CN110607286B

CN110607286B - Application of grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 in synthesis of ergothioneine

Info

Publication number: CN110607286B
Application number: CN201910773782.5A
Authority: CN
Inventors: 林俊芳; 余颖豪; 郭丽琼; 廖寒露; 李浩莹
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-08-21
Filing date: 2019-08-21
Publication date: 2021-02-19
Anticipated expiration: 2039-08-21
Also published as: CN110607286A

Abstract

The invention discloses an application of grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 in ergothioneine synthesis, and belongs to the field of biosynthesis biology. The invention utilizes a homologous comparison method to find out possible ergothioneine synthetase genes Gfegt1 and Gfegt2 in the grifola frondosa, and utilizes the extracted grifola frondosa RNA to carry out reverse transcription to form cDNA for cloning and obtaining. The invention constructs a vector for promoting expression of two genes, namely Gfegt1 and Gfegt2, by using a yeast promoter, and converts the vector into saccharomyces cerevisiae EC1118, then the vector is absorbed by the saccharomyces cerevisiae by adding a substrate, the ergothioneine is catalytically synthesized in vivo through reaction, and the generation of an ergothioneine product is detected by High Performance Liquid Chromatography (HPLC) after extraction, so that the ergothioneine is biosynthesized in vivo. The invention provides a brand new biosynthesis way for the production of ergothioneine.

Description

Application of grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 in synthesis of ergothioneine

Technical Field

The invention belongs to the field of biosynthesis biology, and particularly relates to application of grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 in synthesis of ergothioneine.

Background

Ergothioneine (EGT) is an antioxidant substance and plays important roles in maintaining redox homeostasis, signal transduction, cell metabolism and the like in bacteria and fungi. Tanret (1909) first discovered this substance in the ergot (Claviceps purpurea) and named it. Ergothioneine is a derivative of histidine trimethylbetaine, a thiol group is attached to the second C atom of the imidazole ring, and exists in the form of thione, which has a lower standard reduction potential than that of thiol, so that ergothioneine is more stable than other thiol compounds (such as glutathione) (Cheah et al, 2012).

Ergothioneine has a variety of antioxidant and cell-protective functions in vitro and in a few in vivo, including free radical scavenging, anti-radiation, anti-inflammation, repair of neuronal damage, etc. (Cheah et al, 2012), but cannot be synthesized in animals, and can only be obtained from bacteria or fungi. Ergothioneine is present in Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium smegmatis (Mycobacterium smegmatis), cyanobacteria (Certain cyanobacterium), Methylobacterium (Methylobacterium), and most non-yeast fungi including filamentous fungi such as Neurospora crassa (Cumming et al, 2018). Most edible fungi contain abundant ergothioneine, wherein the content of Boletus edulis (Boletus edulis) is high, and 7.27mg of ergothioneine is contained in per gram of dry weight (kalalas et al, 2017).

A gene cluster formed by five synthetase genes in bacteria, egtABCDE, and two synthetase genes in fungi, Egt1, 2, constitute two different synthetic pathways. In the bacterial synthetic pathway, egtA catalyzes the production of gamma-cysteamine-glutamic acid from glutamic acid and cysteine; egtD catalyzes the methylation of histidine to form histidine betaine; egtB combines the sulfur atom of gamma-cysteamine-glutamic acid with the carbon atom on imidazole ring of histidine betaine to form a compound containing a sulfoxide structure under the assistance of oxygen and ferrous ions; egtC catalyzes the cleavage of glutamic acid on histidine betaine cysteine sulfoxide; egtE then catalyzes the cleavage of the CS bond, ultimately forming ergothioneine. In the fungal synthetic pathway, egt1 catalyzes the methylation of histidine to form a CS bond with cysteine, forming histidine betaine cysteine sulfoxide; egt2 cleaves the CS bond of the precursor to form ergothioneine. Therefore, only two enzymes are needed to participate in the synthesis reaction, so that the biosynthesis pathway of the fungus is far simpler than that of the bacterium, and more convenience is provided for the production and application research of the ergothioneine.

At present, it has been reported that ergothioneine is fermented in large quantities by constructing an E.coli (Escherichia coli) engineering strain containing egtABCDE, and the yield reaches 24mg per liter of fermentation broth (Osawa et al, 2018). However, the egt1 and 2 researches on fungi are mainly protein structure researches (Hu et al, 2014, Irani et al, 2018), and only based on neurospora crassa, and no more fermentation-based applications and related researches based on edible fungi exist at present. And the handsome (2018) clones three genes of ergothioneine in the flammulina velutipes and verifies the activity of the three genes. Therefore, the research and application of the egt1 and 2 target genes in the edible fungi have good prospects.

Disclosure of Invention

In order to overcome the defects of the ergothioneine biosynthesis pathway in the prior art, the invention mainly aims to provide the application of grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 in the ergothioneine synthesis. The invention can synthesize ergothioneine from the beginning only by two genes.

Another objective of the invention is to provide Grifola frondosa ergothioneine genes Gfegt1 and Gfegt 2.

Another object of the present invention is to provide a protein encoded by the above-mentioned grifola frondosa ergothioneine gene.

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

the invention provides an application of grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 in synthesis of ergothioneine.

Preferably, the grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 are applied to the in vitro combined biosynthesis of ergothioneine.

Further preferably, the grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 are applied to the synthesis of ergothioneine in saccharomyces cerevisiae.

Preferably, the saccharomyces cerevisiae is saccharomyces cerevisiae EC 1118.

The nucleotide sequence of the grifola frondosa ergothioneine gene Gfegt1 is shown as SEQ ID NO. 1, and the coded amino acid thereof is shown as SEQ ID NO. 2.

The nucleotide sequence of the grifola frondosa ergothioneine gene Gfegt2 is shown as SEQ ID NO. 3, and the coded amino acid thereof is shown as SEQ ID NO. 4.

The invention also provides a protein Gfegt1 coded by the grifola frondosa ergothioneine gene Gfegt1, and the amino acid sequence of the protein Gfegt1 is shown as SEQ ID NO:2, respectively.

The nucleotide sequence of the grifola frondosa ergothioneine gene Gfegt1 for coding the protein is preferably shown as SEQ ID NO. 1.

The invention also provides a protein Gfegt2 coded by the grifola frondosa ergothioneine gene Gfegt2, and the amino acid sequence of the protein Gfegt2 is shown as SEQ ID NO:4, respectively.

The nucleotide sequence of the grifola frondosa ergothioneine gene Gfegt2 for coding the protein is preferably shown as SEQ ID NO. 3.

The invention also provides an expression vector and a host bacterium containing the gene and a primer for amplifying any segment of the gene.

A recombinant expression vector is obtained by taking pYES2 as a starting vector and sequentially inserting Gfegt2, pPGK, Gfegt1 and KanMX 4; or using pRS42k as starting vector, and sequentially inserting TEF1p, Gfegt1, CYC1t, TEF1p, Gfegt2 and CYC1t to obtain the vector.

A recombinant bacterium contains the recombinant expression vector.

Wherein, the enzyme GfEGT1 can catalyze substrates such as histidine (His), S-adenosylmethionine (SAM) and the like to generate histidine trimethyl inner salt, and then the latter and cysteine (Cys), ferrous ions and the like are used as substrates to generate histidine trimethyl inner salt cysteine sulfoxide under the catalysis of the enzyme GfEGT 1.

The GfEGT2 enzyme can catalyze histidine trimethyl inner salt cysteine sulfoxide to generate ergothioneine on substrates such as coenzyme pyridoxal phosphate (PLP).

The mechanism of the invention is as follows:

grifola frondosa (Grifola frondosus), also known as Grifola frondosa, belongs to the class Hymenomycetes, Basidiomycotina, order Aphyllophorales, and is a natural rare edible and medicinal fungus. Grifola frondosa is rich in various nutrient substances such as protein, vitamins and the like, and is developed into novel health care products and skin care products in recent years. The genes in a grifola frondosa database are compared by using Ncgt 1(NCU04343) and Ncgt 2(NCU11365) in Neurospora crassa by using a Blast tool of an NCBI website to find unidentified genes with high similarity to known sequences, which are correspondingly named as Gfegt1(GenBank: OBZ71212.1 and OBZ71213.1) (two genes in the database are combined into one gene) and Gfegt2(GenBank: OBZ72541.1), specific primers are designed, cDNA reverse transcribed from RNA in the grifola is extracted as a template for cloning, two genes in a synthetic ergothioneine pathway are obtained, and the two genes are expressed in a saccharomyces cerevisiae EC1118 to realize in-vivo biosynthesis of the ergothioneine. The invention provides a brand new biosynthesis way for the production of ergothioneine.

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

(1) cloning of Gfegt1 and Gfegt2 genes. The method comprises the steps of finding out possible ergothioneine synthetase genes Gfegt1 and Gfegt2 in the grifola frondosa by utilizing a homologous comparison method, extracting grifola frondosa RNA, and performing reverse transcription to obtain cDNA for cloning to obtain the gene. The nucleotide sequence of the grifola frondosa ergothioneine gene Gfegt1 has 41.56 percent of similarity with the nucleotide sequence of Neurospora crassa gene Negt 1, and the amino acid sequence of the grifola frondosa ergothioneine gene Gfegt1 has 32.72 percent of similarity with the amino acid sequence of Neurospora crassa gene Negt 1 by sequence comparison and analysis by using DNMAN software; the nucleotide sequence of the grifola frondosa ergothioneine gene Gfegt2 has 46.34 percent of similarity with the nucleotide sequence of Neurospora crassa gene Nregt 2, and the amino acid sequence of the grifola frondosa ergothioneine gene Gfegt2 has 32.74 percent of similarity with the amino acid sequence of Neurospora crassa gene Nregt 2.

(2) Functional verification of Gfegt1 and Gfegt2 genes. A vector for promoting expression of Gfegt1 and Gfegt2 genes by using a yeast promoter is constructed and transformed into Saccharomyces cerevisiae EC1118, and then the ergothioneine is catalytically synthesized by adding a substrate to allow the Saccharomyces cerevisiae to absorb the vector and carrying out in-vivo reaction. The extraction was followed by High Performance Liquid Chromatography (HPLC) to detect the formation of ergothioneine products.

Drawings

FIG. 1 is an electrophoretogram of total RNA of Grifola frondosa.

FIG. 2 is an electrophoretogram of the results of RT-PCR amplification of Grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 from Grifola frondosa cDNA.

FIG. 3 is a map of pYES2-Gfegt1-Gfegt2 expression plasmid.

FIG. 4 shows the results of HPLC detection of the ergothioneine standard in example 1.

FIG. 5 is the results of detection of ergothioneine by HPLC after fermentation of the wild-type s.cerevisiae EC1118(WT) in example 1; wherein a is the original size and b is the enlarged ordinate size.

FIG. 6 shows the results of detection of ergothioneine by HPLC after fermentation of the modified Saccharomyces cerevisiae EC1118(pYES2-Gfegt1-Gfegt2) in example 1; wherein a is the original size and b is the enlarged ordinate size.

FIG. 7 is a map of pRS42k-Gfegt1-Gfegt2 expression plasmid.

FIG. 8 shows the results of HPLC detection of the ergothioneine standard in example 2.

FIG. 9 shows the results of detection of ergothioneine by HPLC after fermentation of the wild-type s.cerevisiae EC1118(WT) in example 2.

FIG. 10 shows the results of detection of ergothioneine by HPLC after fermentation of the modified Saccharomyces cerevisiae EC1118(pRS42k-Gfegt1-Gfegt2) in example 2.

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

1.1 materials and methods

1.1.1 strains and plasmids

Grifola frondosa strain is purchased from Beijing Ji mushroom Garden science and technology Co., Ltd; the pMD18T plasmid was purchased from Takara, Inc. (Beijing) of medical science and technology, Inc., and E.coli DH5 α was purchased from Shanghai Weidi, Biotechnology, Inc., for gene cloning; saccharomyces cerevisiae EC1118 and the corresponding pYES2 plasmid: saccharomyces cerevisiae EC1118 was obtained from Lallemand, and pYES2 plasmid was obtained from Invitrogen; pRS42k is given by the teaching of Christof taxi and Michael Knop and is also disclosed in the literature as "System of centromeric, epigeal, and integral vector based on drug resistance markers for Saccharomyces cerevisiae [ J ]. Biotechniques 2006,40(1): 73-78." for gene expression.

1.1.2 preparation of culture Medium and reagents

PDB culture medium: used for culturing the grifola frondosa hyphae. Potato (peeled) 200g, glucose 20g, MgSO₄·7H₂O 1.5g，KH₂PO₄3g, adding distilled water to a constant volume of 1L, naturally adjusting pH, adding agar 20g (added into solid culture medium), and sterilizing with high pressure steam at 121 deg.C for 30 min.

LB culture medium: used for culturing Escherichia coli. 10g of tryptone, 5g of yeast extract, 10g of NaCl and distilled water are added to a constant volume of 1L, the pH value is 7.4, and the mixture is sterilized by high-pressure steam at 121 ℃ for 30 min.

YPD medium: the method is used for culturing the yeast. Peptone 20g, yeast extract 10g, glucose 20g, distilled water constant volume to 1L, natural pH, 115 deg.C high pressure steam sterilization for 15 min.

YPG medium: the method is used for induction culture of yeast. 20g of peptone, 10g of yeast extract, 20g of galactose and distilled water, wherein the volume is fixed to 1L, the pH value is natural, and the autoclave is sterilized for 15min at 115 ℃.

10 × STE Buffer: 50mM Tris, 10mM EDTA, 1M NaCl, pH8.0, autoclaved at 121 ℃ for 30 min.

3M NaAc: taking 24.6g of CH₃COONa·3H₂O in ddH₂Adjusting pH to 5.5 with glacial acetic acid, adding ddH₂And O is metered to 100 mL. Sterilizing with high pressure steam at 121 deg.C for 30 min.

Phenol/chloroform/isoamyl alcohol (25/24/1): 25mL of phenol, 24mL of chloroform and 1mL of isoamyl alcohol were mixed.

0.1M CaCl₂: taking 1.11g of CaCl₂Soluble in ddH₂And the volume of the mixture is reduced to 100 mL. Sterilizing with high pressure steam at 121 deg.C for 30 min.

50 × TAE: 242g of Tris, Na are taken₂EDTA·2H₂O37.2 g in 800mL ddH₂O, then 57.1mL glacial acetic acid is added, and ddH is added₂And O is metered to 1L.

1% agarose gel: taking 0.2g of agaroseSoluble in ddH₂O and the volume is up to 20 mL.

100mg/mL Amp: 0.1g of ampicillin (Amp) dry powder was dissolved in 1mL of ddH₂O, filtration through a 0.22 μm microporous membrane.

100mg/mL G418: 0.1G of geneticin (G418) dry powder was dissolved in 1mL of ddH₂O, filtration through a 0.22 μm microporous membrane.

10 × TE Buffer: 100mM Tris, 10mM EDTA, pH8.0, autoclaved at 121 ℃ for 30 min.

1M LiAc: 6.6g of LiAc was dissolved in ddH₂Adjusting pH to 8.0 with glacial acetic acid, adding ddH₂And O is metered to 100 mL. Sterilizing with high pressure steam at 121 deg.C for 30 min.

50% PEG 4000: 50g of PEG4000 plus ddH was taken₂And O is metered to 100 mL.

0.5M histidine: 0.7758g histidine in 10mL ddH₂And (4) in O.

1M cysteine hydrochloride salt: 1.750g cysteine hydrochloride in 10mL ddH₂And (4) in O.

0.2M S-ademetionine: 0.8000g ademetionine in 10mL ddH₂And (4) in O.

0.1M pyridoxal phosphate: 0.2651g pyridoxal phosphate dissolved in 10mL ddH₂And (4) in O.

0.1M FeSO₄·7H₂O：0.2780g FeSO₄·7H₂O dissolved in 10mL ddH₂And (4) in O.

1.1.3 enzymes and kits

The reverse transcription kit Transcript One-Step gDNA Removal and cDNA Synthesis SuperMix (AT311) was purchased from Beijing holotype gold organisms.

DNA polymerase KOD Fx (KFX-101) for amplifying a gene of interest was purchased from Toyobo, DNA polymerase PrimeStar Max (R045A) for constructing plasmids was purchased from Takara, and DNA polymerase 2 XTaqMix for adding A bases was purchased from Tokyo Biotech Co., Ltd., Guangzhou.

Restriction endonucleases Hind III, EcoR I were purchased from Thermo Fisher Scientific.

Kit for homologous recombination Clon express Ultra One Step Cloning Kit (C115) was purchased from Biotech Inc. of Nanjing Novowed.

The Kit HiPure Plasmid Mini Kit and the nucleic acid purification and recovery Kit HiPure Gel Pure DNA Mini Kit for Plasmid extraction were purchased from Meiji organisms.

1.1.4 primer sequence information

1.2 functional study of biosynthetic genes for ergothioneine

1.2.1 acquisition of the ergothioneine synthase Gene

The STE method is adopted to extract the RNA of the grifola frondosa, and the concrete method refers to tax paste (2008). The total RNA obtained by extraction is subjected to reverse transcription by using a Transcript One-Step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit to obtain cDNA. The Grifola frondosa cDNA is taken as a template, primers Gfegt1-F & Gfegt1-R and Gfegt2-F & Gfegt2-R are adopted to respectively amplify Gfegt1 and Gfegt2 target genes by using DNA polymerase KOD Fx, and a reaction system and reaction parameters are as follows:

and (3) PCR reaction system:

grifola frondosa cDNA template	1μL
		Gfegt1-F&Gfegt1-R or Gfegt2-F&Gfegt2-R	4μL
2×PCR Buffer for KOD FX	25μL
		2mM dNTPs	10μL
KOD FX	1μL
		dd H₂O	9μL
Total up to	50μL

PCR reaction parameters: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 10sec, annealing at Gfegt 155 ℃ for 30sec, extension at 68 ℃ for 2min36sec, followed by returning to the annealing step for a total of 30 cycles, and final extension at 72 ℃ for 5 min; pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 10sec, annealing at Gfegt 255 ℃ for 30sec, extension at 68 ℃ for 1min24sec, followed by a return to the annealing step for a total of 30 cycles, and final extension at 72 ℃ for 5 min.

And (3) carrying out Gel electrophoresis identification on the PCR product by using 1% agarose, and then recovering nucleic acid by using a HiPure Gel Pure DNA Mini Kit, wherein the steps are described in the specification.

1.2.2 ligation of cloning vector to target Gene

The band of interest amplified by KOD FX is a blunt-ended fragment, and in order to be ligated to pMD18T, it is necessary to add A bases to the fragment 3' using DNA polymerase 2 XTAQix, and the reaction system and reaction parameters are as follows:

base addition system A:

gfegt2 or Gfegt1	25μL
		2×TaqMix	25μL
Total up to	50μL

Addition of A base reaction parameters: incubate at 70 ℃ for 30 min.

Adding A base fragment, carrying out Gel electrophoresis identification by 1% agarose, and recovering nucleic acid by using a HiPure Gel Pure DNA Mini Kit, wherein the steps are described in the specification.

After recovering the A base fragment, the fragment is connected with pMD18T, and the specific reaction system and reaction parameters are as follows:

pMD18T ligation system:

pMD18T	1μL
		adding A base fragment	4μL
Solution I	5μL
		Total up to	10μL

pMD18T ligation reaction parameters: incubate at 16 ℃ for 2 h.

The ligated product was converted into 0.1M CaCl₂Coli DH 5. alpha. competent cells were prepared, and colonies were identified using DNA polymerase 2 XTaqMix, and the reaction system and reaction parameters were as follows:

colony identification reaction system:

2×TaqMix	5μL
		RV-M	1μL
M13-47	1μL
		dd H of 1 colony₂O	3μL
Total up to	10μL

Colony identification PCR reaction parameters were as follows: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30sec, annealing at Gfegt 155 ℃ for 30sec, extension at 68 ℃ for 2min and 36sec, followed by returning to the annealing step for a total of 30 cycles, and final extension at 72 ℃ for 5 min; pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30sec, annealing at Gfegt 255 ℃ for 30sec, extension at 68 ℃ for 1min24sec, followed by a return to the annealing step for a total of 30 cycles, and final extension at 72 ℃ for 5 min.

After the PCR product is identified by gel electrophoresis through 1% agarose, a bacterial colony corresponding to a purposeful band is selected and sent to Tianyihui biological technology limited company for sequencing. Colonies with correct sequencing results are stored for later use.

1.2.3 construction and transformation of expression plasmids

1.2.3.1 additional homology arms of Gene fragments

The method for constructing the vector is a homologous recombination method, a multi-fragment plasmid is connected into an expression vector pYES2 by utilizing a homologous recombinase, DNA polymerase PrimeStar Max is adopted by adding a homologous arm, and primers pYES2-Gfegt2-F & pYES2-Gfegt2-R/pYES2-Gfegt1-F & pYES2-Gfegt1-R/pYES2-KanMX4-F & pYES2-KanMX4-R/Sc-pPGK-F & Sc-pPGK-R are utilized to amplify genes Gfegt1 and Gfegt2 with homologous arms, G418 resistance genes with homologous arms, a pPGK promoter reaction system with homologous arms and reaction parameters are as follows: wherein, the G418 resistance gene KanMX4 is derived from pRS42 k; GenBank accession No. X59720 of pPGK promoter.

PCR reaction system with homologous arm:

PCR reaction parameters with homology arms: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10sec, annealing at 55 ℃ for 15sec, pYES2-Gfegt272 ℃ extension for 7sec/pYES2-Gfegt1 extension for 13sec/pYES2-KanMX4 extension for 7sec/Sc-pPGK extension for 5sec, followed by returning to the annealing step for a total of 30 cycles and final extension at 72 ℃ for 5 min.

1.2.3.2 double digestion of vectors

The cells were shaken overnight and the Plasmid pYES2 Plasmid was extracted using the Plasmid extraction Kit HiPure Plasmid Mini Kit. The plasmid was subsequently double digested with Fastdigest EcoR I and Fastdigest Hind III, the reaction system and parameters were as follows:

a double enzyme digestion reaction system:

10×FastDigest Buffer	2μL
		EcoR I	1μL
Hind III	1μL
		form panel	1μg
dd H₂O	Make up to 20 mu L
		Total up to	20μL

Double enzyme digestion reaction parameters: incubating at 37 deg.C for 40min, inactivating at 80 deg.C for 5min, and cooling in ice bath at 4 deg.C for 5 min.

And (3) carrying out Gel electrophoresis identification on the double digestion products by using 1% agarose, and then recovering nucleic acid by using a HiPure Gel Pure DNA Mini Kit, wherein the steps are detailed in the specification.

1.2.3.3 construction of expression plasmids

The homologous recombinase for constructing the vector adopts Clonexpress Ultra One Step Cloning Kit, and a vector fragment and a multigene fragment are subjected to homologous recombination to construct a plasmid with a circular structure. The specific reaction system and parameters are as follows:

homologous recombination reaction system:

2×ClonExpress Mix	5μL
		double enzyme digestion pYES2	114ng
pYES2-Gfegt2	42ng
		pYES2-Gfegt1	78ng
pYES2-KanMX4	42ng
		Sc-pPGK	27ng
dd H₂O	Make up to 10 mu L
		Total up to	10μL

Homologous recombination reaction parameters: incubate at 50 ℃ for 30 min.

The recombined plasmid is transformed into 0.1M CaCl₂Coli DH 5. alpha. competent cells were prepared and colonies were identified using DNA polymerase 2 XTaqMix, procedure referenced 1.2.2. The expression vector pYES2-Gfegt2-pPGK-Gfegt1-KanMX4, abbreviated as pYES2-Gfegt2-Gfegt1, is obtained by construction.

1.2.4 transformation and characterization of Saccharomyces cerevisiae

The selected saccharomyces cerevisiae is EC1118 wine brewing yeast, and the adopted method is a LiAc conversion method. The specific transformation steps are as follows: (1) culturing EC1118 Saccharomyces cerevisiae to OD using YPD medium₆₀₀0.4-0.6; (2) centrifuging the bacterial liquid without using a small amount of LiAc/TE for resuspension, and then adding plasmid pYES2-Gfegt2-pPGK-Gfegt1-KanMX4 for mixing; (3) incubating the mixture in a water bath at 30 ℃ for about half an hour; (4) adding 50% PEG4000 2 times of the volume of the mixed solution, incubating the mixed solution in water bath at 30 ℃ for about half an hour, and performing heat shock in water bath at 42 ℃ for 20 min; (5) adding YPD medium to the mixture for reconstitutionSu, followed by plating on YPD plates containing G418(100mg/mL) and culturing at 30 ℃. (6) Identifying a saccharomyces cerevisiae single colony by using DNA polymerase KOD Fx, wherein a saccharomyces cerevisiae colony identification reaction system and parameters are as follows:

a saccharomyces cerevisiae colony identification reaction system:

2×PCR Buffer for KOD FX	12.5μL
		2mM dNTPs	5μL
pYES2-T7/pYES2-R	2μL
		KOD FX	0.5μL
dd H containing 1 colony₂O mixed solution	5μL
		Total up to	25μL

Identifying PCR reaction parameters of saccharomyces cerevisiae colonies: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 10sec, annealing at 55 ℃ for 30sec, and extension at 68 ℃ for 6min, followed by a return to the annealing step for a total of 30 cycles, and final extension at 72 ℃ for 5 min.

1.2.5 characterization of synthetase function

1.2.5.1 Induction fermentation of the modified Strain

The plasmid constructed in the experiment has two types of promoters, a Gal promoter is induced and expressed by corresponding to a YPG culture medium, and a pPGK promoter is induced and expressed by corresponding to a YPD culture medium, so the following culture strategies are adopted in the fermentation culture: (1) selecting a saccharomyces cerevisiae EC1118 modified strain to a 50mL YPD culture medium, performing shake culture (30 ℃, 200rpm), inducing the expression of Gfegt1, and enriching thalli; (2) after 2 days of culture, the cells and YPD medium were centrifuged, 50mL of YPG medium was added, and shaking culture (30 ℃ C., 200rpm) was carried out to induce expression of Gfegt2 while adding substrate for reaction, as follows:

0.5M histidine	2mL
		1M cysteine hydrochloride	1mL
0.2M S-adenosylmethionine	1mL
		0.1M pyridoxal phosphate	1mL
0.1M FeSO₄·7H₂O	1mL

1.2.5.2 ergothioneine product detection

The culture after 7 days of fermentation was centrifuged, and the supernatant was subjected to impurity removal filtration using a 0.22 μm organic phase filter head, followed by HPLC analysis.

HPLC conditions reference method of Liu (2016):

HILIC Amphiion II column (5 μm, 4.6X 250 mm); the detection wavelength is 254nm, the flow rate is 1mL/min, the column temperature is 40 ℃, the sample injection amount is 20 mu L, and the mobile phase is acetonitrile: 20mM ammonium acetate (85: 15, pH 6.5). Ergothioneine standard dissolved in dd H₂And (4) in O. The concentrations of the ergothioneine standard substances are respectively 24mg/L, 48mg/L, 96mg/L, 144mg/L, 192mg/L and 240 mg/L. Working standard curves were made and samples were tested using the same conditions.

1.3 results and analysis

1.3.1 extraction and reverse transcription of Grifola frondosa RNA

In this experiment, the STE method was used to extract the RNA of Grifola frondosa, and the results are shown in FIG. 1, in which 28S rRNA in the lane is twice as bright as 18S rRNA for the subsequent reverse transcription. The case of reverse transcription shows success or failure only by amplifying the gene of interest by PCR.

1.3.2 amplification of the Gene of interest

As can be seen from the lane 1 in FIG. 2, a corresponding band appears between 2000-3000 bp, and the brightness is good, and the band can be preliminarily judged to be Gfegt1, and then sequenced by a sequencing company to be a 2580bp complete reading frame. As can be seen from lane 2 of FIG. 2, a corresponding band appears between 1200-2000 bp, and the brightness is good, and the band can be preliminarily judged to be Gfegt1, and then sequenced by a sequencing company to be a 1434bp complete reading frame.

1.3.3 construction of engineered Strain

The expression vector was constructed by the same group method, and the construction results are shown in FIG. 3, in which pGAL1 is a galactose promoter and pPGK is a phosphoglycerol promoter. There were no mutations after sequencing by sequencing company. This plasmid was subsequently transformed into s.cerevisiae EC1118 by the heat shock method.

1.3.4HPLC detection of product formation.

The ergothioneine standard curve is:

ergothioneine content (mg/L) ═ 0.00003 peak area-1.1101 (R)²＝0.9951)；

The peak time of the ergothioneine standard is 34.590min (figure 4), no obvious peak type appears in the wild type fermentation culture solution (figure 5) near the peak time, and the modified strain has an obvious peak at 34.818min (figure 6), which is consistent with the peak time of the standard, so that two enzymes expressed in the modified strain have enzyme activity and can convert a substrate into the ergothioneine. According to the prepared ergothioneine standard curve, the final concentration of the ergothioneine in the fermentation liquor is 11.4 mg/L.

Example 2

2.1 materials and methods (cf. 1.1 materials and methods)

2.2 functional study of biosynthetic genes for ergothioneine

2.2.1 acquisition of the ergothioneine synthase Gene, refer to 1.2.1 acquisition of the ergothioneine synthase gene of example 1.

2.2.2 ligation of cloning vector to Gene of interest, reference example 1, 1.2.2 ligation of cloning vector to Gene of interest.

2.2.3 construction and transformation of expression plasmids

2.2.3.1 Gene fragment addition homology arm

The method for constructing the vector is a homologous recombination method, a multi-fragment plasmid is connected into an expression vector pRS42k by utilizing a homologous recombinase, adding homology arms by using DNA polymerase PrimeStar Max and using a primer (1) Sal1-TEF1p-F & TEF1p-Gfegt1-R/(2) Gfegt1-F & Gfegt1-CYC1t-R/(3) CYC1t-F & CYC1t-TEF1p-R/(4) TEF1p-F & TEF1p-Gfegt2-R/(5) Gfegt2-F & Gfegt2-CYC1t-R/(6) CYC1t-F & CYC1t-EcoRI-R to respectively amplify a TEF1p promoter with a Sal1 site and a homology arm, a target gene Gfegt1 with a homology arm, a CYC1t with a homology arm, a TEF1 promoter with a homology arm, a TEF1p with a homology arm, a DNA polymerase reaction system with a target gene reaction parameter of the GfegEcoRI t:

PCR reaction system with homologous arm:

PCR reaction parameters with homology arms: pre-denaturation at 94 ℃ for 2min, denaturation at 98 ℃ for 10sec, annealing at 55 ℃ for 15sec, extension of all fragments at 72 ℃ for 20sec, followed by a return to the annealing step for a total of 30 cycles, and final extension at 72 ℃ for 5 min.

2.2.3.2 double digestion of vector

Overnight shaking the bacteria, and extracting pRS42k Plasmid with a Plasmid extraction Kit HiPure Plasmid Mini Kit. The plasmid was subsequently double digested with FastDigest EcoR I and FastDiest Sal I, the reaction system and parameters were as follows:

a double enzyme digestion reaction system:

10×FastDigest Buffer	2μL
		EcoR I	1μL
Sal I	1μL
		form panel	1μg
dd H₂O	Make up to 20 mu L
		Total up to	20μL

2.2.3.3 construction of expression plasmids

homologous recombination reaction system:

2×ClonExpress Mix	5μL
		double digestion pRS42k	114ng
Sal1-TEF1p-Gfegt1	6ng
		Gfegt1-CYC1t	42ng
CYC1t-TEF1p	6ng
		TEF1p-Gfegt2	6ng
Gfegt2-CYC1t	78ng
		CYC1t-EcoRI	6ng
dd H₂O	Make up to 10 mu L
		Total up to	10μL

Homologous recombination reaction parameters: incubate at 50 ℃ for 30 min.

The recombined plasmid is transformed into 0.1M CaCl₂Coli DH 5. alpha. competent cells were prepared and colonies were identified using DNA polymerase 2 XTaqMix, procedure referenced 1.2.2. Constructing to obtain an expression vector pRS42k-TEF1p-Gfegt1-CYC1t-TEF1p-Gfegt2-CYC1t, which is abbreviated as pRS42k-Gfegt1-Gfegt 2.

2.2.4 transformation and characterization of Saccharomyces cerevisiae

The selected saccharomyces cerevisiae is EC1118 wine brewing yeast, and the adopted method is a LiAc conversion method. The specific transformation steps are as follows: (1) culturing EC1118 Saccharomyces cerevisiae to OD using YPD medium₆₀₀0.4-0.6; (2) centrifuging the bacterial liquid, resuspending the bacterial liquid without using a small amount of LiAc/TE, and then adding plasmid pRS42k-TEF1p-Gfegt1-CYC1t-TEF1p-Gfegt2-CYC1t for mixing; (3) incubating the mixture in a water bath at 30 ℃ for about half an hour; (4) adding 50% PEG4000 2 times of the volume of the mixed solution, incubating the mixed solution in water bath at 30 ℃ for about half an hour, and performing heat shock in water bath at 42 ℃ for 20 min; (5) YPD medium was added to the mixture for recovery, followed by plating on a YPD plate containing G418(100mg/mL) and culturing at 30 ℃. (6) Identifying a saccharomyces cerevisiae single colony by using DNA polymerase KOD Fx, wherein a saccharomyces cerevisiae colony identification reaction system and parameters are as follows:

a saccharomyces cerevisiae colony identification reaction system:

2×PCR Buffer for KOD FX	12.5μL
		2mM dNTPs	5μL
RV-M/M13-47	2μL
		KOD FX	0.5μL
dd H containing 1 colony₂O mixed solution	5μL
		Total up to	25μL

2.2.5 characterization of synthetase function

2.2.5.1 induced fermentation of the modified strains

The plasmid constructed in this experiment has a type of promoter, and the TEF1p promoter is induced to express in YPD medium regardless of the type of terminator. (1) Selecting a saccharomyces cerevisiae EC1118 modified strain to 50mL YPD culture medium, and performing shake culture (30 ℃, 200rpm) to enrich the thallus; (2) after 2 days of culture, substrate was added for reaction, and the system was as follows:

2.2.5.2 ergothioneine product detection

Centrifuging the culture solution after 7 days fermentation, collecting all thallus, adding 10mL of 50% ethanol, extracting ergothioneine with 50 deg.C hot water bath for 2 hr, centrifuging, collecting supernatant, removing impurities with 0.22 μm organic phase filter head, filtering, and performing HPLC analysis.

HPLC conditions reference method of Liu (2016):

2.3 results and analysis

2.3.1 extraction and reverse transcription of Grifola frondosa RNA

2.3.2 amplification of the Gene of interest

2.3.3 construction of engineered Strain

The expression vector was constructed by the same group method, and the construction results are shown in FIG. 7, in which TEF1p is a translation elongation factor promoter and CYC1t is a cytochrome C terminator. There were no mutations after sequencing by sequencing company. This plasmid was subsequently transformed into s.cerevisiae EC1118 by the heat shock method.

2.3.4HPLC detection of product formation.

The ergothioneine standard curve is:

ergothioneine content (mg/L) ═ 0.00003 peak area-1.1101 (R)²＝0.9951)

The peak time of the ergothioneine standard is 30.056min (figure 8), no obvious peak type appears in the wild type fermentation culture solution (figure 9) near the peak time, the modified strain has an obvious peak at 30.007min (figure 10), and the peak time is matched with the peak time of the standard, which shows that two enzymes expressed in the modified strain have enzyme activity and can convert a substrate into the ergothioneine. According to the prepared ergothioneine standard curve, the final concentration of the ergothioneine in the fermentation liquor is 6.6 mg/L.

Reference documents:

Cheah,I.K.and B.Halliwell.Ergothioneine；antioxidant potential,physiological function and role in disease[J].Biochimica et Biophysica Acta,2012,1822(5):784-793.

Cumming,B.M.,K.C.Chinta,V.P.Reddy and A.J.C.Steyn.Role of Ergothioneine in Microbial Physiology and Pathogenesis[J].Antioxid Redox Signal 2018,28(6):431-444.

Hu,W.,H.Song,A.Sae Her,D.W.Bak,N.Naowarojna,S.J.Elliott,L.Qin,X.Chen and P.Liu.Bioinformatic and biochemical characterizations of C-S bond formation and cleavage enzymes in the fungus Neurospora crassa ergothioneine biosynthetic pathway[J].Organic Letters,2014,16(20):5382-5385.

Irani,S.,N.Naowarojna,Y.Tang,K.R.Kathuria,S.Wang,A.Dhembi,N.Lee,W.Yan,H.Lyu,C.E.Costello,P.Liu and Y.J.Zhang.Snapshots of C-S Cleavage in Egt2Reveals Substrate Specificity and Reaction Mechanism[J].Cell Chemical Biology,2018,25(5):519-529e514.

Kalaras,M.D.,J.P.Richie,A.Calcagnotto and R.B.Beelman.Mushrooms:A rich source of the antioxidants ergothioneine and glutathione[J].Food Chemistry.2017,233:429-433.

Liu,Q.,W.Zhang,H.Wang,Y.Li,W.Liu,Q.Wang,D.Liu,N.Chen and W.Jiang.Validation of a HILIC Method for the Analysis of Ergothioneine in Fermentation Broth[J].J Chromatogr Sci,2016,54(6):934-938.

Osawa,R.,T.Kamide,Y.Satoh,Y.Kawano,I.Ohtsu and T.Dairi.Heterologous and High Production of Ergothioneine in Escherichia coli[J].Journal of Agricultural and Food Chemistry,2018,66(5):1191-1196.

Tanret,C.Sur une base nouvelle retiree du seigle ergote,l'ergothioneine[J].Rend.Acad.Sci..1909,149:222-224.

Taxis,C.and M.Knop.System of centromeric,episomal,and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae[J].Biotechniques 2006,40(1):73-78.

no tax, Zheng Xiao Bing, Linjunfang, Guoliqiong, simple and high quality edible mushroom total RNA extraction method [ J ] edible mushroom academic newspaper, 2018(15):35-39.

Application of Junfang, Linshuoxing, Yangxueqin, Guoqiong needle mushroom genes Fvegt1, Fvegt2 and Fvegt3 in synthesis of ergothioneine, and patent application No. 201811332635.6,2018

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 grifola frondosa ergothioneine genes Gfegt1 and Gfegt2 in ergothioneine synthesis

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2580

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt1 nucleotide sequence

<400> 1

atgtcgaccc tccaggattt cttccatatc gtggaccttc gtgccaacca gccaacactt 60

gcttccagcg tcatccatga acaagttgtt tccggtctct cgcaacctgc gggccagaaa 120

tggcttccca caatgctcct ctacgatgag aggggattga ggctgtacga tgccattacg 180

acagaggcgc ccgaatacta tttgtttccc gccgaggaag agatcctgaa gaaccggtct 240

tccgatattg tgcgggtcat gcatgcgcgg aacgggaatg cagagtcagt tgaagaggtc 300

gtcgttgagc ttggtgctgg tgctttaagg aagacatccc acatcctccg cgctctctca 360

cagcacagta tgtcttccgt ccagtactac gccctcgacc ttgagaagcg cgaactcgaa 420

cgcactctca agacactaca tgactctgaa attggagcag agatcaagga taaggtctct 480

actaagggct tgtgcggaac atatgacgac ggcctcaagt tcatcgccga gggtggcctg 540

gaaggacgca acgatcttga acggatcact actgaagtct ccgagcaata taagctcgag 600

agggttggcg gtgacgattc gcctagatct gcgtcttcct cgaggacacc tacgacggag 660

acagatgtca cacctccatc gacccctggt tttaaccagc cgcttcatat tctcttcctc 720

ggttcatcgc tcggcaactt cactcgtggt gaggatgctg cattcttacg atccttgccg 780

ttgcgacctg gttcaggcga tacattgctc ctgggcctcg accacgacaa tgaagcccat 840

cagattgagc tcgcatataa tgaccccaaa ggtatcacca agaatttcat tatgaacggc 900

ttgaaatgtg caggaagagc tcttggggac gagcacctct ttgatgaaga taaatgggag 960

tatgtcgcga tgtacaacga agaactccgt cgtcatgagg cttactacaa gtcaacgtgt 1020

gagcaaacgg ttgtggacac aaagactaag aagtgtctcc cgttcgaagc agacgagctc 1080

gtccgcatcg aggtttccta caagttctct gagcgagacg cgtacactct ttttactgac 1140

gccaatctcc gccccattca acgctggatg gacagcgctg ggcagtattc tctctggctg 1200

ctagagcgac ctaagttcac gttccctcta ctgcgctcgc cttccgccat tgatgagaag 1260

ggtgtggttt cttctccttt cggcatgcca gcaatggacg agtggcacac aatgtgggcg 1320

gcttgggact ttatcactag acagatgata cccccttcga tgctcttcca gaagccgatc 1380

gatctacgtc atatatgcct gttctattgc gggcatattc ctgcgttcct gtccattcat 1440

atttcgaagc ttcttgagga gccggacacc gaacctgtgg agttcaaata tatttttgaa 1500

cgagggatcg atccaattgt ggatgacccc accaagtgcc accctcactc tgaggttccg 1560

cagcatgacg aggattggcc ttcgctcggg agcattcttg aataccaatc tagggtgcgc 1620

gagcgagtga tgaaattata ccgcgatatc cagtctggga aagttacgct cacgaggaag 1680

atagccagag tcttgtttat gacactcgaa cacgaagctt tccatgctga gacactcttg 1740

tatatgttgt tgcagcgcgc gggcacgggc acattgcctc ctacgggctt cagtccgcca 1800

gtctggtctg tcctcgccga gtcttgggaa cgcctccctg ctccgcatac tcccaccgtg 1860

acgctcggtc cggagacact gacggtcgga catgatgaca gcgaagcgga tgacaatacc 1920

accgacgtgg ctggacatga gtttggctgg gacaacgagc accccaaaag gaccgtgcat 1980

gttccggaat tcaagatcga gtggcgccct gttacgaacg gagagttcta cgagttctac 2040

attggagaag gcaaggaaca agtgcagttg cctgccagct gggtggagat cgacggggag 2100

atgctggtgc gcaccttcta cggaccagtc ccgatgaaag tggcaaaaga ctggccggtc 2160

atcacgtcct acgataatct ctctacctac gccagcgtca agggcggccg catccccacc 2220

gagcctgaac tccgcttgtt cctcgacaag ttcgagtgcg gatacgaagg cggggcaaat 2280

attggcttcc gcaactggca ccctattccg gcgactatgg gcggggtgaa agatggccgg 2340

ggacacaacg gaggtgtctg ggagtggacg tcgacggtgt tcgagaaaca tgacggtttt 2400

gtgccgtcca agctgtatcc gggatattcg atggatttct tcgataccca ccatcaaatt 2460

gtgatcggag gctcctacgc cactattccc cgtctcgcgg agcggcgtac cttgcgtaac 2520

tactaccaac acaactaccc ctacgcgtgg gtcggcgctc ggattgcgta tgatgtgtaa 2580

<210> 2

<211> 859

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt1 amino acid sequence

<400> 2

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

1 5 10 15

Gln Pro Thr Leu Ala Ser Ser Val Ile His Glu Gln Val Val Ser Gly

20 25 30

Leu Ser Gln Pro Ala Gly Gln Lys Trp Leu Pro Thr Met Leu Leu Tyr

35 40 45

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

50 55 60

Glu Tyr Tyr Leu Phe Pro Ala Glu Glu Glu Ile Leu Lys Asn Arg Ser

65 70 75 80

Ser Asp Ile Val Arg Val Met His Ala Arg Asn Gly Asn Ala Glu Ser

85 90 95

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

100 105 110

Ser His Ile Leu Arg Ala Leu Ser Gln His Ser Met Ser Ser Val Gln

115 120 125

Tyr Tyr Ala Leu Asp Leu Glu Lys Arg Glu Leu Glu Arg Thr Leu Lys

130 135 140

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

145 150 155 160

Thr Lys Gly Leu Cys Gly Thr Tyr Asp Asp Gly Leu Lys Phe Ile Ala

165 170 175

Glu Gly Gly Leu Glu Gly Arg Asn Asp Leu Glu Arg Ile Thr Thr Glu

180 185 190

Val Ser Glu Gln Tyr Lys Leu Glu Arg Val Gly Gly Asp Asp Ser Pro

195 200 205

Arg Ser Ala Ser Ser Ser Arg Thr Pro Thr Thr Glu Thr Asp Val Thr

210 215 220

Pro Pro Ser Thr Pro Gly Phe Asn Gln Pro Leu His Ile Leu Phe Leu

225 230 235 240

Gly Ser Ser Leu Gly Asn Phe Thr Arg Gly Glu Asp Ala Ala Phe Leu

245 250 255

Arg Ser Leu Pro Leu Arg Pro Gly Ser Gly Asp Thr Leu Leu Leu Gly

260 265 270

Leu Asp His Asp Asn Glu Ala His Gln Ile Glu Leu Ala Tyr Asn Asp

275 280 285

Pro Lys Gly Ile Thr Lys Asn Phe Ile Met Asn Gly Leu Lys Cys Ala

290 295 300

Gly Arg Ala Leu Gly Asp Glu His Leu Phe Asp Glu Asp Lys Trp Glu

305 310 315 320

Tyr Val Ala Met Tyr Asn Glu Glu Leu Arg Arg His Glu Ala Tyr Tyr

325 330 335

Lys Ser Thr Cys Glu Gln Thr Val Val Asp Thr Lys Thr Lys Lys Cys

340 345 350

Leu Pro Phe Glu Ala Asp Glu Leu Val Arg Ile Glu Val Ser Tyr Lys

355 360 365

Phe Ser Glu Arg Asp Ala Tyr Thr Leu Phe Thr Asp Ala Asn Leu Arg

370 375 380

Pro Ile Gln Arg Trp Met Asp Ser Ala Gly Gln Tyr Ser Leu Trp Leu

385 390 395 400

Leu Glu Arg Pro Lys Phe Thr Phe Pro Leu Leu Arg Ser Pro Ser Ala

405 410 415

Ile Asp Glu Lys Gly Val Val Ser Ser Pro Phe Gly Met Pro Ala Met

420 425 430

Asp Glu Trp His Thr Met Trp Ala Ala Trp Asp Phe Ile Thr Arg Gln

435 440 445

Met Ile Pro Pro Ser Met Leu Phe Gln Lys Pro Ile Asp Leu Arg His

450 455 460

Ile Cys Leu Phe Tyr Cys Gly His Ile Pro Ala Phe Leu Ser Ile His

465 470 475 480

Ile Ser Lys Leu Leu Glu Glu Pro Asp Thr Glu Pro Val Glu Phe Lys

485 490 495

Tyr Ile Phe Glu Arg Gly Ile Asp Pro Ile Val Asp Asp Pro Thr Lys

500 505 510

Cys His Pro His Ser Glu Val Pro Gln His Asp Glu Asp Trp Pro Ser

515 520 525

Leu Gly Ser Ile Leu Glu Tyr Gln Ser Arg Val Arg Glu Arg Val Met

530 535 540

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

545 550 555 560

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

565 570 575

Glu Thr Leu Leu Tyr Met Leu Leu Gln Arg Ala Gly Thr Gly Thr Leu

580 585 590

Pro Pro Thr Gly Phe Ser Pro Pro Val Trp Ser Val Leu Ala Glu Ser

595 600 605

Trp Glu Arg Leu Pro Ala Pro His Thr Pro Thr Val Thr Leu Gly Pro

610 615 620

Glu Thr Leu Thr Val Gly His Asp Asp Ser Glu Ala Asp Asp Asn Thr

625 630 635 640

Thr Asp Val Ala Gly His Glu Phe Gly Trp Asp Asn Glu His Pro Lys

645 650 655

Arg Thr Val His Val Pro Glu Phe Lys Ile Glu Trp Arg Pro Val Thr

660 665 670

Asn Gly Glu Phe Tyr Glu Phe Tyr Ile Gly Glu Gly Lys Glu Gln Val

675 680 685

Gln Leu Pro Ala Ser Trp Val Glu Ile Asp Gly Glu Met Leu Val Arg

690 695 700

Thr Phe Tyr Gly Pro Val Pro Met Lys Val Ala Lys Asp Trp Pro Val

705 710 715 720

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

725 730 735

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

740 745 750

Cys Gly Tyr Glu Gly Gly Ala Asn Ile Gly Phe Arg Asn Trp His Pro

755 760 765

Ile Pro Ala Thr Met Gly Gly Val Lys Asp Gly Arg Gly His Asn Gly

770 775 780

Gly Val Trp Glu Trp Thr Ser Thr Val Phe Glu Lys His Asp Gly Phe

785 790 795 800

Val Pro Ser Lys Leu Tyr Pro Gly Tyr Ser Met Asp Phe Phe Asp Thr

805 810 815

His His Gln Ile Val Ile Gly Gly Ser Tyr Ala Thr Ile Pro Arg Leu

820 825 830

Ala Glu Arg Arg Thr Leu Arg Asn Tyr Tyr Gln His Asn Tyr Pro Tyr

835 840 845

Ala Trp Val Gly Ala Arg Ile Ala Tyr Asp Val

850 855

<210> 3

<211> 1434

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt2 nucleotide sequence

<400> 3

atgacggcca tagatctagg tgcagcgtct gccgacgaga cccagaagaa cacttatgat 60

gcaacccaaa agccgcctcc tttcggtcat gccttgaagc cttactgggc atttgatcca 120

aaatatgtaa atctaaacca cggctcctat ggatcattgc ctctgcctgt tctattttct 180

tgcacccaga atacgattct cgcagagcgg aatcccgaca aattccaccg cgtcacgtat 240

atgcctatgc tccaggagtc caggaaacgc gtggcagaac tggttggtgc tgaacacgac 300

gagatcgtgc tcgtgcctaa tgccactcac ggcttgaaca ccgtgctcag aaattttgag 360

tggaagcaag gcgacgtcat tattggagca tcgacgacat atggtgccat ctctcgcacc 420

atccaatacc tcgcggatcg atcggaacag ccaagacccg aagcatatag cattcagtat 480

acgttcccca tgtcgcacgc agagatcctc gatgccttcc gtgcacgcgt gcgggagatc 540

aagcagctcc atgcgagcac cgaattcagc gacgcgccgt tggagtcgct gggctacgag 600

gaaggcagga agaagaacaa gttcgtcgca gttatagact cggtgactgc caaccctggg 660

gtcctcatgc cctggaaaga gatggtccgc gtctgcaggg aagaaggcat ctggtctgtt 720

gtagatgctg ctcatagcat cgggcaggaa acggatatca atctcagcga agcgaggcct 780

gatttctgga tatccaactg tcataagtgg ctttacgcaa aacggggctg tgccacctta 840

tatgtgccca aacgtaacca gtatatcatc aagtcttcta ttccgacttc acacgcgtat 900

gtctcgccta ccgacacaga acaggccctg caattccggg atgggtatga cacgaatttc 960

atcctgcagc atgaatggac agggacgatg gacttcattc catacttgag cgtctctgca 1020

gcgctcgact tccgcaactg gctgggaggg gaggccgcca tcaacgggta ctgccacaag 1080

ctcgccatgg ccggcggcga gaggctcgcc agcgtgatgg gcacgaaggt catggacaag 1140

accggcgagc tcacgctcaa catgacgaac gtcctactgc cgctccccgt ggagactacg 1200

aagggcgagg tgtattctgg ggaggtcctg tccgcgattt acagccaact cagggagaag 1260

ctgctgtacg agtggaacac ctacgcggca cactacttcc acgcgggcgg ctggtggtgc 1320

cggtgcagcg cacaggtctg gaacgaggaa tcggactttg agtatctggg aaaggcattc 1380

aatgcaatct gcaaggaaat caaggatacc cttctcgcag agaagcgtaa ttag 1434

<210> 4

<211> 477

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt2 amino acid sequence

<400> 4

Met Thr Ala Ile Asp Leu Gly Ala Ala Ser Ala Asp Glu Thr Gln Lys

1 5 10 15

Asn Thr Tyr Asp Ala Thr Gln Lys Pro Pro Pro Phe Gly His Ala Leu

20 25 30

Lys Pro Tyr Trp Ala Phe Asp Pro Lys Tyr Val Asn Leu Asn His Gly

35 40 45

Ser Tyr Gly Ser Leu Pro Leu Pro Val Leu Phe Ser Cys Thr Gln Asn

50 55 60

Thr Ile Leu Ala Glu Arg Asn Pro Asp Lys Phe His Arg Val Thr Tyr

65 70 75 80

Met Pro Met Leu Gln Glu Ser Arg Lys Arg Val Ala Glu Leu Val Gly

85 90 95

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

100 105 110

Asn Thr Val Leu Arg Asn Phe Glu Trp Lys Gln Gly Asp Val Ile Ile

115 120 125

Gly Ala Ser Thr Thr Tyr Gly Ala Ile Ser Arg Thr Ile Gln Tyr Leu

130 135 140

Ala Asp Arg Ser Glu Gln Pro Arg Pro Glu Ala Tyr Ser Ile Gln Tyr

145 150 155 160

Thr Phe Pro Met Ser His Ala Glu Ile Leu Asp Ala Phe Arg Ala Arg

165 170 175

Val Arg Glu Ile Lys Gln Leu His Ala Ser Thr Glu Phe Ser Asp Ala

180 185 190

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

195 200 205

Val Ala Val Ile Asp Ser Val Thr Ala Asn Pro Gly Val Leu Met Pro

210 215 220

Trp Lys Glu Met Val Arg Val Cys Arg Glu Glu Gly Ile Trp Ser Val

225 230 235 240

Val Asp Ala Ala His Ser Ile Gly Gln Glu Thr Asp Ile Asn Leu Ser

245 250 255

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

260 265 270

Ala Lys Arg Gly Cys Ala Thr Leu Tyr Val Pro Lys Arg Asn Gln Tyr

275 280 285

Ile Ile Lys Ser Ser Ile Pro Thr Ser His Ala Tyr Val Ser Pro Thr

290 295 300

Asp Thr Glu Gln Ala Leu Gln Phe Arg Asp Gly Tyr Asp Thr Asn Phe

305 310 315 320

Ile Leu Gln His Glu Trp Thr Gly Thr Met Asp Phe Ile Pro Tyr Leu

325 330 335

Ser Val Ser Ala Ala Leu Asp Phe Arg Asn Trp Leu Gly Gly Glu Ala

340 345 350

Ala Ile Asn Gly Tyr Cys His Lys Leu Ala Met Ala Gly Gly Glu Arg

355 360 365

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

370 375 380

Thr Leu Asn Met Thr Asn Val Leu Leu Pro Leu Pro Val Glu Thr Thr

385 390 395 400

Lys Gly Glu Val Tyr Ser Gly Glu Val Leu Ser Ala Ile Tyr Ser Gln

405 410 415

Leu Arg Glu Lys Leu Leu Tyr Glu Trp Asn Thr Tyr Ala Ala His Tyr

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt1-F

<400> 5

atgtcgaccc tccaggattt cttc 24

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt1-R

<400> 6

ttacacatca tacgcaatcc gagcg 25

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt2-F

<400> 7

atgacggcca tagatctagg tgc 23

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt2-R

<400> 8

ctaattacgc ttctctgcga gaagg 25

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pYES2-Gfegt2-F

<400> 9

gactcactat agggaatatt aatgacggcc atagatctag g 41

<210> 10

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pYES2-Gfegt2-R

<400> 10

aaagcaatta cagtcccggg ctagtggtgg tggtggtggt gattacgctt ctctgcgag 59

<210> 11

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pYES2-Gfegt1-F

<400> 11

tacaacaaat ataaaacatt aattaaatgt cgaccctcca ggatttc 47

<210> 12

<211> 59

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pYES2-Gfegt1-R

<400> 12

acgaggcaag ctaaacagat ctttagtggt ggtggtggtg gtgcacatca tacgcaatc 59

<210> 13

<211> 52

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pYES2-KanMX4-F

<400> 13

tttagcttgc ctcgtccccg ccgggtcacc cggccagcga catggaggcc ca 52

<210> 14

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pYES2-KanMX4-R

<400> 14

gatggatatc tgcagaatta gtatagcgac cagcattcac 40

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Sc-pPGK-F

<400> 15

actgtaattg cttttagttg tg 22

<210> 16

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Sc-pPGK-R

<400> 16

tgttttatat ttgttgtaaa aagtagata 29

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> RV-M

<400> 17

gagcggataa caatttcaca cagg 24

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> M13-47

<400> 18

cgccagggtt ttcccagtca cgac 24

<210> 19

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt2-CYC1t-R

<400> 19

acataactaa ttacatgact agtggtggtg gtggtggtga ttacgcttct ctgcgag 57

<210> 20

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Gfegt1-CYC1t-R

<400> 20

gacataacta attacatgat tagtggtggt ggtggtggtg cacatcatac gcaat 55

<210> 21

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TEF1p-Gfegt2-R

<400> 21

acctagatct atggccgtca tggttgttta tgttcgg 37

<210> 22

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TEF1p-Gfegt1-R

<400> 22

gaaatcctgg agggtcgaca tggttgttta tgttcg 36

<210> 23

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Sal1-TEF1p-F

<400> 23

ccccctcgag gtcgaccagc gacatggagg ccca 34

<210> 24

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CYC1t-EcoRI-R

<400> 24

cccgggctgc aggaattcgc aaattaaagc cttc 34

<210> 25

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CYC1t-TEF1p-R

<400> 25

tgggcctcca tgtcgctggc aaattaaagc cttc 34

<210> 26

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TEF1p-F

<400> 26

cagcgacatg gaggcccag 19

<210> 27

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CYC1t-F

<400> 27

tcatgtaatt agttatgtc 19

<210> 28

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pYES2-T7

<400> 28

taatacgact cactataggg 20

<210> 29

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pYES2-R

<400> 29

tcggttagag cggatgtg 18

Claims

1. Grifola frondosa ergothioneine geneGfegt1AndGfegt2the application of the ergothioneine in the joint synthesis is characterized in that:

the grifola frondosa ergothioneine geneGfegt1The coded amino acid is shown as SEQ ID NO. 2;

the grifola frondosa ergothioneine geneGfegt2The coded amino acid is shown in SEQ ID NO. 4.

2. Use according to claim 1, characterized in that: the grifola frondosa ergothioneine geneGfegt1AndGfegt2the application of the ergothioneine in the in vitro combined biosynthesis.

3. Use according to claim 2, characterized in that: the grifola frondosa ergothioneine geneGfegt1AndGfegt2the application of the ergothioneine in vivo synthesis of saccharomyces cerevisiae.

4. Use according to any one of claims 1 to 3, characterized in that:

the grifola frondosa ergothioneine geneGfegt1The nucleotide sequence of (A) is shown as SEQ ID NO. 1;

the grifola frondosa ergothioneine geneGfegt2The nucleotide sequence of (A) is shown in SEQ ID NO. 3.

5. Grifola frondosa ergothioneine geneGfegt1An encoded protein Gfegt1, characterized by: the amino acid sequence is shown as SEQ ID NO:2, respectively.

6. Grifola frondosa ergothioneine gene encoding the protein of claim 5Gfegt1The method is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 1.

7. Grifola frondosa ergothioneine geneGfegt2An encoded protein Gfegt2, characterized by: the amino acid sequence is shown as SEQ ID NO:4, respectively.

8. Grifola frondosa ergothioneine gene encoding the protein of claim 7Gfegt2The method is characterized in that: the nucleotide sequence is shown in SEQ ID NO. 3.

9. A recombinant expression vector characterized by: the pYES2 as the starting vector is inserted into the vector of claim 1Gfegt2、pPGKThe method of claim 1Gfegt1、KanMX4Obtaining; alternatively, pRS42k was used as a starting vector and inserted in orderTEF1pThe method of claim 1Gfegt1、CYC1t、TEF1pThe method of claim 1Gfegt2、CYC1tAnd (4) obtaining.

10. A recombinant bacterium, which is characterized in that: comprising the recombinant expression vector of claim 9.