Nucleic acid molecule, ergothioneine synthetase and method for synthesizing ergothioneine

Abstract

The invention discloses a nucleic acid molecule, an ergothioneine synthetase and a method for synthesizing ergothioneine, and relates to the technical field of ergothioneine biosynthesis. According to the present invention, 13 coding genes of ergothioneine synthesis related enzymes are respectively connected with a promoter, the coding genes and the identified EGT element gene are subjected to combined co-expression, the co-expressed vector is integrated into the host cell or the strain through gene editing, the heterologous expression of the 13 EGT genes is achieved in the host cell or the strain, and the ergothioneine synthesis related enzymes can be expressed, good catalytic activity is achieved, and cells or strains with different ergothioneine yields are obtained through screening. Particularly, after the nucleic acid molecule as shown in SEQ ID NO.5 and the CpEgt2 gene are co-expressed, compared with a control strain, the yield of ergothioneine is remarkably increased. After the nucleic acid molecule as shown in SEQ ID NO.10 and the NcEgt1 gene are co-expressed, the yield of ergothioneine is also greatly increased.

Description

Technical Field

[0001] The present invention relates to the technical field of ergothioneine biosynthesis, and in particular to a nucleic acid molecule, an ergothioneine synthase and a method for synthesizing ergothioneine. Background Art

[0002] L-ergothioneine (EGT) is a rare natural chiral amino acid with good stability and solubility. In cells, ergothioneine can participate in the scavenging of free radicals, anti-inflammation, anti-ultraviolet radiation, induction of neuronal differentiation and other functions. It has the characteristics of good absorption, long half-life and good safety in clinical practice. It has broad application prospects in the fields of cosmetics, food, medicine, etc. At present, many countries in the world are entering an aging society. In the "Decade of Healthy Aging" plan proposed by the United Nations, "providing primary health care services for the needs of the elderly" has become one of the focuses. The huge market demand for anti-aging has made ergothioneine with good biological activity a hot spot for research and application development [1-3].

[0003] The biological sources and synthesis technology of ergothioneine have been developed as follows: ergothioneine is mainly derived from fungi, which can protect fungal conidia from peroxides. Humans cannot synthesize ergothioneine by themselves, and it must be ingested from the outside and enter the human body through the intestinal transporter OCTN1. At present, the chemical synthesis of ergothioneine still faces many challenges. For example, the acidity of the α-carbon easily causes the reaction to undergo racemization, which makes the price of chemically synthesized products high, partially limiting the application of ergothioneine. The main raw materials of the more mainstream biological extraction method are the fruiting bodies of edible fungi, ergot, animal tissues, etc., but the overall content of these raw materials is still very low, and there are many impurities, and there are problems such as drug residues, high extraction costs, and long raw material growth cycles, which have great limitations for industrialization. Therefore, the use of gene editing technology and deep fermentation processes with more mature microbial heterologous hosts and ergothioneine biosynthetic elements with higher enzyme activity is the current trend of ergothioneine biosynthesis [4-6].

[0004] The biosynthetic pathway of EGT was first identified in Mycobacterium smegmatis. The biosynthesis of EGT includes five steps: histidine is catalyzed by EgtD (SAM-dependent histidine methyltransferase) to form hercynine, cysteine ​​is catalyzed by EgtA (γ-glutamylcysteine ​​synthetase) γ-Glu-Cys, hercynine and γ-Glu-Cys are successively catalyzed by EgtB (mononuclear non-heme iron enzyme), EgtC (amidotransferase), and EgtE (PLP-binding C-S lyase) to finally form ergothioneine (EGT). Later studies found a more concise eukaryotic biosynthetic pathway in Neurosporacrassa, which only requires two biosynthetic genes, Egt1 and Egt2. Among them, the NcEgt1 gene is a multi-domain protein that has the functions of the SAM-dependent methyltransferase of EgtD and EgtB (mononuclear non-heme iron enzyme), which can catalyze the conversion of histidine to Hercynine and further catalyze its synthesis of γ-Cys-HER with cysteine. The NcEgt2 gene encodes a PLP-binding C–S lyase that can directly catalyze the conversion of Cys-HER to ergothioneine. After comparison, it was found that the Egt1 and Egt2 genes have homologous genes in many ascomycetes, such as ergot and Schizosaccharomyces pombe, but relatively few EGT genes have been identified in basidiomycetes. Currently, five groups of EGT eukaryotic biosynthesis genes have been identified, including SpEgt1 and SpEgt2 from Schizosaccharomyces pombe; NcEgt1 and NcEgt2 from Neurospora crassa; CpEgt1 and CpEgt2 from Claviceps africana; FvEgt1, FvEgt2, FvEgt3 from Flammulina velutipes; and TrEgt1 and TrEgt2 from Trichoderma reesei[7-10].

[0005] At present, most of the reported ergothioneine synthesis genes are from ascomycetes, but rarely reported in basidiomycetes, which are more abundant in nature, especially in some edible fungi with publicly available genome sequences.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a nucleic acid molecule, an ergothioneine synthase and a method for synthesizing ergothioneine, so as to realize the heterologous industrial synthesis of ergothioneine.

[0008] The "EGT gene" in the present invention is equivalent to the gene encoding ergothioneine synthesis.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides an ergothioneine synthase having an amino acid sequence as shown in any one of SEQ ID NOs. 14-26.

[0011] In order to provide more diversified thioneine synthesis coding gene, and solve the limitation faced by thioneine industrial production, the inventor obtained 13 enzymes and related coding genes related to thioneine synthesis from a variety of edible fungi (mushroom N1, mushroom N2, mushroom N3, mushroom N4, mushroom N5, mushroom N6, mushroom N7, mushroom N8). The coding genes of these 13 thioneine synthesis related enzymes are connected to promoters respectively, and combined co-expression is carried out with identified EGT element genes, and the co-expressed vector is integrated into host cells or strains by gene editing, and the heterologous expression of 13 EGT genes is realized in host cells or strains, and thioneine synthase can be expressed, and the cells or strains with different thioneine yields are obtained by screening. Especially after the nucleic acid molecules as shown in SEQ ID NO.5 and CpEgt2 gene co-expression, compared with the control strain, the yield of thioneine is significantly improved. After the nucleic acid molecules as shown in SEQ ID NO.10 and NcEgt1 gene co-expression, the yield of thioneine is greatly improved.

[0012] Therefore, the development of these 13 ergothioneine synthases not only expanded the diversity of EGT synthesis elements, but also obtained new elements for efficient EGT synthesis, which will help to further develop ergothioneine biosynthetic cells or strains with higher yields.

[0013] In a second aspect, the present invention provides a nucleic acid molecule encoding ergothioneine synthase, which encodes the above-mentioned ergothioneine synthase.

[0014] The term "nucleic acid molecule encoding ergothioneine synthase" may be a polynucleotide encoding a mutant protein of the present invention, or may be a polynucleotide further comprising additional coding and / or non-coding sequences.

[0015] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or mutant proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the mutant protein encoded by it.

[0016] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. Then the DNA sequence can be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0017] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0018] In an optional embodiment, the above-mentioned nucleic acid molecule includes a first nucleic acid molecule, and the nucleotide sequence of the first nucleic acid molecule is shown in any one of SEQ ID NO.1-13.

[0019] The development of 13 EGT genes not only expanded the diversity of EGT synthesis elements, but also obtained new elements for efficient EGT synthesis, which will help to further develop ergothioneine biosynthetic cells or strains with higher yields.

[0020] In a third aspect, the present invention provides a nucleic acid molecule encoding ergothioneine synthase, the nucleic acid molecule comprising a first nucleic acid molecule and a second nucleic acid molecule, the nucleotide sequence of the first nucleic acid molecule being as shown in any one of SEQ ID NO.1-13, and the second nucleic acid molecule being selected from any one of the following EGT gene clusters: SpEgt1 and SpEgt2, NcEgt1 and NcEgt2, CpEgt1 and CpEgt2, FvEgt1 and FvEgt2, and TrEgt1 and TrEgt2. Any one of the EGT eukaryotic synthesis genes in the above-mentioned EGT gene cluster is selected to be combined with the first nucleic acid molecule, all within the protection scope of the present invention. For example, the second nucleic acid molecule is selected from SpEgt1, FvEgt1 or TrEgt2.

[0021] In an alternative embodiment, the second nucleic acid molecule is selected from any one of the following EGT eukaryotic synthesis genes in the EGT gene cluster: NcEgt1 and NcEgt2, and CpEgt1 and CpEgt2.

[0022] In an optional embodiment, the nucleic acid molecule includes: a first nucleic acid molecule as shown in any one of SEQ ID NOs. 1-7, CpEgt2 or NcEgt2;

[0023] Or the nucleic acid molecule includes the first nucleic acid molecule shown in any one of SEQ ID NO. 8-13, NcEgt1 or CpEgt1.

[0024] The first nucleic acid molecule shown in SEQ ID NO.1-7 is obtained by the inventor by performing homology search on ergothioneine synthase CpEGT2 (Genbank: CCE33140.1) from ergot fungus and the genomes of various edible fungi to obtain a protein sequence similar to CpEGT2. After the protein sequence was codon optimized for Trichoderma reesei, the first nucleic acid molecule shown in SEQ ID NO.1-7 was obtained.

[0025] The first nucleic acid molecule shown in SEQ ID NO.8-13 is obtained by the inventor by performing homology search on the ergothioneine synthase NcEGT1 of Neurospora crassa (NCBI ACCESSION XP_956324.3) and the genomes of various edible fungi to obtain a protein sequence similar to NcEGT1. After the protein sequence was codon optimized for Trichoderma reesei, the first nucleic acid molecule shown in SEQ ID NO.8-13 was obtained.

[0026] In an optional embodiment, the nucleic acid molecule includes: a nucleic acid molecule as shown in SEQ ID NO.5 and CpEgt2; or a nucleic acid molecule as shown in SEQ ID NO.10 and NcEgt1.

[0027] After the nucleic acid molecule as shown in SEQ ID NO.5 and CpEgt2 are combined, they are integrated into host cells to increase the yield of thioneine by 21.7%, and after the nucleic acid molecule as shown in SEQ ID NO.10 and NcEgt1 are combined, they are integrated into host cells to increase the yield of thioneine by 43.5%. Therefore, the nucleic acid molecule provided by the present invention has an application prospect of industrialized efficient synthesis of thioneine.

[0028] In a fourth aspect, the present invention further provides an expression cassette, which comprises the above-mentioned nucleic acid molecule.

[0029] In other embodiments, the expression cassette further comprises a promoter and a terminator.

[0030] In a preferred embodiment of the present invention, the expression cassette comprises: a first nucleic acid molecule as shown in any one of SEQ ID NOs. 1-7 and any one of the following EGT eukaryotic synthetic genes: CpEgt2 and NcEgt2;

[0031] Or the expression cassette comprises: a nucleic acid molecule as shown in any one of SEQ ID NOs. 8-13 and any one of the following EGT eukaryotic synthesis genes: NcEgt1 and CpEgt1;

[0032] In an optional embodiment, the expression cassette comprises: a nucleic acid molecule as shown in any one of SEQ ID NOs. 1-7 and CpEgt2; or a nucleic acid molecule as shown in any one of SEQ ID NOs. 8-13 and NcEgt1;

[0033] In an optional embodiment, the expression cassette comprises: a nucleic acid molecule as shown in SEQ ID NO.5 and CpEgt2; or a nucleic acid molecule as shown in SEQ ID NO.10 and NcEgt1.

[0034] In a fifth aspect, the present invention further provides a vector, which includes the above-mentioned nucleic acid molecule or the above-mentioned expression cassette. The vector includes but is not limited to an expression vector, a shuttle vector and an integration vector.

[0035] In the present invention, the term "expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid or vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0036] In an alternative embodiment, the expression vector further includes a ribosome binding site for translation initiation and a transcription terminator.

[0037] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0038] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0039] In a sixth aspect, the present invention further provides a cell or strain, which comprises the above-mentioned nucleic acid molecule, the above-mentioned expression cassette or vector.

[0040] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, plant cells (such as ginseng cells).

[0041] In a preferred embodiment of the present invention, the strain is selected from Agrobacterium, Escherichia coli or Trichoderma reesei; in addition, the above cells or strains can be selected from other chassis cells or strains.

[0042] In an optional embodiment, the Trichoderma reesei is a Trichoderma reesei with glycolysis repression effect; the Trichoderma reesei with glycolysis repression effect is obtained by three rounds of screening: ultraviolet, chemical mutagenesis, and ultraviolet. The mutagen used in chemical mutagenesis is selected from N-nitroguanidine.

[0043] In an alternative embodiment, the Trichoderma reesei with glycolysis repression effect includes but is not limited to the MC3 strain or the RutC30 strain.

[0044] Compared with the wild-type Trichoderma reesei, when the starting cell or strain is selected from any one of the above two types, the yield of exogenous ergothioneine can be significantly increased. RutC30 is a commercially available strain purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), No. 56765; MC3 is obtained by laboratory screening.

[0045] Trichoderma reesei is a food-grade safety strain certified by the US FDA. As a production strain, this strain does not produce endotoxins similar to those produced by Escherichia coli. Compared with brewer's yeast fermentation, the fermentation process of Trichoderma reesei does not produce a large amount of heat, reducing the investment cost of cooling equipment. Compared with large fungi, Trichoderma reesei has a shorter fermentation cycle, and as a model strain with a well-developed cellulase enzyme system, this bacterium can directly utilize cellulose, making it possible to convert bran, corn cobs, etc. into ergothioneine.

[0046] In a seventh aspect, the present invention also provides the use of a nucleic acid molecule, an ergothioneine synthase, an expression cassette, a vector or a cell or a strain in synthesizing ergothioneine.

[0047] In an optional embodiment, the application includes: using a gene editing method to integrate a nucleic acid molecule, an expression cassette or a vector into a cell or a strain, and culturing the cell or strain into which the target gene is heterologously inserted.

[0048] The gene editing technology is, for example, selected from CRISPR / Cas9 technology, zinc-finger nuclease (ZFN, zinc-fingernucleases) technology or transcription activator-likeeffector nuclease (TALEN, transcriptionactivator-likeeffector nucleases) technology.

[0049] The nucleic acid molecule, expression cassette or vector is integrated into the cbh1 site of Trichoderma reesei. The cbh1 gene encodes the cellobiohydrolase with the highest expression in Trichoderma reesei, and its production can reach 50% of the total amount of extracellular secretory proteins of Trichoderma reesei. Therefore, the inventors chose to insert the nucleic acid molecule, expression cassette or vector into this site, firstly to remove the protein secretion burden of the gene, and at the same time to use the cbh1 promoter to highly express the target gene of the present invention.

[0050] In an eighth aspect, the present invention also provides a method for synthesizing ergothioneine, comprising:

[0051] Cultivating the above-mentioned cells or strains;

[0052] In an optional embodiment, the culture temperature is 28°C±1°C, and the culture is carried out in SDB medium at 200±20 rpm for 30-40 hours, and then fermentation culture is carried out.

[0053] The present invention has the following beneficial effects:

[0054] The present invention develops 13 EGT genes, which not only expands the diversity of EGT synthesis elements, but also obtains new elements for efficient synthesis of EGT, which is helpful to further develop ergothioneine biosynthetic cells or strains with higher yield.

[0055] The inventor is connected to the coding gene of 13 thioneine synthesis related enzymes with promoter respectively, and carries out combined co-expression with identified EGT element gene, and the co-expressed vector is integrated into host cell or strain by gene editing, and the heterologous expression of 13 EGT genes is realized in host cell or strain, and thioneine synthase can be expressed, with good catalytic activity. On the above basis, the inventor further screens and obtains cells or strains with different thioneine yields. Especially after the co-expression of nucleic acid molecules and CpEgt2 genes as shown in SEQ ID NO.5, compared with the control strain, the yield of thioneine is significantly improved. After the co-expression of nucleic acid molecules and NcEgt1 genes as shown in SEQ ID NO.10, the yield of thioneine is also greatly improved. Its catalytic activity exceeds the EGT element combination yield 1.0g / L identified at present. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0057] Figure 1The HPLC test result diagram of the Trichoderma reesei heterologous expression transformation strains EGTMR01-MC3△10 to EGTMR07-MC3△10 of the edible fungus EGT1 element;

[0058] Figure 2 This is a graph showing the HPLC detection results of the Trichoderma reesei heterologous expression transformation strains EGTMR08-MC3△10 to EGTMR13-MC3△10 of the edible fungus EGT1 element. DETAILED DESCRIPTION

[0059] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0060] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc., 1987). Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0061] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0062] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0063] In order to provide more diversified thioneine synthesis encoding genes and solve the limitations faced by the industrial production of thioneine, the present invention obtains 13 enzymes related to thioneine synthesis and related encoding genes from edible mushrooms mushroom N1, mushroom N2, mushroom N3, mushroom N4, mushroom N5, mushroom N6, mushroom N7 and mushroom N8, and the nucleotide sequences (SEQ ID NOs: 1-13) thereof are respectively connected to the promoter of Trichoderma reesei and combined with the identified EGT element gene for co-expression, wherein the Egt1 gene is co-expressed with the CpEgt2 gene, and the Egt2 gene is co-expressed with the NcEgt1 gene, and the co-expressed plasmid is integrated into the cbh1 site of Trichoderma reesei through CRISPR / Cas9 gene editing, so that the heterologous expression of 13 EGT genes is realized in Trichoderma reesei, and a total of 13 Trichoderma reesei transformants EGTMR01-MC3△10 to EGTMR13-MC3△10 are obtained, and a control strain EGTMR14-MC3△10 is obtained. Subsequently, all the recombinant Trichoderma strains were fermented for 96 hours to obtain a fermentation broth containing ergothioneine. The aqueous solution containing ergothioneine was obtained by boiling the bacterial broth, and the ergothioneine production was measured after filtration and dilution. By comparison, it was found that the EGT1 element derived from mushroom N5 and the EGT2 element derived from mushroom N4 had the best catalytic activity, with the highest shake flask yields reaching 1.34 g / L and 1.51 g / L, respectively. The catalytic capacity exceeded the combined yield of the EGT elements identified so far by 1.0 g / L.

[0064] Example 1

[0065] This embodiment provides methods for obtaining the protein sequences of ergothioneine synthase EGT1 and EGT2 encoding genes of various edible fungi and obtaining the encoding sequences after codon optimization according to Trichoderma reesei.

[0066] The identified Neurospora crassa ergothioneine synthase NcEGT1 (Protein ID: XP_956324.3) and the ergothioneine synthase CpEGT2 from Claviceps (Genbank: CCE33140.1) were used to perform homology searches with the genomes of various edible fungi, and protein sequences similar to NcEGT1 and CpEGT2 were found in mushroom N1, mushroom N2, mushroom N3, mushroom N4, mushroom N5, mushroom N6, mushroom N7, and mushroom N8, respectively. The protein sequence was codon-optimized for Trichoderma reesei, and 13 corresponding CDS encoding genes (SEQ ID NO. 1-13) were obtained, namely N1Egt1, N2Egt1, N3Egt1, N4Egt1, N5Egt1, N6Egt1, N7Egt1, N1Egt2, N2Egt2, N4Egt2, N8Egt2, N5Egt2, and N7Egt2, which were synthesized by Suzhou Hongxun Biotechnology Co., Ltd. The similarity between them and the currently identified EGT biosynthetic genes is as follows:

[0067] EGT1 Component Source Highest similarity EGT2 Component Source Highest similarity N1Egt1 Mushroom N1 32.91％ N1Egt2 Mushroom N1 40.99％ N2Egt1 Mushroom N2 31.44％ N2Egt2 Mushroom N2 37.95％ N3Egt1 Mushroom N3 32.70％ N4Egt2 Mushroom N4 39.48％ N4Egt1 Mushroom N4 32.29％ N8Egt2 Mushroom N8 48.70％ N5Egt1 Mushroom N5 33.76％ N5Egt2 Mushroom N5 41.84％ N6Egt1 Mushroom N6 34.11％ N7Egt2 Mushroom N7 43.70％ N7Egt1 Mushroom N7 34.11％

[0068] Example 2

[0069] This example constructs expression vectors for the CDS sequences of ergothioneine synthase EGT1 and EGT2 encoding genes of various edible fungi.

[0070] (1) Strains and plasmids

[0071]

[0072]

[0073] The MC3-Δ10 strain is obtained by knocking out the cbh2, xyn2, egl1, egl2, egl3, bxl1, egl4, cip1, cel74a, and bgl1 genes on the basis of the MC3 strain. The construction method of the strain refers to the public text of patent application number 2023104120463.

[0074] (2) Using the synthesized CDS sequence as a template, the designed primers for ergothioneine synthase EGT1 and EGT2 of various edible fungi were used for PCR amplification using the high-fidelity enzyme PrimeStar. The amplification system was 50 μL, and the reaction system was: 5*PrimeStar Buffer 10 μL, 2 mM dNTPs 4 μL, Primer F 1.25 μL, Primer R 1.25 μL, Template 0.5 μL, PrimeStar 0.5 μL, and ddH2O was added to 50 μL. The PCR program was: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 30 s, 56℃ annealing for 15 s, Egt1 primer extension at 68℃ for 3 min, 35 cycles; Egt2 primer extension at 68℃ for 2 min, 35 cycles.

[0075] (3) The method for constructing the expression vector of CDS sequence of ergothioneine synthase EGT1 and EGT2 encoding genes of various edible fungi is as follows:

[0076] The target bands related to EGT1 amplified by PCR were connected to the expression vector CpEgt2-pMD18T vector, and the target bands related to EGT2 amplified by PCR were connected to the expression vector NcEgt1-pMD18T vector, and the identified NcEgt1 and CpEgt2 were connected to the pMD18T vector. The ligation products were transformed into Escherichia coli Top10, spread on LB solid plates supplemented with ampicillin (Amp: 100 mg / L) for screening, and cultured in a constant temperature incubator at 37°C overnight until a single colony grew. Single clones were picked for colony PCR verification, and positive clones were selected for sequencing verification.

[0077] (4) The XEgt1-CpEgt2-pMD18T plasmid and the NcEgt1-XEgt2-pMD18T plasmid (where XEgt1 represents N1Egt1, N2Egt1, N3Egt1, N4Egt1, N5Egt1, N6Egt1, and N7Egt1, and XEgt2 represents N1Egt2, N2Egt2, N4Egt2, N8Egt2, N5Egt2, and N7Egt2) were extracted from Escherichia coli verified by sequencing.

[0078] Example 3

[0079] In this example, CRISPR / Cas9 technology was used to heterologously insert genes encoding ergothioneine synthases EGT1 and EGT2 of various edible fungi into Trichoderma reesei.

[0080] The Trichoderma reesei MC30 was cultured in a plate using PDA solid medium (200 g / L potato pieces, 20 g / L glucose, 20 g / L agarose, natural pH) and cultured at 28°C for 5-7 days before using physiological saline (0.85% NaCl + 0.02% Tween-80) to prepare a spore suspension of Trichoderma reesei MC30 for 10 min. 8 / mL, spread 150-200 μL of the washed spore suspension on a PDA plate supplemented with 5 mM Uridine, and culture at 28°C until the spores germinate.

[0081] Use the lysing enzyme (Sigma#L1412 lysing enzymes) dissolved in the protoplast solution 1 to lyse the germinated mycelium at 28°C for 2h. Filter the lysed solution to obtain a protoplast suspension. Centrifuge at 2200rpm at 4°C for 10min, remove the supernatant, resuspend the protoplasts with 4mL of protoplast solution 2, centrifuge at 2200rpm at 4°C for 10min, remove the supernatant, and resuspend the protoplasts with 200μL of protoplast solution 2.

[0082] Prepare RNP solution: 10*active buffer 30μL, 10μL gRNA, Cas9 6μg, incubate at 37℃ for 15min. Mix 46μL RNP solution, 10μL expression vector 20-40μg in Example 2 and 56μL 2× protoplast solution 2 and add to the protoplasts resuspended with 200μL protoplast solution 2, then add 75μL protoplast solution 3, mix gently and ice bath for 20min, then add 2mL protoplast solution 3 at room temperature, mix gently, place at 25℃ for 20min, add 4mL protoplast solution 2 at room temperature, mix gently and pour into 50mL upper culture medium, and evenly pour into 3 1.5% agar aqueous solution solid culture medium plates. 28℃ culture medium, after 3-4 days, pick the transformant for genome verification, and select the positive strain for PDA plate culture.

[0083] In this embodiment: the preparation method of protoplast solutions 1, 2, 3 and the upper culture medium is as follows:

[0084] Protoplast solution 1: 1.2 M sorbitol; 0.1 M KH2PO4; pH = 5.5;

[0085] Protoplast solution 2: 1 M sorbitol; 50 mM CaCl2; 10 mM Tris-HCl; pH = 7.5;

[0086] Protoplast solution 3: 25% PEG6000; 50 mM CaCl2; 10 mM Tris-HCl; pH = 7.5;

[0087] Upper culture medium (1L): 1M sorbitol; 4.06mM MgSO4.7H2O; 73.48mM KH2PO4; 45.41mM (NH4)2SO4; 10.20mM trisodium citrate; 50.46mM glucose·H2O; 17.98μMFeSO4·7H2O; 9.47μM MnSO4·H2O; 4.87μM ZnSO4·7H2O; 9.24μM CoCl2; 1.2% agarose.

[0088] Example 4

[0089] In this example, the genes encoding ergothioneine synthases EGT1 and EGT2 from various edible fungi were integrated into Trichoderma reesei to heterologously synthesize ergothioneine.

[0090] The positive strains of XEgt1-CpEgt2 or NcEgt1-XEgt2 correctly inserted into the designated site (see Table 1 for strain number) were picked up, and cultured in PDA solid medium at 28°C for 6-7 days. Then, 108 spore suspensions of Trichoderma reesei XEgt1-CpEgt2-MC30 and NcEgt1-XEgt2-MC30 were prepared using physiological saline (0.85% NaCl + 0.02% Tween-80). 200 μL of the spore suspension was added to 10 mL of SDB liquid medium (1% Tryptone; 1% Yeast extract; 4% glucose) and seed solution was prepared at 28°C, 200 rpm, and 36 h. Take 1mL of seed liquid and add it to 10mL of fermentation medium (3% lactose; 0.5% (NH4)2SO4; 1% KH2PO4; 0.05% MgSO4.7H2O; 0.02% CaCl2; 2% corn steep; 0.1% v / v traceelements; pH 5.5) (trace elements: 0.5% FeSO4·7H2O; 0.16% MnSO4·H2O; 0.14% ZnSO4·7H2O; 0.37% CoCl2·6H2O), 28℃, 200rpm, 96h. Collect the bacterial liquid, boil it in water bath for 10min, centrifuge and collect the supernatant, repeat once, take the total supernatant and filter it for HPLC detection.

[0091] The HPLC detection conditions were: detection wavelength 254 nm, flow rate 1 mL / min, mobile phase (pure acetonitrile, 0.1% formic acid), and single sample analysis time of about 20 min.

[0092] HPLC results are as follows Figure 1 and Figure 2As shown, wherein, the EGT standard product peaks at 3.22min, and the strains of heterologously expressed edible fungus ergothioneine synthase EGT1 and EGT2 encoding genes peak at specific times, respectively, which is basically consistent with the peak time of the EGT standard product. The starting strain detected a trace of EGT product at 3.24min. It can be seen from this that the edible fungus ergothioneine synthase EGT1 and EGT2 of heterologous expression can participate in the synthesis of ergothioneine.

[0093] The EGT yield of the Trichoderma reesei transformed strain obtained above is shown in Table 1:

[0094] Table 1. Transformed strain numbers and EGT yields obtained by heterologous expression of EGT elements from edible fungi in Trichoderma reesei

[0095]

[0096]

[0097] In summary, the present invention develops 13 EGT genes, which not only expands the diversity of EGT synthesis elements, but also obtains new elements for efficient synthesis of EGT, which is helpful to further develop higher-yield ergothioneine biosynthetic cells or strains.

[0098] The inventors connect the coding genes of 13 thioneine synthesis-related enzymes to promoters respectively, and combine and co-express with identified EGT element genes, integrate the co-expressed vector into host cells or strains by gene editing, realize heterologous expression of 13 EGT genes in host cells or strains, and can express thioneine synthase, with good catalytic activity. And screen and obtain cells or strains with different thioneine yields. Especially after the co-expression of nucleic acid molecules and CpEgt2 genes as shown in SEQ ID NO.5, compared with the control strain, the yield of thioneine is significantly improved, and its shaking bottle maximum yield can reach 1.34g / L. After the co-expression of nucleic acid molecules and NcEgt1 genes as shown in SEQ ID NO.10, the yield of thioneine is also greatly improved, and its shaking bottle maximum yield can reach 1.51g / L. Its catalytic activity exceeds the EGT element combination yield 1.0g / L identified at present, so it has good industrial application prospects.

[0099] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0100] References:

[0101] [1]Ergothioneine;antioxidant potential,physiological function androle in disease.BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OFDISEASE.2012.1822,784-793.

[0102] [2]Ergothioneine,recent developments.Redox Biol.2021.42.101868.

[0103] [3]Oral Administration of the Food-Derived Hydrophilic AntioxidantErgothioneine Enhances Object Recognition Memory in Mice.Curr MolPharmacol.2021.14,220-233.

[0104] [4]The Neurospora crassa mutant NcEgt-1identifies an ergothioneinebiosynthetic gene and demonstrates that ergothioneine enhances conidialsurvival and protects against peroxide toxicity during conidialgermination.Fungal genetics and biology:FG&B.2012.49.2,160-172.

[0105] [5]Genetic and metabolomic dissection of the ergothioneine andselenoneine biosynthetic pathway in the fission yeast,S.pombe,andconstruction of an overproduction system.PLoS One.2014.9,e97774.

[0106] [6]The current status of biotechnological production and theapplication of a novel antioxidant ergothioneine.Critical Reviews inBiotechnology.2021.41,580-593.

[0107] [7]Genetic and metabolomic dissection of the ergothioneine andselenoneine biosynthetic pathway in the fission yeast,S.pombe,andconstruction of an overproduction system.PLoS One 9,2014,e97774.0097774.

[0108] [8]Engineering the Yeast Saccharomyces cerevisiae for the Productionof L-(+)-Ergothioneine.Frontiers in Bioengineering and Biotechnology 7,2019,00262.

[0109] [9]The Biosynthetic Pathway of Ergothioneine in Culinary-MedicinalWinter Mushroom,Flammulina velutipes(Agaricomycetes),Int J Med Mushrooms 22,2020,033826.

[0110]

[10] Toward more efficient ergothioneine production using the fungalergothioneine biosynthetic pathway.Microb Cell Fact 2022,21,76。

Claims

1. An ergothioneine synthase, characterized in that It has an amino acid sequence as shown in any one of SEQ ID NOs. 14-26.

2. A nucleic acid molecule encoding ergothioneine synthetase, characterized in that, It encodes the ergothioneine synthase according to claim 1; Preferably, the nucleic acid molecule comprises a first nucleic acid molecule, and the nucleotide sequence of the first nucleic acid molecule is shown in any one of SEQ ID NOs. 1-13.

3. A nucleic acid molecule encoding ergothioneine synthetase, characterized in that, The nucleic acid molecule comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein the nucleotide sequence of the first nucleic acid molecule is shown in any one of SEQ ID NOs. 1-13; the second nucleic acid molecule is selected from any one of the following EGT eukaryotic synthetic genes in any one of the EGT gene clusters: SpEgt1 and SpEgt2, NcEgt1 and NcEgt2, CpEgt1 and CpEgt2, FvEgt1 and FvEgt2, and TrEgt1 and TrEgt2; Preferably, the second nucleic acid molecule is selected from any one of the following EGT eukaryotic synthesis genes in any one of the EGT gene clusters: NcEgt1 and NcEgt2, and CpEgt1 and CpEgt2; Preferably, the nucleic acid molecule comprises: a first nucleic acid molecule as shown in any one of SEQ ID NOs. 1-7, CpEgt2 or NcEgt2; Or the nucleic acid molecule includes the first nucleic acid molecule as shown in any one of SEQ ID NOs. 8-13, NcEgt1 or CpEgt1; Preferably, the nucleic acid molecule comprises: a nucleic acid molecule as shown in SEQ ID NO.5 and CpEgt2; or a nucleic acid molecule as shown in SEQ ID NO.10 and NcEgt1.

4. An expression cassette, characterized in that It comprises the nucleic acid molecule according to any one of claims 2 to 3.

5. The expression cassette according to claim 4, characterized in that The expression cassette comprises: a first nucleic acid molecule as shown in any one of SEQ ID NO.1-7 and any one of the following EGT eukaryotic synthetic genes: CpEgt2 and NcEgt2; Or the expression cassette comprises: a nucleic acid molecule as shown in any one of SEQ ID NOs. 8-13 and any one of the following EGT eukaryotic synthesis genes: NcEgt1 and CpEgt1; Preferably, the expression cassette comprises: a nucleic acid molecule as shown in any one of SEQ ID NOs. 1-7 and CpEgt2; or a nucleic acid molecule as shown in any one of SEQ ID NOs. 8-13 and NcEgt1; Preferably, the expression cassette comprises: a nucleic acid molecule as shown in SEQ ID NO.5 and CpEgt2; or a nucleic acid molecule as shown in SEQ ID NO.10 and NcEgt1.

6. A carrier, characterized in that It comprises the nucleic acid molecule according to any one of claims 2-3 or the expression cassette according to any one of claims 4-5.

7. A cell or strain, characterized in that It comprises the nucleic acid molecule according to any one of claims 2 to 3, the expression cassette according to any one of claims 4 to 5, or the vector according to claim 6.

8. The cell or strain according to claim 7, characterized in that The strain is selected from Agrobacterium, Escherichia coli or Trichoderma reesei; Preferably, the Trichoderma reesei is a Trichoderma reesei with glycolysis repression effect; Preferably, the Trichoderma reesei with glycolysis inhibition effect is selected from MC3 strain or RutC30 strain;.

9. An application of the ergothioneine synthase according to claim 1, the nucleic acid molecule according to any one of claims 2-3, the expression cassette according to any one of claims 4-5, the vector according to claim 6 or the cell or strain according to any one of claims 7-8 in synthesizing ergothioneine; Preferably, the application includes: Integrate the nucleic acid molecule of any one of claims 2 to 3, the expression cassette of any one of claims 4 to 5, or the vector of claim 6 into a cell or strain using a gene editing method, and cultivate the cell or strain heterologously inserted with the target gene; Preferably, the nucleic acid molecule according to any one of claims 2 to 3, the expression cassette according to any one of claims 4 to 5, or the vector according to claim 6 is integrated into the cbh1 site of Trichoderma reesei; Preferably, the gene editing technology is selected from CRISPR / Cas9 technology, zinc finger endonuclease or transcription activator-like effector nuclease technology.

10. A method for synthesizing ergothioneine, characterized in that, It includes: Cultivating the cell or strain according to any one of claims 7 to 8; Preferably, the culture temperature is 28°C±1°C, and the culture is carried out in SDB medium at 200±20 rpm for 30-40 h, and then fermentation culture is carried out.

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