

The nucleotide sequence of the ergothioneine synthase PEGT2 encoding gene of Oyster mushroom is shown in SEQ ID NO: 4;

The nucleotide sequence of the ergothioneine synthase PEGT1 encoding gene of the Bailing mushroom is shown in SEQ ID NO: 6;

The nucleotide sequence of the ergothioneine synthase PEGT2 encoding gene of the Bailing mushroom is shown in SEQ ID NO: 8;

The nucleotide sequence of the ergothioneine synthase PEGT1 encoding gene of Pleurotus eryngii is shown in SEQ ID NO: 10;

The nucleotide sequence of the gene encoding the ergothioneine synthase PEGT2 of Pleurotus eryngii is shown in SEQ ID NO: 12.

4. Pleurotus edible fungus ergothioneine synthase PEGT1, is characterized in that:

The amino acid sequence of described oyster mushroom ergothioneine synthase PEGT1 is a protein composed of the amino acid sequence shown in SEQ ID NO: 1;

The amino acid sequence of the described Bailing mushroom ergothioneine synthase PEGT1 is a protein composed of the amino acid sequence shown in SEQ ID NO: 5;

The amino acid sequence of the ergothioneine synthase PEGT1 of Pleurotus eryngii is a protein composed of the amino acid sequence shown in SEQ ID NO: 9.

5. the coding gene of the Pleurotus edible fungus ergothioneine synthase PEGT1 according to claim 4, it is characterized in that: the nucleotide sequence of the coding gene of the described oyster mushroom ergothioneine synthase PEGT1 is as SEQ ID NO : the nucleotide sequence shown in 2;

The nucleotide sequence of the encoding gene of the described Bailing mushroom ergothioneine synthase PEGT1 is the nucleotide sequence shown in SEQ ID NO: 6;

The nucleotide sequence of the gene encoding the ergothioneine synthase PEGT1 of Pleurotus eryngii is the nucleotide sequence shown in SEQ ID NO: 10.

6. Pleurotus edible fungus ergothioneine synthase PEGT2, characterized in that: its amino acid sequence is as follows: the protein composed of the amino acid sequence shown in SEQID NO:7;

The Pleurotus edible mushroom is Bailing mushroom.

7 . The gene encoding the ergothioneine synthase PEGT2 of Pleurotus edible fungus according to claim 6 , wherein the nucleotide sequence is as follows: the nucleotide sequence shown in SEQ ID NO: 8. 8 .

8. the coding gene of Pleurotus edible fungus ergothioneine synthase PEGT2, is characterized in that:

The Pleurotus edible mushroom is oyster mushroom or king oyster mushroom;

The nucleotide sequence of the coding gene of the described oyster mushroom ergothioneine synthase PEGT2 is the nucleotide sequence shown in SEQ ID NO: 4;

The nucleotide sequence of the gene encoding the ergothioneine synthase PEGT2 of Pleurotus eryngii is the nucleotide sequence shown in SEQ ID NO: 12.

9. A recombinant vector, expression cassette or recombinant bacteria containing the encoding gene of claim 5 or 7 or 8.

10. The application of the recombinant vector, expression cassette or recombinant bacteria of claim 9 in synthesizing ergothioneine.

11. The application according to claim 10, characterized in that:

Constructing a recombinant expression vector containing the encoded gene of claim 5 or 7 or 8, transforming the recombinant expression vector into Escherichia coli BL21 (DE3), inducing expression of the obtained genetically engineered bacteria, washing with PBS to collect the induced expression cells Finally, mix the engineering bacteria containing PEGT1 and the engineering bacteria with PEGT2, dissolve and mix with PBS, add a reaction substrate, and then react after crushing to obtain a fermentation broth containing ergothioneine.