CN117512027A - ScEgt1 gene derived from schizophyllum commune and application of coded protein thereof in synthesis of ergothioneine - Google Patents
ScEgt1 gene derived from schizophyllum commune and application of coded protein thereof in synthesis of ergothioneine Download PDFInfo
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 83
- 229940093497 ergothioneine Drugs 0.000 title claims abstract description 61
- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 title claims abstract description 57
- 241000222481 Schizophyllum commune Species 0.000 title claims abstract description 13
- 230000015572 biosynthetic process Effects 0.000 title abstract description 20
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- 238000000034 method Methods 0.000 claims description 16
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 241000187480 Mycobacterium smegmatis Species 0.000 description 1
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- 101100065105 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) egt-1 gene Proteins 0.000 description 1
- 101100172079 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) egt-2 gene Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
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- IMBKASBLAKCLEM-UHFFFAOYSA-L ferrous ammonium sulfate (anhydrous) Chemical compound [NH4+].[NH4+].[Fe+2].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O IMBKASBLAKCLEM-UHFFFAOYSA-L 0.000 description 1
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Y201/01—Methyltransferases (2.1.1)
- C12Y201/01044—Dimethylhistidine N-methyltransferase (2.1.1.44)
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Abstract
The invention discloses an application of ScEgt1 gene derived from schizophyllum commune and a coded protein thereof in ergothioneine synthesis. The invention provides application of complete protein consisting of ScEgt1 protein (SEQ ID No. 1) and PoEgt2 protein (SEQ ID No. 2) in preparation of ergothioneine. The invention constructs the heterologous synthesis engineering strain of the ergothioneine by expressing the ergothioneine synthetase ScEgt1 from schizophyllum commune and PoEgt2 from Pleurotus ostreatus in escherichia coli BL21 (DE 3). The invention is helpful for further enriching ergothioneine biosynthesis elements, and lays a foundation for the subsequent construction of high-yield engineering strains through metabolic engineering and synthetic biology means.
Description
Technical Field
The invention relates to the field of synthetic biology, in particular to an ScEgt1 gene derived from schizophyllum commune and application of a coded protein thereof in ergothioneine synthesis.
Background
Ergothioneine (EGT) is a natural sulfur-containing amino acid found in fungi and has strong antioxidant capacity. The EGT has good water solubility and stability, and has wide application prospect in the fields of foods, beverages, dietary supplements, cosmetics and the like.
Currently, the main production modes of ergothioneine are natural biological extraction, chemical synthesis and biological synthesis. The extraction method has low extraction content and more impurities, and limits the industrialized application of ergothioneine; the chemical method has the disadvantages of expensive raw materials, difficult guarantee of safety and inapplicability to industrialized mass production. In recent years, the biosynthesis of ergothioneine has received extensive attention due to its low cost, readily available raw materials, environmental friendliness and other advantages. Biological synthesis of ergothioneine is primarily by bacterial and fungal pathways: bacterial pathways were originally discovered in the prokaryote mycobacterium smegmatis, with histidine, cysteine, glutamic acid, S-adenosylmethionine (SAM) as key intermediate metabolites, requiring five steps of reactions, involving a total of five key genes; the fungal pathway (FIG. 1) was originally found in Neurospora crassa and uses histidine, cysteine and SAM as key intermediate metabolites, requiring three steps of reaction, and two key genes in total. In bacterial pathways, the synthesis of ergothioneine requires gamma-glutaminocysteine (γgc) as a substrate, and forms a competitive relationship with the synthesis of glutathione, another important regulatory metabolite in cells, affecting the rate of ergothioneine production. Compared with the bacterial approach, the fungal approach avoids the competitive relationship between ergothioneine and glutathione, and only two key genes need to be introduced when the engineering strain is constructed, so that the subsequent expression regulation and control are relatively simple. Furthermore, the natural accumulation of ergothioneine in the fungus, which is a fungal synthetic pathway, is relatively high in many natural synthetic hosts. Therefore, the current engineering strain research mainly surrounds a fungus approach with simpler approach, and the identification, screening and combination optimization work of key enzyme genes is carried out. Unfortunately, most of the key enzyme candidate genes selected for these efforts were the earliest reported genes responsible for identifying the Egt1 and Egt2 genes in MAICON, whereas the natural high-yielding host-derived candidate genes and their cognate genes were relatively few. The relative lack of key biosynthetic elements is one of the limiting factors in current heterologous biosynthesis of ergothioneine. Therefore, the ergothioneine synthetase from different species is screened and excavated, which is helpful to further enrich the ergothioneine biosynthesis elements and lays a foundation for the subsequent construction of high-yield engineering strains through metabolic engineering and synthetic biology means.
Disclosure of Invention
The invention aims to provide an application of ScEgt1 gene derived from schizophyllum commune and a coded protein thereof in ergothioneine synthesis.
In a first aspect, the invention claims the use of a protein set or a specific protein for the preparation of ergothioneine;
the complete set of proteins consists of protein A and protein B;
the specific protein is the protein A;
the protein A is a protein as shown in any one of the following (A1) to (A4):
(A1) A protein with an amino acid sequence shown as SEQ ID No. 1;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence defined in the step (A1) and is derived from schizophyllum commune and has the same function;
(A3) A protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in (A1) or (A2) and having the same function and derived from schizophyllum commune;
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
The protein B is a protein as shown in any one of the following (B1) to (B4):
(B1) The amino acid sequence is a protein shown as SEQ ID No. 2;
(B2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence defined in the step (B1) and is derived from Pleurotus ostreatus and has the same function;
(B3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (B1) to (B2) and having the same function derived from Pleurotus ostreatus;
(B4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (B1) to (B3).
Wherein, the protein with the amino acid sequence shown in SEQ ID No.1 is named ScEgt1; the protein with the amino acid sequence shown in SEQ ID No.2 is named PoEgt2.
In (A2) and (B2), the substitution and/or deletion and/or addition of one or several amino acid residues means substitution and/or deletion and/or addition of not more than ten amino acid residues.
In the above protein, the tag refers to a polypeptide or protein which is fusion expressed together with the target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In the above proteins, homology refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
In the above protein, the homology of 95% or more may be at least 96%, 97% or 98% identical. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical.
In a second aspect, the invention claims the use of a set of nucleic acid molecules or a specific nucleic acid molecule for the preparation of ergothioneine;
the complete set of nucleic acid molecules consists of nucleic acid molecule A and nucleic acid molecule B;
the nucleic acid molecule A is a nucleic acid molecule encoding the protein A described in the first aspect;
the nucleic acid molecule B is a nucleic acid molecule encoding the protein B described in the first aspect;
the specific nucleic acid molecule claimed in the present invention is a nucleic acid molecule encoding a specific protein as described in the first aspect hereinbefore (i.e. the protein a) (i.e. the nucleic acid molecule a).
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, and the like.
Further, the nucleic acid molecule a may be a DNA molecule as shown in any one of the following (a 1) to (a 3):
(a1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 3;
(a2) A DNA molecule which hybridizes under stringent conditions with a DNA molecule as defined in (a 1) and which encodes a protein a as described in the first aspect;
(a3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (a 1) or (a 2) and encoding the protein a of the first aspect;
the nucleic acid molecule B is a DNA molecule as shown in any one of the following (B1) to (B3):
(b1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 4;
(b2) A DNA molecule which hybridizes under stringent conditions with a DNA molecule as defined in (B1) and which encodes the protein B of the first aspect;
(b3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (B1) or (B2) and encoding the protein B of the first aspect;
the specific nucleic acid molecule is a DNA molecule as set forth in any one of (a 1) to (a 3) above.
Wherein, the DNA molecule with the nucleotide sequence shown as SEQ ID No.3 is ScEgt1 gene, and codes ScEgt1 protein shown as SEQ ID No. 1; the DNA molecule with the nucleotide sequence shown as SEQ ID No.4 is a PoEgt2 gene, and encodes the PoEgt2 protein shown as SEQ ID No. 2.
In the above nucleic acid molecule, the stringent conditions may be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.
In the above nucleic acid molecules, homology refers to the identity of nucleotide sequences. The identity of nucleotide sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of nucleotide sequences is searched for and calculated, and then the value (%) of identity can be obtained.
In the nucleic acid molecule, the homology of 95% or more may be at least 96%, 97% or 98% identical. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical.
Further, the nucleic acid molecules defined in (a 2) and (a 3) are preferably derived from schizophyllum commune (s.communication).
Further, the nucleic acid molecules defined in (b 2) and (b 3) are preferably derived from Pleurotus ostreatus (Pleurotus ostreatus).
In a third aspect, the invention claims the use of a biomaterial in the preparation of ergothioneine;
the biological material is any one of the following:
(c1) A recombinant vector comprising both the nucleic acid molecule a and the nucleic acid molecule B described in the second aspect;
(c2) Recombinant bacterium comprising both the nucleic acid molecule A and the nucleic acid molecule B according to the second aspect.
To facilitate identification and screening of recombinant bacteria, the recombinant vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds capable of producing color changes, antibiotic markers having resistance, etc., which can be expressed in the recipient bacteria.
The recombinant vector may be a bacterial plasmid, phage, yeast plasmid, or retrovirus packaging plasmid, etc.
The recombinant bacterium may be a prokaryotic cell or a eukaryotic cell.
In particular, the prokaryotic cell may be a bacterium; the eukaryotic cell may be a yeast, a filamentous fungus, a higher fungus, or a microalgae.
More specifically, the bacterium may be E.coli.
In a specific embodiment of the present invention, the promoters in the recombinant vector that initiate transcription of the nucleic acid molecule a and the nucleic acid molecule B are respectively T7 promoters and the termination sequences are respectively T7 terminators. The recombinant vector is specifically a recombinant vector obtained by inserting the nucleic acid molecule A (SEQ ID No. 3) between the cleavage sites NdeI and BamHI of the pET-28a (+) vector and inserting the nucleic acid molecule B (SEQ ID No. 4) between the cleavage sites BamHI and XhoI of the pET-28a (+) vector.
In a specific embodiment of the present invention, the recombinant bacterium is specifically obtained by introducing the recombinant vector into escherichia coli.
In a fourth aspect, the invention claims the use of an engineering bacterium in the preparation of ergothioneine;
the engineering bacteria are prepared by the method comprising the following steps: modifying the receptor bacteria to express the complete set of proteins (namely ScEgt1 protein and PoEgt2 protein) in claim 1, wherein the modified receptor bacteria are the engineering bacteria.
Further, the method may comprise the steps of: introducing the nucleic acid set described in the second aspect (i.e., the ScEgt1 gene and the PoEgt2 gene) into the recipient strain to obtain recombinant strain expressing the protein set described in the first aspect, i.e., the engineering strain.
In the above method, the recipient bacterium may be a prokaryotic cell or a eukaryotic cell.
In particular, the prokaryotic cell may be a bacterium; the eukaryotic cell may be a yeast, a filamentous fungus, a higher fungus, or a microalgae.
More specifically, the bacterium may be E.coli.
In a specific embodiment of the invention, the recipient bacterium is E.coli BL21 (DE 3).
In a fifth aspect, the invention claims the use of a kit for the preparation of ergothioneine;
the kit is as follows (d 1) or (d 2):
(d1) Consisting of a protein set as described in the first aspect above and all or part of the following: histidine, cysteine, methionine or S-adenosylmethionine;
(d2) Consists of the engineering bacteria described in the fourth aspect and all or part of the following: histidine, cysteine, methionine or S-adenosylmethionine.
The kit may further comprise Fe as required 2+ And/or pyridoxal phosphate.
In the first to fifth aspects described above, the preparation of ergothioneine may be either an in vitro synthesis process or a microbial fermentation process.
Further, the in vitro synthesis method exerts its catalytic action of the synthetase in the form of crude enzyme solution, crude enzyme solution lyophilized powder, pure enzyme or cells of the protein set described in the first aspect.
In a sixth aspect, the invention claims a method of preparing ergothioneine.
The method for preparing ergothioneine claimed by the invention can comprise the following steps: the production of ergothioneine from a substrate catalyzed by a protein set as described in the first aspect hereinbefore or an engineered bacterium as described in the fourth aspect hereinbefore;
wherein the substrate may be histidine, cysteine, and methionine or S-adenosylmethionine.
Further, the final concentration of histidine, cysteine and methionine/S-adenosylmethionine added to the reaction system of the method may be 10mM.
Further, the reaction conditions in the method may be 25℃for 9 hours.
Further, in the method, after the reaction is finished, the method may further include the following steps: heating the reaction solution at 90deg.C for 30min, centrifuging (such as at 4deg.C for 15 min), collecting supernatant, and filtering with 0.22 μm filter membrane.
In a seventh aspect, the invention claims a kit for the preparation of ergothioneine.
The kit of parts to be claimed in the present invention may be (d 1) or (d 2) as follows:
(d1) Consisting of a protein set as described in the first aspect above and all or part of the following: histidine, cysteine, methionine or S-adenosylmethionine;
(d2) Consists of the engineering bacteria described in the fourth aspect and all or part of the following: histidine, cysteine, methionine or S-adenosylmethionine.
The kit may further comprise Fe as required 2+ And/or pyridoxal phosphate.
Experiments prove that the ergothioneine synthetase ScEgt1 from schizophyllum commune (S.communication) H4-8 and PoEgt2 from Pleurotus ostreatus (Pleurotus ostreatus) are expressed in escherichia coli BL21 (DE 3) to construct the heterologous synthesis engineering strain of the ergothioneine. The invention enriches the ergothioneine biosynthesis elements and lays a foundation for the subsequent construction of high-yield engineering bacteria by metabolic engineering and synthetic biology means.
Drawings
FIG. 1 shows the biosynthetic fungal pathway of ergothioneine.
FIG. 2 shows the results of UPLC-MS/MS detection in example 4. (A) is ergothioneine standard; (B) Whole cell catalysis was performed for 9h as in example 3 for the strain containing the control plasmid pET28a (+) and the results of the treated reaction supernatant were sampled; (C) The whole cell was catalyzed for 9 hours as in example 3 for the strain containing the recombinant expression vector pET-ScEgt1-PoEgt2, and the results of the reaction supernatant after the treatment were sampled.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Quantitative data in the examples below are the mean of at least three replicates.
Example 1 acquisition of amino acid sequences of ergothioneine synthase ScEgt1 and PoEgt2 and bioinformatics analysis
The amino acid sequence of PEgt1 (Protein ID: XP_ 036627813.1) reported by Pleurotus ostreatus Pleurotus ostreatus PC is put in NCBI database and subjected to homology search by BLAST to find the corresponding Protein similar to the Protein in schizophyllum commune, named ScEgt1 (SEQ ID No. 1); the amino acid sequence of PEgt2 (Protein ID: KAG 9224528.1) reported by Pleurotus ostreatus Pleurotus ostreatus PC was placed in NCBI database and subjected to homology search using BLAST to find a corresponding Protein similar to that in Pleurotus ostreatus, designated PoEgt2 (SEQ ID No. 2).
EXAMPLE 2 construction of expression vectors for genes encoding ergothioneine synthase ScEgt1 and PoEgt2
(1) Strains and plasmids
Coli DH 5. Alpha. Was purchased from Beijing gold full company for gene cloning; coli BL21 (DE 3) was purchased from Beijing gold full company for gene expression; the expression vector pET28a (+) is preserved in a laboratory and is used for constructing a fungal ergothioneine synthetase encoding gene expression vector.
(2) Acquisition of ergothioneine synthase Gene
All gene synthesis and cloning operations were performed by Jin Weizhi biotechnology (Beijing) Inc.:
the encoding gene of the ScEgt1 protein (SEQ ID No. 1) is optimized according to the codon preference of escherichia coli to obtain an optimized ScEgt1 gene (SEQ ID No. 3), and the optimized ScEgt1 gene is sent to Jin Weizhi biotechnology (Beijing) limited company for synthesis and connected between NdeI and BamHI of an enzyme cleavage site of a pET-28a (+) vector to construct a pET-ScEgt1 plasmid. The coding gene of the PoEgt2 protein (SEQ ID No. 2) is optimized according to the codon preference of escherichia coli to obtain an optimized PoEgt2 gene (SEQ ID No. 4), and the optimized PoEgt2 gene is sent to Jin Weizhi biotechnology (Beijing) limited company for synthesis and is connected between the enzyme cutting site BamHI and XhoI of the pET-ScEgt1 vector together with a 5' -end flanking sequence ' gaaggagatgggca ', so as to construct a pET-ScEgt1-PoEgt2 plasmid.
The structure of the recombinant expression vector pET-ScEgt1-PoEgt2 is described as follows: the optimized ScEgt1 gene (SEQ ID No. 3) was inserted between the NdeI and BamHI cleavage sites of the pET-28a (+) vector, and the optimized PoEgt2 gene (SEQ ID No. 4) was inserted between the BamHI and XhoI cleavage sites, to obtain a recombinant vector.
Example 3 preparation of genetically engineered bacteria Using the recombinant expression vector constructed in example 2 and Whole cell catalytic production of ergothioneine
(1) Culture medium and reagent
LB medium: is used for culturing escherichia coli. 10g of tryptone, 5g of yeast extract, 10g of NaCl, distilled water to a volume of 1L, and 15g of agar powder to be added into an LB solid medium, and sterilizing for 20min by high-pressure steam at 121 ℃.
5 XM 9 medium: na (Na) 2 HPO 4 33.9g，KH 2 PO 4 15g，NaCl 2.5g，NH 4 Cl 5g, distilled water to a constant volume of 1L, and high-pressure steam sterilization at 121 ℃ for 20min.
20% glucose: 20g of glucose was weighed out in distilled water and the volume was set to 100mL. And sterilizing with steam at 115 deg.C for 30min.
1M MgSO 4 : 1.21g MgSO was taken 4 Dissolved in dd H 2 O and constant volume to 10mL,0.22 μm microporous membrane filtration.
1M CaCl 2 : 1.11g CaCl was taken 2 Dissolved in dd H 2 O and constant volume to 10mL,0.22 μm microporous membrane filtration.
M9 medium: is used for whole cell catalysis. 5 XM 9 Medium 200mL,20% glucose solution 50mL,1M MgSO 4 2mL，CaCl 2 100μL，dd H 2 O is fixed to 1000mL.
0.1M potassium phosphate ion buffer (KPI) buffer: 6mL of 1M KH was taken separately 2 PO 4 And 94mL of 1M K 2 HPO 4 Distilled water is used for constant volume to 1L and is preserved at 4 ℃.
0.25M histidine: 3.88g histidine was dissolved in 100mL dd H 2 O.
0.5M cysteine: 6.05g cysteine was dissolved in 100mL dd H 2 O.
0.2M methionine: 2.98g methionine in 100mL dd H 2 O.
1M IPTG:2.383g IPTG was dissolved in 10mL dd H 2 O.
Pyridoxal phosphate 0.1M: 0.2651g pyridoxal phosphate is dissolved in 10mL dd H 2 O.
0.1M Fe(NH 4 ) 2 ·(SO 4 ) 2 ·6H 2 O:0.3921g of ferrous ammonium sulphate hexahydrate is dissolved in 10mL dd H 2 In O。
Whole cell catalytic medium: is used for whole cell catalysis. 5 XM 9 Medium 200mL,20% glucose solution 50mL,1M MgSO 4 2mL，1M CaCl 2 100. Mu.L, 40mL of 0.25M histidine, 20mL of 0.5M cysteine, 50mL of 0.2M methionine, sterile dd H 2 O is fixed to 1000mL.
(2) Method for preparing ergothioneine by whole-cell catalysis method
The plasmid pET-ScEgt1-PoEgt2 containing the fungal ergothioneine synthase gene prepared in example 2, which selects the IPTG inducible promoter (pET 28a (+) harbor), was transferred into E.coli BL21 (DE 3). Culturing recombinant bacteria with correct sequencing verification in LB culture medium for 2.5h, and culturing to obtain thallus OD 600 When the value reaches 0.6-0.8, IPTG is added to a final concentration of 0.5mM, pyridoxal phosphate is added to a final concentration of 1mM, fe (NH) 4 ) 2 ·(SO 4 ) 2 ·6H 2 The final O concentration was 0.1mM, and the target enzyme proteins (i.e., scEgt1 protein and PoEgt2 protein) on pET-ScEgt1-PoEgt2 were induced (16 ℃ C., 18 h). After the cells were collected by centrifugation (4 ℃ C., 6000r/min, 20 min), the cells were resuspended in M9 medium and collected by centrifugation again. And adding 100mL of whole cell catalytic culture medium into the finally collected thalli to re-suspend to obtain a whole cell suspension, carrying out a whole cell preparation reaction (25 ℃, 220 r/min) of ergothioneine, taking 1mL of reaction solution for corresponding time, and preserving at-20 ℃.
Treatment of ergothioneine reaction solution: heating 1mL of reaction solution in a metal bath at 90 ℃ and 700r/min for 30min, centrifuging at 4 ℃ for 15min, and taking a supernatant, and filtering with a 0.22 mu m filter membrane for later use.
The experiment was also carried out with an empty control group into which pET28a (+) was introduced into E.coli BL21 (DE 3).
Example 4 ergothioneine product detection
Ultra performance liquid chromatography-mass spectrometry (UPLC-MS) detection conditions: UPLC-MS analysis was performed using an ultra-high performance liquid chromatography system (Nexera 30A, shimadzu, japan, kyoto) and a mass spectrometer (triple TOF 6600, applied biosystems SCHEX, USA). UPLC condition: metabolites were identified using ZIC-HILIC columns (100X 2.1mm,3.5 μm, merck, germany). Then respectively taking 10mM ammonium acetate and 100% acetonitrile as mobile phases A and B, wherein the flow rate is 0.2mL/min, and the gradient is as follows: 0-3min,90% B;3-6min,90-60% B;6-25min,60-50% B;25-30min,50% B;30-30.5min,50-90% B;30.5-38min,90% B (% represents volume percent). MS conditions: electrospray ionization source negative ion mode, ion voltage 4500V, declustering voltage 80V, ion source temperature 550 ℃, air curtain pressure 35psi, atomization pressure 55psi, heater pressure 55psi. Each scan cycle includes 1 TOF-MS full scan and 15 MS/MS scans. The mass range of TOF-MS is m/z63-1000, and the mass range of MS/MS is m/z 30-1000.
100 μl of the treated ergothioneine reaction supernatant of example 3 was mixed with 100% acetonitrile by equal volume, centrifuged at 4deg.C for 30min, and the supernatant was subjected to subsequent ultra-high liquid chromatography-mass spectrometry detection.
As a result, as shown in FIG. 2, in the result of whole cell catalysis of the strain containing the recombinant expression vector pET-ScEgt1-PoEgt2 for 9 hours (FIG. 2 (C), rt 13.807 min), an MRM ion pair having substantially the same retention time as that of the ergothioneine standard (FIG. 2 (A), rt 13.833 min) was detected, and the content of ergothioneine was 62.73mg/L; whereas no apparent ergothioneine MRM ion pair was detected in the whole cell catalyzed 9h of the strain containing only control plasmid pET28a (+) (FIG. 2 (B)). This suggests that the catalytic synthesis of ergothioneine by E.coli can be achieved by introducing the ScEgt1 gene and the PoEgt2 gene.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, it will be appreciated that the invention may 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.
Claims (10)
1. Use of a protein set or a specific protein in the preparation of ergothioneine;
the complete set of proteins consists of protein A and protein B;
the specific protein is the protein A;
the protein A is a protein as shown in any one of the following (A1) to (A4):
(A1) A protein with an amino acid sequence shown as SEQ ID No. 1;
(A2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence defined in the step (A1) and is derived from schizophyllum commune and has the same function;
(A3) A protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in (A1) or (A2) and having the same function and derived from schizophyllum commune;
(A4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3);
the protein B is a protein as shown in any one of the following (B1) to (B4):
(B1) The amino acid sequence is a protein shown as SEQ ID No. 2;
(B2) A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence defined in the step (B1) and is derived from Pleurotus ostreatus and has the same function;
(B3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (B1) to (B2) and having the same function derived from Pleurotus ostreatus;
(B4) A fusion protein obtained by ligating a tag to the N-terminus and/or C-terminus of the protein defined in any one of (B1) to (B3).
2. Use of a nucleic acid molecule set or a specific nucleic acid molecule for the preparation of ergothioneine;
the complete set of nucleic acid molecules consists of nucleic acid molecule A and nucleic acid molecule B;
the nucleic acid molecule A is a nucleic acid molecule encoding the protein A of claim 1;
the nucleic acid molecule B is a nucleic acid molecule encoding the protein B of claim 1;
the specific nucleic acid molecule is a nucleic acid molecule encoding the specific protein of claim 1.
3. The use according to claim 2, characterized in that:
the nucleic acid molecule A is a DNA molecule as shown in any one of the following (a 1) to (a 3):
(a1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 3;
(a2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (a 1) and which encodes the protein a of claim 1;
(a3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (a 1) or (a 2) and encoding the protein a of claim 1;
the nucleic acid molecule B is a DNA molecule as shown in any one of the following (B1) to (B3):
(b1) A DNA molecule with a nucleotide sequence shown as SEQ ID No. 4;
(b2) A DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (B1) and which encodes the protein B of claim 1;
(b3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in (B1) or (B2) and encoding the protein B of claim 1;
the specific nucleic acid molecule is a DNA molecule as set forth in any one of (a 1) to (a 3) above.
4. The application of biological material in the preparation of ergothioneine;
the biological material is any one of the following:
(c1) A recombinant vector comprising both the nucleic acid molecule a according to claim 2 or 3 and the nucleic acid molecule B;
(c2) Recombinant bacterium comprising both the nucleic acid molecule a according to claim 2 or 3 and the nucleic acid molecule B.
5. Application of engineering bacteria in preparation of ergothioneine;
the engineering bacteria are prepared by the method comprising the following steps: modifying the receptor bacteria to express the complete set of protein in claim 1, wherein the modified receptor bacteria are the engineering bacteria.
6. The use according to claim 5, characterized in that: the method comprises the following steps: introducing the complete set of nucleic acid molecules of claim 2 or 3 into the recipient bacterium to obtain recombinant bacterium expressing the complete set of protein of claim 1, namely the engineering bacterium.
7. Use according to claim 5 or 6, characterized in that: the recipient bacterium is escherichia coli.
8. Application of the complete product in the preparation of ergothioneine;
the kit is as follows (d 1) or (d 2):
(d1) Consisting of a protein set as claimed in claim 1 and all or part of the following: histidine, cysteine, methionine or S-adenosylmethionine;
(d2) Consists of the engineering bacterium as claimed in any one of claims 5 to 7 and all or part of the following: histidine, cysteine, methionine or S-adenosylmethionine.
9. A process for preparing ergothioneine, comprising the steps of: catalyzing the substrate to produce ergothioneine with the protein set of claim 1 or the engineered bacterium of any one of claims 5-7;
the substrate is histidine, cysteine, methionine or S-adenosylmethionine.
10. A kit for the preparation of ergothioneine, characterized by: the kit is as follows (d 1) or (d 2):
(d1) Consisting of a protein set as claimed in claim 1 and all or part of the following: histidine, cysteine, methionine or S-adenosylmethionine;
(d2) Consists of the engineering bacterium as claimed in any one of claims 5 to 7 and all or part of the following: histidine, cysteine, methionine or S-adenosylmethionine.
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