CN110551697B - Application of ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus in synthesis of ergothioneine - Google Patents
Application of ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus in synthesis of ergothioneine Download PDFInfo
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Abstract
The invention discloses application of ergothioneine synthetases PEGT1 and PEGT2 of Pleurotus ostreatus in synthesis of ergothioneine. 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 invention firstly expounds the ergothioneine biosynthesis pathway of pleurotus edible fungi, and shows that the edible fungi has a simpler synthesis pathway, namely, two genes Pegt1 and Pegt2 participate in the synthesis of the ergothioneine, and meanwhile, the gene engineering cells constructed by the invention are safe and stable, have short production period and show the important value of the edible fungi in the application and development of the ergothioneine.
Description
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 (11)
1. The application of the ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus in the synthesis of ergothioneine is characterized in that:
the Pleurotus ostreatus edible fungi is Pleurotus ostreatus, Pleurotus nebrodensis or Pleurotus eryngii;
the amino acid sequence of the oyster mushroom ergothioneine synthase PEGT1 is shown as SEQ ID NO: 1 is shown in the specification;
the amino acid sequence of the oyster mushroom ergothioneine synthase PEGT2 is shown as SEQ ID NO: 3 is shown in the specification;
the amino acid sequence of the pleurotus nebrodensis ergothioneine synthase PEGT1 is shown as SEQ ID NO: 5 is shown in the specification;
the amino acid sequence of the pleurotus nebrodensis ergothioneine synthase PEGT2 is shown as SEQ ID NO: 7 is shown in the specification;
the amino acid sequence of the pleurotus eryngii ergothioneine synthase PEGT1 is shown as SEQ ID NO: 9 is shown in the figure;
the amino acid sequence of the pleurotus eryngii ergothioneine synthase PEGT2 is shown as SEQ ID NO: shown at 11.
2. Use according to claim 1, characterized in that:
the application of the ergothioneine synthetase PEGT1 and PEGT2 of Pleurotus ostreatus in the in vitro combined biosynthesis of ergothioneine.
3. Use according to claim 1 or 2, characterized in that:
the nucleotide sequence of the coding gene of the oyster mushroom ergothioneine synthase PEGT1 is shown as SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of the coding gene of the oyster mushroom ergothioneine synthase PEGT2 is shown as SEQ ID NO: 4 is shown in the specification;
the nucleotide sequence of the coding gene of the ergothioneine synthase PEGT1 of the pleurotus nebrodensis is shown as SEQ ID NO: 6 is shown in the specification;
the nucleotide sequence of the coding gene of the ergothioneine synthase PEGT2 of the pleurotus nebrodensis is shown as SEQ ID NO: 8 is shown in the specification;
the nucleotide sequence of the coding gene of the pleurotus eryngii ergothioneine synthase PEGT1 is shown as SEQ ID NO: 10 is shown in the figure;
the nucleotide sequence of the coding gene of the pleurotus eryngii ergothioneine synthase PEGT2 is shown as SEQ ID NO: shown at 12.
4. The ergothioneine synthase PEGT1 of Pleurotus ostreatus is characterized in that:
the Pleurotus ostreatus edible fungi is Pleurotus ostreatus, Pleurotus nebrodensis or Pleurotus eryngii;
the amino acid sequence of the oyster mushroom ergothioneine synthase PEGT1 is shown as SEQ ID NO: 1, and the protein is composed of the amino acid sequence shown in the specification;
the amino acid sequence of the pleurotus nebrodensis ergothioneine synthase PEGT1 is shown as SEQ ID NO: 5, and the protein is composed of the amino acid sequence shown in the specification;
the amino acid sequence of the pleurotus eryngii ergothioneine synthase PEGT1 is shown as SEQ ID NO: 9, and the amino acid sequence shown in the figure.
5. The gene encoding ergothioneine synthase PEGT1 of Pleurotus ostreatus of claim 4, wherein: the nucleotide sequence of the coding gene of the oyster mushroom ergothioneine synthase PEGT1 is shown as SEQ ID NO: 2;
the nucleotide sequence of the coding gene of the ergothioneine synthase PEGT1 of the pleurotus nebrodensis is shown as SEQ ID NO: 6;
the nucleotide sequence of the coding gene of the pleurotus eryngii ergothioneine synthase PEGT1 is shown as SEQ ID NO: 10, or a nucleotide sequence shown in the figure.
6. The ergothioneine synthase PEGT2 of Pleurotus ostreatus is characterized in that: the amino acid sequence is shown as follows: SEQ ID NO: 7;
the Pleurotus ostreatus is Pleurotus nebrodensis.
7. The gene encoding ergothioneine synthase PEGT2 of Pleurotus ostreatus of claim 6, wherein: the nucleotide sequence is shown as follows: SEQ ID NO: 8.
8. The coding gene of ergothioneine synthase PEGT2 of Pleurotus ostreatus is characterized in that:
the Pleurotus ostreatus edible fungi is Pleurotus ostreatus or Pleurotus eryngii;
the nucleotide sequence of the coding gene of the oyster mushroom ergothioneine synthase PEGT2 is SEQ ID NO: 4;
the nucleotide sequence of the coding gene of the pleurotus eryngii ergothioneine synthase PEGT2 is SEQ ID NO: 12.
9. A recombinant vector, expression cassette or recombinant bacterium comprising the coding gene of claim 5, 7 or 8.
10. Use of the recombinant vector, expression cassette or recombinant bacterium of claim 9 for the synthesis of ergothioneine.
11. Use according to claim 10, characterized in that:
constructing a recombinant expression vector containing the coding gene of claim 5, 7 or 8, transforming the recombinant expression vector into escherichia coli BL21(DE3), performing induced expression on the obtained genetically engineered bacteria, washing and collecting thalli after 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 and reacting to obtain a fermentation liquid containing ergothioneine.
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