CN114107326A

CN114107326A - Two ergothioneine synthetic proteins and application thereof in ergothioneine synthesis

Info

Publication number: CN114107326A
Application number: CN202010903383.9A
Authority: CN
Inventors: 董志扬; 陈秀珍; 何永志; 陈智慧; 张山; 董亮
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-09-01
Filing date: 2020-09-01
Publication date: 2022-03-01

Abstract

The invention discloses two ergothioneine synthetic proteins and application thereof in ergothioneine synthesis. The two ergothioneine synthetic proteins disclosed by the invention are respectively named as Tregt1 and Tregt2, and Tregt1 is a protein with an amino acid sequence of sequence 2; tregt2 is a protein whose amino acid sequence is seq id No. 4. Experiments prove that the Tregt1 and Tregt2 can be used for preparing ergothioneine, the yield can reach 4.5g/L in 144 hours, the yield is 3.46 times of the highest production level reported at present, and the time is shortened by 72 hours. The Tregt1 and Tregt2 and the coding genes thereof have wide application prospects.

Description

Two ergothioneine synthetic proteins and application thereof in ergothioneine synthesis

Technical Field

The invention relates to two ergothioneine synthetic proteins and application thereof in ergothioneine synthesis in the field of biotechnology.

Background

Ergothioneine (2-mercapto-L-histidine trimethyl inner salt, Ergothionine, ERG) is a rare natural mercapto imidazole-containing amino acid, has strong antioxidant capacity and cell protection function, and can be used for scavenging hydroxy free radical (. OH), hypochlorous acid (HOCl), peroxynitrite (ONOO-), activating antioxidase, chelating divalent metal ion (such as Cu)²⁺) The health-care product plays an important role in preventing ultraviolet injury, inhibiting hemoglobin oxidation, resisting inflammation, resisting aging, inhibiting tumor generation and the like. The ergothioneine has unique and diversified biological functions, so that the ergothioneine has wide application prospect in the industries of medicine, food, health care products, cosmetics and the like.

Some fungi, actinomycetes bacteria, unicellular cyanobacteria can synthesize ergothioneine, animals and plants can not synthesize ergothioneine, and the ergothioneine can only be obtained through diet or from the surrounding environment. Ergothioneine is mainly produced by chemical synthesis and natural extraction. The ergothioneine is extracted by fermenting edible fungus hypha, the fermentation period is long, and the yield is low; and the safety of the product obtained by chemical synthesis cannot be ensured. Although biosynthesis of ergothioneine has been achieved in various systems such as Escherichia coli, Bacillus, Aspergillus oryzae, and Saccharomyces cerevisiae, the efficiency and yield of ergothioneine synthesis are still poor in economic competitiveness, and it is difficult to achieve wide application. Therefore, it is necessary to find new genes and new methods for further increasing the ergothioneine production.

Trichoderma reesei (Trichoderma reesei) is a food-safe strain approved by the FDA in the United states and widely used for the production of cellulase, but no ergothioneine synthesis has been reported.

Disclosure of Invention

The invention mainly aims to provide application of a group of Trichoderma reesei genes tregt1 and tregt2 in synthesis of ergothioneine.

The invention firstly provides a protein set which can be used for producing ergothioneine, the protein set consists of proteins with the names of Tregt1 and Tregt2, the Tregt1 and the Tregt2 are both from Trichoderma reesei (Trichoderma reesei), and the Tregt1 is A1), A2) or A3 as follows:

A1) the amino acid sequence is the protein of sequence 2;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N terminal or/and the C terminal of A1) or A2);

the Tregt2 is C1), C2), or C3) as follows:

C1) the amino acid sequence is the protein of sequence 4;

C2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4 in the sequence table and has the same function;

C3) c1) or C2) at the N-terminus or/and the C-terminus.

In order to facilitate the purification of the protein in A1) or C1), the amino terminus or the carboxyl terminus of the protein consisting of the amino acid sequence shown in sequence 2or 4 in the sequence listing is labeled as shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 2 and having the same function. The protein of C2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 4 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The proteins in A2) and C2) can be artificially synthesized, or obtained by synthesizing the coding genes and then performing biological expression.

The gene encoding the protein of A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID No. 1, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in sequence 1 encodes the protein shown in sequence 2. The gene encoding the protein of C2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID No. 3, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in sequence 3 encodes the protein shown in sequence 4.

The protein set has any one of the following uses:

x1, producing ergothioneine;

x2, preparing ergothioneine products;

x3, preparing an antioxidant or cytoprotective product;

x4, and preparing products for scavenging hydroxyl free radical, hypochlorous acid, peroxynitrite, antioxidase, bivalent metal ion, ultraviolet injury, hemoglobin oxidation, inflammation, aging, and tumor.

The invention also provides a set of biological materials, which are respectively named as biological material 1 and biological material 2, wherein the biological material 1 is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding said Tregt 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a cell line comprising B1) the nucleic acid molecule or a cell line comprising B2) the expression cassette;

the biomaterial 2 is any one of the following D1) to D5):

D1) a nucleic acid molecule encoding said Tregt 2;

D2) an expression cassette comprising the nucleic acid molecule of D1);

D3) a recombinant vector containing the nucleic acid molecule of D1) or a recombinant vector containing the expression cassette of D2);

D4) a recombinant microorganism containing D1) the nucleic acid molecule, or a recombinant microorganism containing D2) the expression cassette, or a recombinant microorganism containing D3) the recombinant vector;

D5) a cell line comprising D1) the nucleic acid molecule or a cell line comprising D2) the expression cassette.

The invention also provides a biological material named biological material 3, wherein the biological material 3 is any one of the following E1) to E4):

E1) an expression cassette comprising the nucleic acid molecule of B1) and the nucleic acid molecule of D1);

E2) a recombinant vector comprising the expression cassette of E1);

E3) a recombinant microorganism containing E1) the expression cassette or a recombinant microorganism containing E2) the recombinant vector;

E4) a cell line comprising the expression cassette described in E1).

In the above biological material, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 1 in the sequence table;

b12) DNA molecule shown in sequence 1 in the sequence table;

b13) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding said Tregt 1;

b14) a cDNA molecule or a DNA molecule hybridizing under stringent conditions to the nucleotide sequence defined in b11) or b12) or b13) and encoding said Tregt 1;

D1) the nucleic acid molecule may be d11) or d12) or d13) or d14) as follows:

d11) the coding sequence is cDNA molecule or DNA molecule of sequence 3 in the sequence table;

d12) DNA molecule shown in sequence 3 in the sequence table;

d13) a cDNA molecule or a DNA molecule having 75% or more identity to a nucleotide sequence defined by d11) or d12) and encoding said Tregt 2;

d14) hybridizes with a nucleotide sequence defined by d11) or d12) or d13) under strict conditions and encodes the cDNA molecule or DNA molecule of the Tregt 2.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequences encoding the Tregt1 and the Tregt2 proteins of the invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides that are artificially modified to have 75% or more identity to the nucleotide sequences of the Tregt1 and the Tregt2 proteins of the invention are derived from and identical to the nucleotide sequences of the invention as long as they encode the Tregt1 and the Tregt2 proteins and have the functions of the Tregt1 and the Tregt2 proteins.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 2or 4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

Hereinbefore, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mMHybridization in EDTA mixed solution, at 65 degrees, 0.1 x SSC, 0.1% SDS rinsing; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

As used herein above, the expression cassette containing a nucleic acid molecule encoding the Tregt1 protein (Tregt1 gene expression cassette) described in B2) refers to a DNA capable of expressing the Tregt1 protein in a host cell, which DNA may include not only a promoter that promotes transcription of the Tregt1 gene but also a terminator that terminates transcription of the Tregt1 gene. D2) The expression cassette containing the nucleic acid molecule encoding the Tregt2 protein (Tregt2 gene expression cassette) refers to DNA capable of expressing the Tregt2 protein in host cells, and the DNA not only can comprise a promoter for starting transcription of the Tregt2 gene, but also can comprise a terminator for stopping transcription of the Tregt2 gene. E1) The expression cassette is DNA capable of expressing Tregt1 and Tregt2 proteins in host cells, and the DNA not only can comprise a promoter for starting the transcription of Tregt1 genes and Tregt2 genes, but also can comprise a terminator for terminating the transcription of Tregt1 genes and Tregt2 genes. Further, the expression cassette may also include an enhancer sequence.

The recombinant vector containing the Tregt1 gene and/or the Tregt2 gene expression cassette can be constructed by using the existing expression vector.

As above, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid can be pBAD/HisA plasmid.

B3) The recombinant vector can be pBAD-tegt1, and pBAD-tegt1 is a recombinant plasmid obtained by inserting the Tregt1 gene into multiple cloning sites of pBAD/HisA plasmid. The pBAD-tegt1 contains a Tregt1 gene shown in a sequence 1 in a sequence table, and can express a Tregt1 protein shown in a sequence 2, and the expression of the Tregt1 gene is driven by a promoter araBAD.

D3) The recombinant vector can be pBAD-tegt2, and pBAD-tegt2 is a recombinant plasmid obtained by inserting the Tregt2 gene into multiple cloning sites of pBAD/HisA plasmid. The pBAD-tegt2 contains a Tregt2 gene shown in a sequence 3 in a sequence table, and can express a Tregt2 protein shown in a sequence 4, and the expression of the Tregt2 gene is driven by a promoter araBAD.

E2) The recombinant vector can be pBAD-tegt1,2, and pBAD-tegt1,2 is a recombinant plasmid obtained by inserting a Tregt1 gene and a Tregt2 gene which are connected by a ribosome binding site RBS of T7 into a pBAD/HisA plasmid. The pBAD-tegt1,2 contains Tregt1 gene and Tregt2 gene shown in sequence 1 and sequence 3 in the sequence table, can express Tregt1 protein shown in sequence 2 and Tregt2 protein shown in sequence 4, and the expression of Tregt1 gene and Tregt2 gene is driven by promoter araBAD.

The microorganism may be a yeast, bacterium, algae or fungus, as hereinbefore described. Wherein the bacteria can be Escherichia coli, such as Escherichia coli BW 25113.

B4) The recombinant microorganism can be BW-pBAD-tegt1, and the BW-pBAD-tegt1 is a recombinant bacterium obtained by introducing the pBAD-tegt1 into Escherichia coli BW 25113.

D4) The recombinant microorganism can be BW-pBAD-tegt2, and the BW-pBAD-tegt2 is a recombinant bacterium obtained by introducing the pBAD-tegt2 into Escherichia coli BW 25113.

E3) The recombinant microorganism can be BW-pBAD-tegt1,2, and the BW-pBAD-tegt1,2 is a recombinant bacterium obtained by introducing the pBAD-tegt1,2 into Escherichia coli BW 25113.

As above, the cell line does not comprise propagation material.

The biomaterial kit has any one of the following uses:

x1, producing ergothioneine;

x2, preparing ergothioneine products;

x3, preparing an antioxidant or cytoprotective product;

Tregt1 or the Tregt2 also belong to the protection scope of the invention.

The biological material 1 or the biological material 2 also belongs to the protection scope of the invention.

The invention also provides any one of the following uses of the protein kit, or the biological material kit or the biological material 3, or the Tregt1 or the Tregt2, or the biological material 1 or the biological material 2:

x1, producing ergothioneine;

x2, preparing ergothioneine products;

x3, preparing an antioxidant or cytoprotective product;

In the application, the product X3 and the product X4 can be medicines, foods, health products or cosmetics.

The present invention also provides a process for the production of ergothioneine, the process comprising: culturing the recombinant microorganism expressing the Tregt1 and/or the Tregt2 to express the Tregt1 and/or the Tregt2, and then continuously culturing the recombinant microorganism in a system containing L-histidine, L-methionine and L-cysteine to obtain the ergothioneine.

In the method, the system containing the L-histidine, the L-methionine and the L-cysteine can be a transformation solution 1 or a transformation solution 2, the transformation solution 1 consists of a solute and a solvent, the solvent is water, and the solute and the concentration of the solute in the transformation solution 1 are respectively 5.8g/L Na₂HPO₄，3g/L KH₂PO₄，0.5g/L NaCl，1g/L NH₄Cl,1g/L L-histidine, 1g/L L-methionine, 1g/L L-cysteine, 20mg/L FeSO₄·7H₂O, 50mmol/L glucose;

the transformation liquid 2 is a culture system which is obtained by feeding materials into an initial system and contains 500g of glucose, 40g of L-cysteine, 40g of L-methionine and 40g of L-histidine in each liter of system,

the initial system contained 10g of glucose per liter, (NH)₄)₂HPO₄ 8g,KH₂PO₄ 13.3g,MgSO₄·7H₂1.2g of O, 1.7g of citric acid, 10mL of trace salt solution, NaOH for adjusting pH and water, and the pH is 7.0;

the trace salt solution consists of a solvent and a solute, wherein the solvent is a 5M hydrochloric acid aqueous solution, and the solute and the concentration of the solute in the trace salt solution are respectively FeSO₄·7H₂O 10g/L,ZnSO₄·7H₂O 2.25g/L,CuSO₄·5H₂O 1g/L,MnSO₄·5H₂O 0.5g/L,Na₂B₄O₇·10H₂O 0.23g/L,CaCl₂·2H₂O2 g/L and (NH)₄)₆Mo₇O₂₄0.1g/L。

In the above method, the recombinant microorganism may be B4) or D4). The culture may be cultured under conditions suitable for growth of the recombinant microorganism, such as 16 ℃ to 30 ℃. The time for said cultivation may be determined according to the growth of said recombinant microorganism or the ergothioneine production, e.g.12-144 h. The culturing may be performed first in a culture system containing arabinose (L-arabinose) to induce the expression of the Tregt1 and the Tregt 2.

The invention also provides a kit consisting of Y1 and Y2:

y1, the protein set, or the biological material set or the biological material 3, or the Tregt1 or the Tregt2, or the biological material 1 or the biological material 2;

y2, the transformation liquid.

The kit has any one of the following uses:

x1, producing ergothioneine;

x2, preparing ergothioneine products;

x3, preparing an antioxidant or cytoprotective product;

Any of the following uses of the kit also fall within the scope of the invention:

x1, producing ergothioneine;

x2, preparing ergothioneine products;

x3, preparing an antioxidant or cytoprotective product;

Experiments prove that the Tregt1 and Tregt2 can be used for preparing ergothioneine, the yield can reach 4.5g/L in 144 hours, the yield is 3.46 times of the highest production level reported at present (Osawa R, Kamid T, Satoh Y, et al. hetelologue and high production of ergothionine in Escherichia coli [ J ]. Journal of Agricultural and Food Chemistry,2017,66: 1191-1196), and the time is shortened by 72 hours. The Tregt1 and Tregt2 and the coding genes thereof have wide application prospects.

Drawings

FIG. 1 is a vector map. A: expression vector pBAD-tegt1, 2; b: expression vector pBAD-tegt 1; c: expression vector pBAD-tegt 2.

FIG. 2 shows the expression of SDS-PAEG protein. Lane M is a protein molecular weight standard; lane 1: negative control histone-like (supernatant); lane 2: BW-pBAD-tegt1,2 cell disruption liquid protein-like (supernatant); lane 3: BW-pBAD-tegt1 cell debris protein sample (supernatant); lane 4: BW-pBAD-tegt2 cell debris protein sample (supernatant); lane 5: negative control histone-like (whole bacteria); lane 6: BW-pBAD-tegt1,2 cell disruption liquid protein-like (whole bacteria); lane 7: BW-pBAD-tegt1 cell disruption liquid protein-like (whole bacteria); lane 8: BW-pBAD-tegt2 cell disruption liquid protein-like (whole bacteria); the upper arrow in the figure indicates the Tregt1 protein and the lower arrow indicates the Tregt2 protein.

FIG. 3 shows the HPLC detection results of ergothioneine synthesis by whole-cell catalysis. A: 20mg/mL ergothioneine standard; b: negative control whole cell catalyzed conversion products; c: BW-pBAD-tegt1,2 whole cell catalyzed post-transformation product; d: BW-pBAD-tegt1 whole cell catalyzed transformation product; e: BW-pBAD-tegt2 whole cell catalyzed post-transformation product.

FIG. 4 shows the results of secondary mass spectrometry of the conversion products. A: 10mg/mL ergothioneine standard; b: BW-pBAD-tegt1,2 whole-cell catalyzed transformation product.

FIG. 5 shows the high density fermentation of E.coli strain BW-pBAD-tegt1, 2.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

PBS buffer (pH 7.0): 380ml of 0.1M sodium dihydrogenphosphate solution was added to 620ml of 0.1M disodium hydrogenphosphate solution to prepare 100mM PBS buffer.

The conversion solution consists of solute and solvent, the solvent is water,the solute and the concentration thereof in the conversion solution are respectively 5.8g/L Na₂HPO₄，3g/L KH₂PO₄，0.5g/L NaCl，1g/L NH₄Cl,1g/L L-histidine, 1g/L L-methionine, 1g/L L-cysteine, 20mg/L FeSO₄·7H₂O, 50mmol/L glucose.

Seed medium (1L): 16g of peptone, 10g of yeast extract, 5g of sodium chloride and pH 7.0.

Fermentation medium (1L): glucose 10g, (NH)₄)₂HPO₄ 8g,KH₂PO₄ 13.3g,MgSO₄·7H₂O1.2 g, citric acid 1.7g and trace salt solution 10mL, the volume is adjusted to 1L by water, and the pH value is adjusted to 7.0 by 5M NaOH.

The trace salt solution consists of solvent and solute, the solvent is 5M hydrochloric acid water solution, and the solute and the concentration thereof in the trace salt solution are respectively FeSO₄·7H₂O 10g/L,ZnSO₄·7H₂O 2.25g/L,CuSO₄·5H₂O 1g/L,MnSO₄·5H₂O 0.5g/L,Na₂B₄O₇·10H₂O 0.23g/L,CaCl₂·2H₂O2 g/L and (NH)₄)₆Mo₇O₂₄ 0.1g/L。

Example 1 the ergothioneine Synthesis proteins Tregt1 and Tregt2 of Trichoderma reesei can synthesize ergothioneine

1. Construction of E.coli expression vectors

pBAD-tegt1，2：

Extracting RNA of Trichoderma reesei (Trichoderma reesei) QM9414(ATCC 26921), performing reverse transcription to obtain cDNA, performing PCR amplification by using primers P1(Tregt1-F, Tregt1-R) and P2(Tregt2-F, Tregt2-R) by using the obtained cDNA as templates, and respectively recording PCR products with correct sequences as Tregt1(2529bp) and Tregt2(1438 bp); PCR amplification is carried out by taking pBAD/HisA plasmid (invitrogen, V430-01) as a template and using a primer pBAD-F, pBAD-R to obtain a DNA fragment with a correct sequence, namely a linearized vector pBADve (3993bp), wherein the sequences of the used primers are as follows:

Tregt1-F：5’-ATGGCGCTCAAGTCAGCC-3’；

Tregt1-R：5’-GGTATATCTCCTTCTTAAAGTTAAACATCAAACATCTCGCACCAACC-3’；

Tregt2-F：5’-TTTAACTTTAAGAAGGAGATATACCATGGCGTCTCTGCCGG-3’；

Tregt2-R：5’-TCAAATCTTCTGGCCACCC-3’；

pBAD-F：5’-AAGGGTGGCCAGAAGATTTGAGGCTGTTTTGGCGGATGAG-3’；

pBAD-R：5’-TCGTGGCTGACTTGAGCGCCATGGTTAATTCCTCCTGTTAGCCC-3’。

using a recombinant cloning kit (Clone)

The MultiS One Step Cloning Kit, Novozan (C113-02)) connects Tregt1, Tregt2 and pBADve in three fragments, transforms them into competent cells Trans1-T1 (phase chemical content Cell, all-round gold (CD501-03)) by calcium chloride chemical transformation, picks up positive clones, extracts plasmids for sequencing, and records the resulting recombinant plasmids with correct sequence as pBAD-tegt1,2 (the vector map is shown in A in FIG. 1), pBAD-tegt1,2, the structure is described as follows: pBAD-tegt1,2 is a recombinant plasmid obtained by inserting Tregt1 gene and Tregt2 gene into pBAD/HisA plasmid, wherein the Tregt1 gene and Tregt2 gene are connected through ribosome binding site RBS of T7, the promoter is araBAD, the screening marker is ampicillin Amp, and the replication origin is pBR322 ori. The pBAD-tegt1,2 contains Trichoderma reesei ergothioneine synthesis protein Tregt1 gene and Tregt2 gene, the sequences of the Tregt1 gene and the Tregt2 gene are respectively

sequences

1 and 3 in a sequence table, and the Tregt1 protein shown in the sequence 2 and the Tregt2 protein shown in the sequence 4 can be expressed.

pBAD-tegt1：

PCR was carried out using the recombinant plasmid pBAD-tegt1,2 as a template and primers P3(Tregt1-F, Tregt1a-R) and P4(pBADA-F, pBAD-R), and the obtained PCR products with correct sequences were designated as Tregt1a (2522bp) and pBADvea (3972bp), respectively, and the sequences of the primers used were as follows:

Tregt1-F：5’-ATGGCGCTCAAGTCAGCC-3’；

Tregt1a-R：5’-TCTCATCCGCCAAAACAGCCTCAAACATCTCGCACCAACCTT-3’；

pBADa-F：5’-GGCTGTTTTGGCGGATGAGA-3’；

pBAD-R：5’-TCGTGGCTGACTTGAGCGCCATGGTTAATTCCTCCTGTTAGCCC-3’。

using a recombinant cloning kit (Clone)

The MultiS One Step Cloning Kit, Novozan (C113-02)) seamlessly splices Tregt1a and pBADvea, transforms into competent cells Trans1-T1 (phase chemical content Cell, all-round gold (CD501-03)) by calcium chloride chemical transformation, picks positive clones, extracts plasmids for sequencing, and records the resulting recombinant plasmid with correct sequence as pBAD-tegt1 (the vector map is shown as B in FIG. 1), and the structure of pBAD-tegt1 is described as follows: the Tregt1 gene is inserted into the multiple cloning site of pBAD/HisA plasmid, the promoter is araBAD, the selection marker is ampicillin Amp, and the replication origin is pBR322 ori. The pBAD-tegt1 contains a Trichoderma reesei ergothioneine synthetic protein Tregt1 gene, the sequence of the Tregt1 gene is a sequence 1 in a sequence table, and the Tregt1 protein shown in a sequence 2 can be expressed.

pBAD-tegt2：

PCR amplification was performed using plasmid pBAD-tegt1,2 as a template and primers P5(Tregt2b-F, Tregt2-R) and P6(pBAD-F, pBADb-R), and the obtained PCR products with correct sequences were designated as Tregt2b (1413bp) and pBADveb (3991bp), respectively, and the sequences of the primers used were as follows:

Tregt2b-F：5’-ATGGCGTCTCTGCCGGTT-3’；

Tregt2-R：5’-TCAAATCTTCTGGCCACCC-3’；

pBAD-F：5’-AAGGGTGGCCAGAAGATTTGAGGCTGTTTTGGCGGATGAG-3’；

pBADb-R：5’-CGAACCGGCAGAGACGCCATGGTTAATTCCTCCTGTTAGCCCA-3’。

using a recombinant cloning kit (Clone)

The Multi S One Step Cloning Kit, Novozam (C113-02)) seamlessly spliced Tregt2b and pBADveb and transformed into competent cells Trans1-T1 by calcium chloride chemical transformation(Phage chemical company Cell, all-type gold (CD501-03)), positive clones were picked, plasmids were extracted and sequenced, and the resulting recombinant plasmid with the correct sequence was designated pBAD-tegt2 (the vector map is shown in FIG. 1C), and the structure of pBAD-tegt2 was described as follows: the Tregt2 gene is inserted into the multiple cloning site of pBAD/HisA plasmid, the promoter is araBAD, the selection marker is ampicillin Amp, and the replication origin is pBR322 ori. The pBAD-tegt2 contains a Trichoderma reesei ergothioneine synthetic protein Tregt2 gene, the sequence of the Tregt2 gene is a sequence 3 in a sequence table, and the Tregt2 protein shown in a sequence 4 can be expressed.

2. Obtaining of recombinant expression bacterium of escherichia coli ergothioneine

The pBAD-tegt1,2, pBAD-tegt1 and pBAD-tegt2 obtained in step 1 were introduced into E.coli BW25113(Thermo Cat # OEC5042), the recombinant bacteria obtained were designated BW-pBAD-tegt1,2, BW-pBAD-tegt1 and BW-pBAD-tegt2, respectively, and BW-pBAD obtained by introducing plasmid pBAD/HisA into E.coli BW25113 was used as a negative control.

3. Induction, expression and detection of proteins

Respectively taking the BW-pBAD-tegt1,2, BW-pBAD-tegt1, BW-pBAD-tegt2 and BW-pBAD obtained in the step 2 as strains to be detected, and carrying out protein expression and detection according to the following steps:

transferring the overnight-cultured bacterial liquid of the strain to be detected into 5mL of LB culture medium containing ampicillin with the final concentration of 100 mu g/mL according to the inoculation amount of 1%, culturing for 1.5h at 37 ℃ and 200rpm, adding L-arabinose (the mass percentage concentration of the L-arabinose in the obtained system is 0.2%), and performing induced fermentation on the obtained system at 16 ℃ and 160rpm for 24h to obtain fermentation liquor.

Collecting the fermentation liquid in a centrifuge tube, centrifuging at 13000rpm for 2min, discarding the supernatant, adding 1mL PBS buffer (pH 7.0) into the thallus precipitate, crushing with an ultrasonic crusher to obtain cell crushed liquid, taking 50 μ L of the cell crushed liquid to prepare a protein sample (whole bacteria), centrifuging the remaining cell crushed liquid at 13000rpm for 2min, taking the supernatant to prepare the protein sample (supernatant), and detecting the protein expression by SDS-PAEG.

The result is shown in figure 2, the trichoderma reesei ergothioneine synthesis proteins Tregt1(94.6kDa) and Tregt2(52.7kDa) are both soluble expressed in the strain BW-pBAD-tegt1,2, wherein the expression level of Tregt1 is higher, and the expression level of Tregt2 is extremely low; the Trichoderma reesei ergothioneine synthesis protein Tregt1(94.6kDa) is soluble expressed in the strain BW-pBAD-tegt1, and the Trichoderma reesei ergothioneine synthesis protein Tregt2(94.6kDa) is soluble expressed in the strain BW-pBAD-tegt 2.

4. Synthesis of ergothioneine by whole-cell catalysis method

Respectively taking the BW-pBAD-tegt1,2, BW-pBAD-tegt1, BW-pBAD-tegt2 and BW-pBAD obtained in the step 2 as strains to be detected, and synthesizing ergothioneine according to the following steps:

4.1 fermentation of the Strain

4.2 bioconversion

The fermentation broth obtained in step 4.1 was collected, centrifuged at 5000g for 5min, the supernatant was discarded, and the cell pellet was added with a transformation solution (containing 100. mu.g/mL ampicillin) to obtain a cell suspension having a cell concentration of 3OD/mL (. lamda. gtoreq.600 nm), and the resulting cell suspension was transformed at 30 ℃ and 200rpm for 16 hours to obtain a transformed product.

4.3 detection of ergothioneine

The conversion product of step 4.2 was collected, centrifuged at 13000rpm for 3min, the supernatant was aspirated, and the volume of the supernatant was reduced to 3: 7 (supernatant: acetonitrile), filtering with a 0.22 μm organic microporous membrane to obtain a filtrate, namely a sample to be detected, dissolving the ergothioneine standard (Shanghai Aladdin Biotechnology GmbH (L134175-10mg)) with 70% (volume percentage) acetonitrile aqueous solution to 10mg/L, and detecting by HPLC and secondary mass spectrometry.

The HPLC detector is Agilent 1260Infinity LC, the detection column is an Agilent ZOBAX-NH2 amino column, the ultraviolet detection wavelength is 254nm, the mobile phase is 70% (V/V) acetonitrile water solution, the flow rate is 1.0mL/min, and the sample volume is 10 muL.

The secondary mass spectrum adopts an LC/MS liquid chromatogram/Triple Quadrupole tandem mass spectrometer (Agilent 1260/6460LC/Triple Quadrupole MS), and the mobile phase adopts acetonitrile: 4mmol/L ammonium acetate (85: 15) and the detection column is an Agilent ZOBAX-NH2 amino column.

As shown in FIG. 3, the HPLC analysis showed that the transformed product obtained after whole-cell catalysis with the strain BW-pBAD-tegt1,2 showed a single strong absorption peak (C in FIG. 3) at the position where the retention time was 10.191min, which was consistent with the position where the single absorption peak was observed with the ergothioneine standard (10.039min) (A in FIG. 3), and no absorption peak was observed at this position with the negative control (B in FIG. 3), and the ergothioneine content was 29.04mg/L according to the standard curve. Secondary mass spectrometry shows that the absorption peak is ergothioneine, and shows that the Trichoderma reesei ergothioneine synthesis proteins Tregt1 and Tregt2 expressed by BW-pBAD-tegt1,2 can synthesize the ergothioneine.

The transformation product obtained after the whole-cell catalysis of BW-pBAD-tegt1 has a single strong absorption peak (D in figure 3) at the position with the retention time of 10.128min, and is consistent with the position of the absorption peak of a standard product of ergothioneine (A in figure 3), but the peak area is obviously smaller than that of the transformation product obtained after the whole-cell catalysis of BW-pBAD-tegt1,2, and the content of the ergothioneine is 9.38mg/L, which indicates that the ergothioneine can be synthesized by only the BW-pBAD-tegt1 which independently expresses Tregt 1.

The whole-cell catalysis of BW-pBAD-tegt2 has no specific chromatographic peak (E in figure 3) at the position of the absorption peak of ergothioneine standard, which indicates that BW-pBAD-tegt2 alone expressing Tregt2 can not synthesize ergothioneine.

The results of the secondary mass spectrometry of the conversion products are shown in FIG. 4.

5. Production of ergothioneine by fermentation tank bottom logistic addition method

And (3) taking the BW-pBAD-tegt1,2 obtained in the step (2) as a strain to be tested, and synthesizing ergothioneine according to the following steps:

inoculating the strain to be detected into 100mL of seed culture medium according to the inoculation amount of 1%, and culturing overnight at 37 ℃ and 200rpm to obtain a seed solution; inoculating the seed solution into 900mL fermentation medium (2L fermentation tank) containing ampicillin (100 μ g/mL), culturing at 37 deg.C under stirring until the concentration of the bacterial solution reaches 30OD (λ 600nm, about 12h), adding L-arabinose (the mass percentage concentration of L-arabinose in the obtained system is 0.2%), inducing the obtained system at 30 deg.C for 12h, and adding substrate solution (mother solution containing L-methionine, L-cysteine and L-histidine at 40g/L, and water as solvent) at flow rate of 4 mL/h/L; the whole process is carried out by adding 50g/100mL glucose aqueous solution at a feeding rate of 4mL/h/L, maintaining the dissolved oxygen at about 20% by adjusting the stirring speed and the ventilation amount, and maintaining the pH to about 7.0 by 2.7M ammonia water and 1M phosphoric acid.

The content of ergothioneine in the supernatant is detected by sampling and detecting OD600 every 12h with the seed liquid inoculation time being 0h, centrifuging and collecting the supernatant, and the ergothioneine standard (L134175-10mg) is used as a standard by an external standard method to be quantified according to HPLC peak area.

The HPLC detection method is the same as that in step 4.

As shown in FIG. 5 and Table 1, the amount of ergothioneine synthesized by BW-pBAD-tegt1,2 during fermentation increased with the increase of fermentation time within 12-144h, due to the addition of inducer during fermentation for 12h, strain BW-pBAD-tegt1,2 synthesized a small amount of ergothioneine and secreted extracellularly by utilizing L-methionine, L-cysteine and L-histidine contained in its own cells, and after 24h, the concentration of ergothioneine continuously increased due to the continuous growth of substrate fed-batch bacteria, and the concentration of extracellular ergothioneine reached 4.5g/L during fermentation for 144 h.

TABLE 1 extracellular Ergothioneine (ERG) yield (mg/L)

Fermentation time (h)	0	12	24	36	48	60	72
								BW-pBAD-tegt1，2	0	0	100	387	1081	1245	1662
Fermentation time (h)	84	96	108	120	132	144
								BW-pBAD-tegt1，2	2305	3405	3804	4199	4322	4490

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> two ergothioneine synthetic proteins and application thereof in ergothioneine synthesis

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 2502

<212> DNA

<213> Trichoderma reesei (Trichoderma reesei)

<400> 1

atggcgctca agtcagccac gaccacgact accaccacac agagggcgct cgacatcatc 60

gacattcaga atgcgcgaat cgaggtcaac ctcaaggacg agatccttgc ccagatgaat 120

cccgagcagg gcccccgcac gctgcccacg ctgctgctct atgacgagcg cggcctgcag 180

ctgtttgaag aaatcacgta tctggacgaa tactacctga ccaactacga gattgagctc 240

ctgaagaaat cggcagccga aatggccagc cagatccccg agggagcaat tgtggtggag 300

ctgggcagcg gaaacctgcg caaagtctgc ctcctcctgc aggcgtttga agatgccaag 360

aagaagattg actacttcgc gctcgacctg tcccagaagg agctcgagcg cacgctggcc 420

gaggcgcccg tctttgagta cgtgagctgc cggggcctgc gagggacgta tgacgacggg 480

tgcgagtggc tgaagcagga ggcgattctg gctcggccaa agtgcatttt gcacctcggc 540

tccagcattg gcaactttac gcgcgacgag gcggcagagt tcctgaggtc atttgcagag 600

gttctgcagc cctcggatct gatgattgtc ggggttgact catgtcagaa cccggacaag 660

gtgtaccatg catacaatga cagcaagggc gtcacacatc aattcgtcct caacggcctc 720

actcacgcaa acgaggttct cggtcaggaa gccttcaacg tcgaagagtg gcaagtcatt 780

ggcgaatacg tgtacgatgt cgacggcggc cgtcaccagg cctttgtgtc gcctctcgag 840

gtcgcctcgg tcctgggaca catcatcaag ccccacgagc gcatcaagat tgagcagagc 900

ttcaagtact ctgacgtcgg cctcgacaag ctgttcaaga cggccggcct cgtcgaggtt 960

gccaaatgga gccgccaggg cgaatacggc cttcacatgc tcaagagagc caagatgccc 1020

ttccccaggc tccgcgagct ttacgccagc gacaccctgc ccacctgggc cgactgggag 1080

aacctctggg cggcctggga cacggtcacc cgcaagatgc tccccgacgc cgagctcaac 1140

gagaagccca tcaagctgcg caacgcctgc atcttctacc tcggccacat cccggccttc 1200

ctcgacatcc agctcaagaa gacgaccaag gccggcggaa ccgagccgct ctacttccac 1260

accatctttg agcgcggcat cgacccggac gtcgacaacc cggaaaagtg ccacgaccac 1320

tccgaggtcc cggacgagtg gcctcccctt gaggacatcc tgaagtatca ggaccgcgtc 1380

cgcgagagac tccgcaagct gtatgccagc cccgatgagc ttgtcggaga cgttcggcgc 1440

gccgtctgga tcggcttcga gcacgaggcg cttcacctcg agacgctgct gtacatgctg 1500

ctgcagagcg acaagacgct tccgccgccg cacacggtgg tgccggactt ccccaagatg 1560

gcgcagaagg cgtatgccgc gcgggtgccg aatcagtggt ttgatgtccc ggagcagacg 1620

attaccattg gcatggacga tcctgaggat gagcacgaat caaaccggca ctttggatgg 1680

gacaacgaga agcctgccag acaggagact gtgcgtgcct ttcaggccaa ggccaggccc 1740

atcaccaacg aggagtacgc caagtacctc tactcttctc acattgagaa cctcccggcc 1800

tcttggtcgg tcatccctcc caactatcac cacaacacca acgccacgac gcccgggaag 1860

cccatcttga gcgagctccc cgagagcttc ctccacgaca aggcggtgcg gaccgtctac 1920

gggctggtgc ccctgcgcta cgccctcgac tggcctgtct ttgcgtcgta cgacgagctt 1980

gccggctgcg cggcgtggat gggcggcagg atcccgacga tggaggaggc caagagcatc 2040

tacgcgtatg tggagaggca aaaggatatt gccaagcaga gcaagctctc caacaaggtt 2100

ccagccgtca acgggcacct cgtcaatgac ggcgtcgaag agactccccc atccaagccc 2160

tccccggcct ccctcttcgt cgacctcagc acaaccaaca ccggcttcct ccactggcac 2220

cccgtccccg tgacccccaa cggcggctcc ctcgccggcc aggccgagct gggcggcgtc 2280

tgggagtgga cgagctccgt gctgcgtccg caccaggggt tccgcccgat gagcctctac 2340

ccgggctaca cggcggactt cttcgacgac aagcacaacg tcgtgctggg cgggtcctgg 2400

gcgacgcatc cccggattgc gggccggaag agctttgtca attggtatca gaggaattat 2460

ctgtatgcct gggttggggc aaggttggtg cgagatgttt ga 2502

<210> 2

<211> 833

<212> PRT

<213> Trichoderma reesei (Trichoderma reesei)

<400> 2

Met Ala Leu Lys Ser Ala Thr Thr Thr Thr Thr Thr Thr Gln Arg Ala

1 5 10 15

Leu Asp Ile Ile Asp Ile Gln Asn Ala Arg Ile Glu Val Asn Leu Lys

20 25 30

Asp Glu Ile Leu Ala Gln Met Asn Pro Glu Gln Gly Pro Arg Thr Leu

35 40 45

Pro Thr Leu Leu Leu Tyr Asp Glu Arg Gly Leu Gln Leu Phe Glu Glu

50 55 60

Ile Thr Tyr Leu Asp Glu Tyr Tyr Leu Thr Asn Tyr Glu Ile Glu Leu

65 70 75 80

Leu Lys Lys Ser Ala Ala Glu Met Ala Ser Gln Ile Pro Glu Gly Ala

85 90 95

Ile Val Val Glu Leu Gly Ser Gly Asn Leu Arg Lys Val Cys Leu Leu

100 105 110

Leu Gln Ala Phe Glu Asp Ala Lys Lys Lys Ile Asp Tyr Phe Ala Leu

115 120 125

Asp Leu Ser Gln Lys Glu Leu Glu Arg Thr Leu Ala Glu Ala Pro Val

130 135 140

Phe Glu Tyr Val Ser Cys Arg Gly Leu Arg Gly Thr Tyr Asp Asp Gly

145 150 155 160

Cys Glu Trp Leu Lys Gln Glu Ala Ile Leu Ala Arg Pro Lys Cys Ile

165 170 175

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

180 185 190

Glu Phe Leu Arg Ser Phe Ala Glu Val Leu Gln Pro Ser Asp Leu Met

195 200 205

Ile Val Gly Val Asp Ser Cys Gln Asn Pro Asp Lys Val Tyr His Ala

210 215 220

Tyr Asn Asp Ser Lys Gly Val Thr His Gln Phe Val Leu Asn Gly Leu

225 230 235 240

Thr His Ala Asn Glu Val Leu Gly Gln Glu Ala Phe Asn Val Glu Glu

245 250 255

Trp Gln Val Ile Gly Glu Tyr Val Tyr Asp Val Asp Gly Gly Arg His

260 265 270

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

275 280 285

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

290 295 300

Asp Val Gly Leu Asp Lys Leu Phe Lys Thr Ala Gly Leu Val Glu Val

305 310 315 320

Ala Lys Trp Ser Arg Gln Gly Glu Tyr Gly Leu His Met Leu Lys Arg

325 330 335

Ala Lys Met Pro Phe Pro Arg Leu Arg Glu Leu Tyr Ala Ser Asp Thr

340 345 350

Leu Pro Thr Trp Ala Asp Trp Glu Asn Leu Trp Ala Ala Trp Asp Thr

355 360 365

Val Thr Arg Lys Met Leu Pro Asp Ala Glu Leu Asn Glu Lys Pro Ile

370 375 380

Lys Leu Arg Asn Ala Cys Ile Phe Tyr Leu Gly His Ile Pro Ala Phe

385 390 395 400

Leu Asp Ile Gln Leu Lys Lys Thr Thr Lys Ala Gly Gly Thr Glu Pro

405 410 415

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

420 425 430

Asn Pro Glu Lys Cys His Asp His Ser Glu Val Pro Asp Glu Trp Pro

435 440 445

Pro Leu Glu Asp Ile Leu Lys Tyr Gln Asp Arg Val Arg Glu Arg Leu

450 455 460

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

465 470 475 480

Ala Val Trp Ile Gly Phe Glu His Glu Ala Leu His Leu Glu Thr Leu

485 490 495

Leu Tyr Met Leu Leu Gln Ser Asp Lys Thr Leu Pro Pro Pro His Thr

500 505 510

Val Val Pro Asp Phe Pro Lys Met Ala Gln Lys Ala Tyr Ala Ala Arg

515 520 525

Val Pro Asn Gln Trp Phe Asp Val Pro Glu Gln Thr Ile Thr Ile Gly

530 535 540

Met Asp Asp Pro Glu Asp Glu His Glu Ser Asn Arg His Phe Gly Trp

545 550 555 560

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

565 570 575

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

580 585 590

Ser His Ile Glu Asn Leu Pro Ala Ser Trp Ser Val Ile Pro Pro Asn

595 600 605

Tyr His His Asn Thr Asn Ala Thr Thr Pro Gly Lys Pro Ile Leu Ser

610 615 620

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

625 630 635 640

Gly Leu Val Pro Leu Arg Tyr Ala Leu Asp Trp Pro Val Phe Ala Ser

645 650 655

Tyr Asp Glu Leu Ala Gly Cys Ala Ala Trp Met Gly Gly Arg Ile Pro

660 665 670

Thr Met Glu Glu Ala Lys Ser Ile Tyr Ala Tyr Val Glu Arg Gln Lys

675 680 685

Asp Ile Ala Lys Gln Ser Lys Leu Ser Asn Lys Val Pro Ala Val Asn

690 695 700

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

705 710 715 720

Ser Pro Ala Ser Leu Phe Val Asp Leu Ser Thr Thr Asn Thr Gly Phe

725 730 735

Leu His Trp His Pro Val Pro Val Thr Pro Asn Gly Gly Ser Leu Ala

740 745 750

Gly Gln Ala Glu Leu Gly Gly Val Trp Glu Trp Thr Ser Ser Val Leu

755 760 765

Arg Pro His Gln Gly Phe Arg Pro Met Ser Leu Tyr Pro Gly Tyr Thr

770 775 780

Ala Asp Phe Phe Asp Asp Lys His Asn Val Val Leu Gly Gly Ser Trp

785 790 795 800

Ala Thr His Pro Arg Ile Ala Gly Arg Lys Ser Phe Val Asn Trp Tyr

805 810 815

Gln Arg Asn Tyr Leu Tyr Ala Trp Val Gly Ala Arg Leu Val Arg Asp

820 825 830

Val

<210> 3

<211> 1413

<212> DNA

<213> Trichoderma reesei (Trichoderma reesei)

<400> 3

atggcgtctc tgccggttcg tcagcgagag gaaggagagg cgagggttgg cgaggatggc 60

ttcaaggtgt ttggcggcga gatgaagaag gactttttgt ttgctcccgg gtggacgaac 120

ctcaaccacg gctcgtacgg caccatcccc agggccatcc aagcaaaact gcgcagctac 180

caagacgaca ttgaggctcg tcccgacccc tttatccgct tcgagcacgc ccgcctgacg 240

gacgaatccc gcgccgccgt cgcgggcgtg ctcaacgtcc ccgtcgagac agtcgtcttc 300

gtcaacaacg cgaccgaggg cgtcaacacc gtcttccgca acatcaagtg ggacgccgac 360

ggcaaggacg tggcgctctg gttctcgacc gtgtacgagg cgtgcggcaa ggcgattgat 420

ttcctgtacg actaccacgg ggacggacgg ctgtcgagcc gggagattga gattgcgtat 480

ccgatcgagg acgacgagat cctgcggcgc ttccggagcg cggtggagca ggtccggagc 540

gaggggaagc gcgccaagat ttgcatcttt gacgtggtgt cgtcgcggcc gggcgtggtg 600

tttccctggg agcgcatggt ggctgcgtgt cgcgagctgg gcgtgctgag cctcgtggac 660

ggcgcgcagg ggatcggcat ggtgaggctg gatctcgggg ccgcggatcc ggatttcttc 720

gtgtcgaatt gtcacaagtg gttgtttacg ccgaggggtt gcgcggtgtt ttacgtgcct 780

gtgcggaatc agccgttgtt gccgtcgacg ctggcgacga gtcatgggta tgcttcgttg 840

acggggaaga ggagggcgcc ggcggggaag catgaagatg atgataatga tgacggttct 900

ttgaagaaga gcgcgtttgt gagcaacttt gagtttacgg ggacgaggga ctatacgccg 960

aatttctgtg tcaaggatgc ggttgcgtat cggagggatg tgctgggtgg ggaggagagg 1020

attttggagt atctgtggga tttgaataag aaggggagta ggcttgttgc ggagaggctg 1080

ggcacggagg tgttggagaa taaagagggg acgttgacga actgcgcgat ggcgaacatt 1140

gccatgcctc tgtggaaggg cgaggcaggc aaggaggggg atgttgttgt gcctgaggag 1200

gatggggatc gggtggttgt gtggatgatg agcacgatgg cgaaggatta caatacgatt 1260

gtgcccatgt tttggctcgg gaagaggttc tgggtgagga tcagtgcgca ggtgtatttg 1320

gatttgggag attatgagta tggcgcggag acgttgaaga agttgattga gagggttggc 1380

aagggggagt ataagggtgg ccagaagatt tga 1413

<210> 4

<211> 470

<212> PRT

<213> Trichoderma reesei (Trichoderma reesei)

<400> 4

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

1 5 10 15

Gly Glu Asp Gly Phe Lys Val Phe Gly Gly Glu Met Lys Lys Asp Phe

20 25 30

Leu Phe Ala Pro Gly Trp Thr Asn Leu Asn His Gly Ser Tyr Gly Thr

35 40 45

Ile Pro Arg Ala Ile Gln Ala Lys Leu Arg Ser Tyr Gln Asp Asp Ile

50 55 60

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

65 70 75 80

Asp Glu Ser Arg Ala Ala Val Ala Gly Val Leu Asn Val Pro Val Glu

85 90 95

Thr Val Val Phe Val Asn Asn Ala Thr Glu Gly Val Asn Thr Val Phe

100 105 110

Arg Asn Ile Lys Trp Asp Ala Asp Gly Lys Asp Val Ala Leu Trp Phe

115 120 125

Ser Thr Val Tyr Glu Ala Cys Gly Lys Ala Ile Asp Phe Leu Tyr Asp

130 135 140

Tyr His Gly Asp Gly Arg Leu Ser Ser Arg Glu Ile Glu Ile Ala Tyr

145 150 155 160

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

165 170 175

Gln Val Arg Ser Glu Gly Lys Arg Ala Lys Ile Cys Ile Phe Asp Val

180 185 190

Val Ser Ser Arg Pro Gly Val Val Phe Pro Trp Glu Arg Met Val Ala

195 200 205

Ala Cys Arg Glu Leu Gly Val Leu Ser Leu Val Asp Gly Ala Gln Gly

210 215 220

Ile Gly Met Val Arg Leu Asp Leu Gly Ala Ala Asp Pro Asp Phe Phe

225 230 235 240

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

245 250 255

Phe Tyr Val Pro Val Arg Asn Gln Pro Leu Leu Pro Ser Thr Leu Ala

260 265 270

Thr Ser His Gly Tyr Ala Ser Leu Thr Gly Lys Arg Arg Ala Pro Ala

275 280 285

Gly Lys His Glu Asp Asp Asp Asn Asp Asp Gly Ser Leu Lys Lys Ser

290 295 300

Ala Phe Val Ser Asn Phe Glu Phe Thr Gly Thr Arg Asp Tyr Thr Pro

305 310 315 320

Asn Phe Cys Val Lys Asp Ala Val Ala Tyr Arg Arg Asp Val Leu Gly

325 330 335

Gly Glu Glu Arg Ile Leu Glu Tyr Leu Trp Asp Leu Asn Lys Lys Gly

340 345 350

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

355 360 365

Glu Gly Thr Leu Thr Asn Cys Ala Met Ala Asn Ile Ala Met Pro Leu

370 375 380

Trp Lys Gly Glu Ala Gly Lys Glu Gly Asp Val Val Val Pro Glu Glu

385 390 395 400

Asp Gly Asp Arg Val Val Val Trp Met Met Ser Thr Met Ala Lys Asp

405 410 415

Tyr Asn Thr Ile Val Pro Met Phe Trp Leu Gly Lys Arg Phe Trp Val

420 425 430

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

435 440 445

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

450 455 460

Lys Gly Gly Gln Lys Ile

465 470

Claims

1. A set of proteins consisting of proteins designated Tregt1 and Tregt2, respectively, the Tregt1 being a1), a2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 2;

the Tregt2 is C1), C2), or C3) as follows:

C1) the amino acid sequence is the protein of sequence 4;

C3) c1) or C2) at the N-terminus or/and the C-terminus.

2. A set of biological material or biological material 3, the set of biological material consisting of the names biological material 1 and biological material 2, respectively, the biological material 1 being any of the following B1) to B5):

B1) a nucleic acid molecule encoding the Tregt1 of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

the biomaterial 2 is any one of the following D1) to D5):

D1) a nucleic acid molecule encoding the Tregt2 of claim 1;

D2) an expression cassette comprising the nucleic acid molecule of D1);

D5) a cell line comprising D1) the nucleic acid molecule or a cell line comprising D2) the expression cassette;

the biological material 3 is any one of the following E1) to E4):

E2) a recombinant vector comprising the expression cassette of E1);

E4) a cell line comprising the expression cassette described in E1).

3. The kit of biological material according to claim 2, wherein: B1) the nucleic acid molecule is b11) or b12) or b13) or b14) as follows:

b12) DNA molecule shown in sequence 1 in the sequence table;

D1) the nucleic acid molecule is d11) or d12) or d13) or d14) as follows:

d12) DNA molecule shown in sequence 3 in the sequence table;

4. The Tregt1 or the Tregt2 of claim 1.

5. The biomaterial 1 or the biomaterial 2 as claimed in claim 2or 3.

6. The protein kit of claim 1, or the biological material kit of claim 2or 3 or the biological material 3, or the Tregt1 or the Tregt2 of claim 1, or the use of any of the following for the biological material 1 or the biological material 2 of claim 2or 3:

x1, producing ergothioneine;

x2, preparing ergothioneine products;

x3, preparing an antioxidant or cytoprotective product;

7. A process for the production of ergothioneine comprising: culturing the recombinant microorganism expressing the Tregt1 and/or the Tregt2 to express the Tregt1 and/or the Tregt2, and then continuously culturing the recombinant microorganism in a system containing L-histidine, L-methionine and L-cysteine to obtain the ergothioneine.

8. The method of claim 7, wherein: the system containing L-histidine, L-methionine and L-cysteine is a transformation solution 1 or a transformation solution 2, the transformation solution 1 consists of a solute and a solvent, the solvent is water, and the solute and the concentration of the solute in the transformation solution 1 are respectively 5.8g/L Na₂HPO₄，3g/L KH₂PO₄，0.5g/L NaCl，1g/L NH₄Cl,1g/L histidine, 1g/L methionine, 1g/L cysteine, 20mg/L FeSO₄·7H₂O, 50mmol/L glucose;

9. A kit consisting of Y1 and Y2:

y1, the protein set of claim 1, or the biological material set of claim 2or 3 or the biological material 3, or the Tregt1 or the Tregt2 of claim 1, or the biological material 1 or the biological material 2 of claim 2or 3;

y2, the transformation solution of claim 7 or 8.

10. Use of a kit according to claim 9 for any of the following applications:

x1, producing ergothioneine;

x2, preparing ergothioneine products;

x3, preparing an antioxidant or cytoprotective product;