Construction method of engineering aspergillus niger for producing ergothioneine

Abstract

The invention discloses a construction method of engineering aspergillus niger for producing ergothioneine. According to the invention, an ergothioneine synthesis system is constructed in aspergillus niger for the first time, that is, neurospora crassa Nc egt1 and Nc egt2 are expressed at the same time; according to the invention, egt1 and egt2 genes derived from fungi are screened, a better ergothioneine synthesis route in aspergillus niger is determined, namely, Trichoderma reesei Tr egt1 and Neurospora crassa Nc egt2 are expressed at the same time, and the total intracellular and extracellular yield of ergothioneine is 818.3 mg / L through shake flask fermentation, so that the ergothioneine has a relatively strong industrial application prospect; according to the method disclosed by the invention, genes in synthesis routes of three precursors of cysteine, histidine and S-adenosylmethionine are enhanced, the content of intracellular cysteine is increased, the growth of thalli is accelerated, and a thought is provided for development of ergothioneine in the field of heterologous synthesis of filamentous fungi.

Description

Technical Field

[0001] The invention relates to the technical field of ergothioneine synthesis, and mainly relates to the construction and optimization of an ergothioneine biosynthesis pathway in Aspergillus niger. Background Art

[0002] Ergothioneine (EGT) is a unique natural amino acid that is usually biosynthesized by bacteria and fungi. As a food-derived antioxidant and cytoprotectant, it has a variety of physiological benefits, such as scavenging free radicals, detoxification, maintaining DNA biosynthesis, normal cell growth and cellular immunity, anti-aging, etc., which makes it have high application value in the fields of cosmetics, food and medicine. The traditional production of EGT is mainly through biological extraction or chemical synthesis. However, these methods are costly and inefficient, making it difficult for large-scale production to meet the growing market demand. Nowadays, the rapid development of synthetic biology has greatly accelerated the research on the production of EGT by microbial fermentation, among which the production of EGT by fermentation using recombinant strains has higher safety application value.

[0003] The engineering production strains constructed by genetic engineering and metabolic engineering are often effective methods for green and efficient synthesis of target products. After metabolic modification, genetically engineered bacteria can be stimulated to have a higher ability to synthesize target products. Based on the theoretical guidance of the EGT biosynthetic pathway and the analysis of key enzymes, heterologous expression of EGT biosynthetic enzymes has been achieved in some model microorganisms (Escherichia coli, Saccharomyces cerevisiae, etc.), and the production of ergothioneine has been achieved. Wang Li et al. used Escherichia coli BL21 (DE3) as the starting strain, and constructed an engineered strain E1-A1 for de novo fermentation and synthesis of EGT by introducing the EGT synthesis gene cluster egtABCDE from Mycobacterium smegmatis and Egt1 from Schizosaccharomyces pombe; van der Hoek et al. heterologously integrated EGT biosynthetic pathway genes from four different sources into Saccharomyces cerevisiae. These genes were combined to form 16 different metabolic pathways, among which Neurospora crassa Nc egt1 and Claviceps purpurea Cp egt2 were integrated as the yeast strains with the highest EGT production screened out.

[0004] So far, there is no report on the use of Aspergillus niger genetic engineering to achieve ergothioneine production. Aspergillus niger is a food production safety strain (GRAS) certified by the U.S. FDA. It has vigorous growth metabolism, simple nutritional requirements, does not produce mycotoxins, and has a clear genetic background. It is an ideal host bacteria for building filamentous fungal cell factories. It is widely used in the field of biomanufacturing because of its advantages such as high yield, high secretion, and high safety. Using Aspergillus niger as a host bacteria for producing EGT can make the product have a higher safety application value, providing safety guarantees for the wide application of EGT in the fields of food, cosmetics, and medicine. Summary of the invention

[0005] The object of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a method for the construction and optimization of an ergothioneine biosynthetic pathway in Aspergillus niger. The present invention expresses ergothioneine synthesis pathways egt1 and egt2 in Aspergillus niger for the first time, and by integrating different combinations of ergothioneine expression enzyme genes in Aspergillus niger, the best ergothioneine biosynthetic pathway genes in Aspergillus niger are screened out, and cysteine, histidine and S-adenosylmethionine synthesis or metabolic pathway branch genes from Aspergillus niger are overexpressed or knocked out at the same time, to achieve efficient synthesis of ergothioneine in Aspergillus niger.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for constructing an engineered Aspergillus niger producing ergothioneine, comprising at least one of the following improvements on the basis of the starting strain:

[0008] (a) expressing fungal genes of ergothioneine synthesis pathway: the genes include Nc egt1 and Nc egt2 from Neurospora crassa, Tre egt1 and Tre egt2 from Trichoderma reesei, Cp egt2 from Claviceps ergotii or Sp egt1 from Schizosaccharomyces pombe;

[0009] (b) strengthening the genes of the ergothioneine precursor cysteine ​​synthesis pathway branch;

[0010] (c) strengthening the ergothioneine precursor histidine synthesis pathway branch gene;

[0011] (d) strengthening the branch genes of the synthesis pathway of ergothioneine precursor S-adenosylmethionine;

[0012] (e) Simultaneously strengthening the genes for the synthesis pathway of the three precursor amino acids of ergothioneine: cysteine, histidine, and S-adenosylmethionine.

[0013] Furthermore, the precursor synthesis pathway branch gene is at least one of the genes cysB, mecB, Cys1, His1, His2, and SAM1.

[0014] Furthermore, the starting strain is Aspergillus niger SH-2.

[0015] According to the Aspergillus niger gene editing method disclosed in the literature "YU LY, LI LX, YAO LL, et al. A Special Phenotype of Aconidial Aspergillus niger SH2 and Its Mechanism of Formation via CRISPRi [J]. Journal of Fungi, 2022, 8 (7): 679.", the two genes pyrG and kusA of the starting strain were knocked out.

[0016] The amino acid sequence of the protein encoded by the gene pyrG is registered in the FungiDB database as An12g03570; the amino acid sequence of the protein encoded by the gene kusA is registered in the FungiDB database as An15g02700. The amino acid sequence of the protein encoded by the gene pyrG and the amino acid sequence of the protein encoded by the gene kusA are shown in SEQ ID NOs: 13 and 14.

[0017] The starting strain is of the same species as the strain with the catalog number of China Industrial Microbiological Culture Collection Center: CICC 2462, and can realize the genetic modification method of this patent.

[0018] Specifically, the present invention introduces thioneine synthesis pathway in the Aspergillus niger strain genome, overexpresses thioneine synthesis pathway expression genes Nc egt1 and Nc egt2 derived from Neurospora crassa, and preliminarily determines the feasibility of heterologous synthesis of thioneine fungal synthesis pathway in Aspergillus niger. Aspergillus niger starting strain SH-2 synthesizes thioneine in trace amounts, and the transformed strain OE1-N1N2 expressing Nc egt1 and Nc egt2 in Neurospora crassa increases thioneine yield, which is 191.7mg / L.

[0019] The NCBI database sequence registration number of the amino acid sequence of the gene Ncegt1 is XP_956324, the NCBI database sequence registration number of the amino acid sequence of the gene Ncegt2 is XP_001728131, the Aspergillus niger genome insertion site is the neutral amylase site amyA, and the FungiDB database sequence registration number is An05g02100 / An12g06930. As shown in SEQ ID NOs: 1 and 2.

[0020] The invention expresses ergothioneine synthesis pathway genes from different fungi sources, wherein the egt1 gene and the egt2 gene are derived from at least one of Trichoderma reesei, Claviceps purpurea and Schizosaccharomyces pombe, and based on egt1 and egt2 from Neurospora crassa, five scientific combination methods are obtained, and the genes are integrated into the neutral amylase site amyA of the Aspergillus niger genome in a knockout repair manner.

[0021] The amino acid sequence of the gene Trichoderma reesei Tr egt1 has an NCBI database sequence registration number of XP_006968620, the amino acid sequence of Trichoderma reesei Tr egt2 has an NCBI database sequence registration number of XP_006968735, the amino acid sequence of C. ergotii Cp egt2 has an NCBI database sequence registration number of CCE33140.1, and the amino acid sequence of Schizosaccharomyces pombe Sp egt1 has an NCBI database sequence registration number of NP_596639.2. As shown in SEQ ID NOs: 3 to 6.

[0022] The expression plasmid of the present invention includes pMD18 or pYep352, including common expression plasmids in the art, and the plasmid construction method used is TAR homologous recombination technology.

[0023] The knockout integration amyA site is to use a CRISPR / Cas9 plasmid vector to knock out or edit the nucleotide sequence of the corresponding gene; the CRISPR / Cas9 plasmid vector contains an Aspergillus autonomous replication element, a Cas9 sequence, an sgRNA sequence, an hph screening marker, etc.; the sgRNA is obtained by sequentially assembling a promoter, a spacer RNA and an sgRNA skeleton; the promoter is at least one of a T7 or U6 promoter.

[0024] When the gene to be knocked out is amyA, the spacer RNA is the sequence from the 579th to the 598th in the forward direction of its nucleotide sequence.

[0025] The present invention provides the use of the above-mentioned optimal implementation of the Aspergillus niger strain in strengthening the synthesis of ergothioneine. The preferred method is to integrate Trichoderma reesei Tre egt1 and Neurospora crassa Nc egt2 in the starting strain. The Aspergillus niger strain OE4-T1N2 overexpressing the Tre egt1 and Nc egt2 genes increases the ergothioneine production to 419.3 mg / L compared to the basic combination strain OE1-N1N2.

[0026] The present invention attempts to utilize the modified Aspergillus niger strain to strengthen the synthesis of ergothioneine by enhancing the intracellular content of amino acids in the ergothioneine synthesis pathway.

[0027] The enhanced precursor supply is to overexpress amino acid precursor synthesis pathway genes to achieve the accumulation of precursor amino acids in the cell. The amino acid precursors are cysteine, histidine, and S-adenosylmethionine.

[0028] The synthetic pathway genes involved in overexpression include mecB, cysB, cys1, his1, his2, and SAM1; the metabolic modification involved is at least one of the above genes; and the overexpression is to use a constitutive promoter to integrate an additional copy on the Aspergillus niger genome.

[0029] The amino acid sequence of the protein encoded by the gene mecB has a FungiDB database sequence accession number of An16g08720, the amino acid sequence of the protein encoded by the gene cysB has a FungiDB database sequence accession number of An02g10750, the amino acid sequence of the protein encoded by the gene Cys1 has a FungiDB database sequence accession number of An12g09880, the amino acid sequence of the protein encoded by the gene His1 has a FungiDB database sequence accession number of An17g01640, the amino acid sequence of the protein encoded by the gene His2 has a FungiDB database sequence accession number of An14g07210, and the amino acid sequence of the protein encoded by the gene SAM1 has a FungiDB database sequence accession number of An02g10660 (as shown in SEQ ID NOs: 7 to 12).

[0030] After the amino acid precursor metabolic pathway gene is overexpressed, the intracellular cysteine ​​content of an Aspergillus niger is accumulated; the precursor supply in ergothioneine synthesis is strengthened, the growth intensity of the engineered bacteria is increased, the bacterial body volume is increased, and the growth is accelerated.

[0031] In the present invention, in an Aspergillus niger, the expression of gene Nc egt1 and gene Nc egt2 can synthesize ergothioneine; in an Aspergillus niger, the expression of gene Nc egt1 and gene Tr egt2 can synthesize ergothioneine; in an Aspergillus niger, the expression of gene Nc egt1 and gene Cp egt2 can synthesize ergothioneine; in an Aspergillus niger, the expression of gene Tr egt1 and gene Tr egt2 can synthesize ergothioneine; in an Aspergillus niger, the expression of gene Nc egt1 and gene Nc egt2 can synthesize ergothioneine, and the synthesis level of ergothioneine can be greatly improved.

[0032] In a strain of Aspergillus niger, the effect of egt1 on ergothioneine synthesis was ranked as Tr egt1>Nc egt1>Spegt1.

[0033] In a strain of Aspergillus niger, the effect of egt2 on ergothioneine synthesis was ranked as Nc egt2>Tr egt2>Cpegt2.

[0034] In an engineered Aspergillus niger, the genes cysB, mecB, and cys1 were simultaneously overexpressed to increase the accumulation of intracellular cysteine.

[0035] Compared with the prior art, the present invention has the following advantages and effects:

[0036] a. In the present invention, two genes of the ergothioneine synthesis pathway are integrated into the Aspergillus niger genome, and ergothioneine is heterologously synthesized in Aspergillus niger for the first time, which is innovative and provides a new host selectivity for the heterologous synthesis research of ergothioneine.

[0037] b. The present invention integrates ergothioneine synthesis pathway genes from different sources into the Aspergillus niger genome, determines a combination that makes the Aspergillus niger ergothioneine synthesis amount expressed relatively high, and the combined analysis is conducive to the realization of efficient heterologous synthesis of ergothioneine by Aspergillus niger.

[0038] c. The present invention obtains an Aspergillus niger engineered strain with enhanced synthesis of cysteine, histidine and S-adenosylmethionine based on the integration of Tr egt1 and Nc egt2. The growth of the engineered strain is accelerated, which provides an application reference for enhancing the supply of precursors in the synthesis direction of ergothioneine. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a map of plasmid pYep352-N1N2.

[0040] Figure 2 This is a schematic diagram of the CRISPR-Cas9 cutting site and the homologous recombination of the integrated fragment.

[0041] Figure 3 It is a peak diagram of HPLC detection results: (Ⅰ) is a diagram of HPLC detection results of ergothioneine at various concentration gradients; (Ⅱ) is a diagram of the standard curve of ergothioneine; (Ⅲ) is a diagram of the HPLC results of the basic strain SH2-WT and the ergothioneine engineering bacteria; (Ⅳ) is a diagram of the HPLC results of Aspergillus niger OE4-T1N2 and the precursor gene overexpression engineering bacteria.

[0042] Figure 4 This is a graph showing the yield of ergothioneine by fermentation of the basic strain SH2-WT and the engineered bacteria.

[0043] Figure 5This is a graph showing the production results of intracellular and extracellular ergothioneine in strain OE4-T1N2 and precursor modified engineered bacteria.

[0044] Figure 6 This is a graph showing the detection of intracellular cysteine ​​content in strain OE7-T1N2-cys. DETAILED DESCRIPTION

[0045] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0046] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0047] The strains, raw materials, reagents and instruments involved in the present invention: Aspergillus niger SH-2△pyr G,△kus A, disclosed in the document "YU LY, LI LX, YAO LL, et al. A Special Phenotype of Aconidial Aspergillus niger SH2 and Its Mechanism of Formation via CRISPRi[J]. Journal of Fungi, 2022, 8(7): 679."; the chemical reagents and drugs involved in the present invention: snail enzyme, cellulase (Beijing Dingguo Changsheng Biotechnology Co., Ltd.), peptone, yeast powder (UK OXOID Company), polyethylene glycol (PEG4000), sorbitol, anhydrous calcium chloride, lysozyme, uridine, Triton X-100 (Sigma Company, USA), agarose (Biowest, Spain), restriction endonuclease (Thermo Fisher Company, USA), Dream Taq Green PCR Master Mix (Nanjing Novigene), PrimerStar HSDNA polymerase, DNA Ligation Kit, DNA marker, FastAP dephosphorylation kit, RNAiso plus, 6× Loading buffer, RNase free water, RNase inhibitor, SYBR Premix EX TaqTM (TaKaRa, Japan), GelRed nucleic acid dye (Biotum, USA), Miracloth filter cloth (Merck, Germany), PCR product purification kit, plasmid large-scale extraction kit (Guangzhou Jiebeisi), Infusion kit (NEBuilder HiFi DNAAssembly Cloning Kit), cysteine ​​detection kit (Beijing Solaibao), T7 in vitro transcription kit, Cas9 protein kit, restriction endonuclease (NEB, USA), amino-free yeast nitrogen source YNB, ampicillin, defective amino acid mixture DO Supplement-Ura (Beijing Puboxin), other chemical reagents and drugs are domestic or imported analytical grade or above; the main instruments and equipment involved in the present invention are:

[0048] NanoDrop1000 biospectrophotometer, PCR instrument, gel imaging system 1500Pro (ThermoFisher, USA), high pressure sterilizer (Panasonic, Japan), DNA gel electrophoresis instrument (Bio-Rad, USA), mold incubator (SHP-450D, Shanghai Senxin Experimental Instrument Co., Ltd.), constant temperature air bath shaker (Suzhou Peiying Experimental Instrument Co., Ltd.), refrigerated high-speed centrifuge, molecular pipette (Eppendorf, Germany), clean bench (SW-CJ-1FD, Suzhou Purification Co., Ltd.), IMS-20 ice machine (Guangzhou Shenhua Biotechnology Co., Ltd.), ultrapure water instrument (Shanghai Lingde Instrument Co., Ltd.), electric tissue grinder (Beijing Tiangen Biochemical Technology Co., Ltd.), high-precision balance (Sartorius, Germany), vortex oscillator (SCILOGEX, USA), ultrasonic cleaner (Ningbo Xinzhi Instrument Co., Ltd.), vacuum filtration pump (DOA-P70-BN, PALL, USA).

[0049] The culture medium involved in the present invention is:

[0050] Hypertonic CD medium: 342.3 g / L sucrose, 2 g / L potassium chloride, 1 g / L potassium dihydrogen phosphate, 3 g / L sodium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate heptahydrate.

[0051] Ordinary CD culture medium: 20 g / L anhydrous glucose, 2 g / L potassium chloride, 1 g / L potassium dihydrogen phosphate, 3 g / L sodium nitrate, 0.5 g / L magnesium sulfate heptahydrate, 0.01 g / L ferrous sulfate heptahydrate.

[0052] YPD medium: 20 g / L anhydrous glucose, 10 g / L yeast powder, 10 g / L peptone.

[0053] Fermentation medium: 40 g / L anhydrous glucose, 20 g / L peptone, 2 g / L sodium nitrate, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, pH 5.5. Sterilize at 115°C for 20 min.

[0054] STC solution: 10 mM Tris-HCl, 1.2 M sorbitol, 50 mM calcium chloride, pH 7.5, first make up to volume and then adjust pH, sterilize by 0.22 μL membrane filtration.

[0055] PEG solution: 60% (w:v) PEG4000, 50mM calcium chloride, 10mM Tris-HCl, pH7.5, first make up to volume and then adjust pH, sterilize at high temperature and high pressure.

[0056] Ergothioneine concentration is measured by high performance liquid chromatography (HPLC), using Agilent 1220 high performance liquid detection system, C18 posts (250 × 4.6mm, 5 μm), UV detector, detection wavelength 257nm, mobile phase ratio: water: methanol=99:1, organic phase is pure methanol, injection flow rate 0.7mL / min, injection volume 5 μL, single sample analysis duration 20min. The thioneine standard samples of 25mg / L, 50mg / L, 100mg / L, 200mg / L, 400mg / L are configured respectively, the corresponding peak area under each concentration measured respectively according to HPLC method, with the concentration of thioneine standard samples as abscissa, corresponding peak area as ordinate, draw standard curve, calculate the output of corresponding thioneine according to the sample peak area measured.

[0057] Example 1: Construction of engineered Aspergillus niger OE1-N1N2 producing ergothioneine

[0058] This embodiment provides a method for constructing an Aspergillus niger strain for producing ergothioneine, comprising the following steps:

[0059] Chemical synthesis (two genes Nc egt1 and Nc egt2 of the ergothioneine fungal biosynthesis pathway of GenScript Company, the amplified fragment was obtained by standard PCR technology principle; the plasmid Yep352 was digested with DNA endonucleases EcoRⅠ and HinⅢ to obtain the vector linear fragment, and the amplified fragment Nc egt1 / Nc egt2, promoter Ptef / PgpdA, terminator Ttef / TtrpC, linear vector, and selection marker pyrG were connected by yeast in vivo TAR cloning technology. The yeast transformants were identified by primer identification to obtain the Saccharomyces cerevisiae transformants successfully connected to the expression plasmid pYep352-N1N2. The schematic diagram of the pYep352-N1N2 vector is shown in Figure 1 . The correct Saccharomyces cerevisiae transformants were cultured and identified, the plasmids in vivo were extracted, and then transformed into Escherichia coli match1 T1 competent cells. The transformation products were spread on LB solid culture medium containing 50 μg / mL ampicillin, and inverted overnight culture was carried out at 37°C for 12 hours to obtain transformants; the transformants were picked and cultured in LB liquid culture medium containing 50 μg / mL ampicillin, and then the plasmids were extracted in large quantities. Escherichia coli containing the knockout neutral amylase plasmid pFC332-PU6-amyA-TU6-hygB were cultured in LB liquid culture medium containing 50 μg / mL ampicillin, and then the plasmids were extracted in large quantities. Aspergillus niger protoplasts were prepared, and the Aspergillus niger transformation was carried out using the PEG-mediated protoplast method. (The primers used in this implementation example are shown in Table 1)

[0060] Table 1 Primer sequences for fragment amplification and transformant PCR identification

[0061]

[0062]

[0063] The in vivo TAR cloning method of yeast includes two parts: preparation of competent cells of Saccharomyces cerevisiae and transformation. The specific steps are as follows:

[0064] The brewer's yeast strain to be prepared was inoculated into YPD liquid medium, cultured at 30°C, 220rpm for two days. The activated seed solution was inoculated into 50ml liquid YPD and cultured to the logarithmic phase (OD=0.6). After the culture was completed, the culture solution was centrifuged at 800g for 8min, the supernatant was removed, and the bacteria were collected. The bacteria were resuspended in 30ml sterile water, centrifuged at 800g for 8min, and the supernatant was removed. The bacteria were resuspended in 1.5ml TE / LiAc and the resuspended solution was transferred to a 1.5ml centrifuge tube. The resuspended solution was centrifuged at 10000g for 15s, the supernatant was removed, and then 600ul TE / LiAc was added to resuspend the bacteria to obtain competent cells. In the 50ul competent cells, plasmids, fragments, 5ul of Carrier DNA (treated at 95℃-105℃ for 5min, quickly ice bathed for 1min, repeated once), and 500ul of PEG / LiAc were added and mixed well, and then reacted in a 30℃ water bath for 30min, and inverted and mixed every 10min. Add 20ul of DMSO and react in a 42℃ water bath for 15min, mixing by inversion every 5min. Centrifuge at 10000g for 15s, remove the supernatant, add 1ml of YPD medium, and culture in a 30℃ shaker for 1h. Centrifuge at 10000g for 15s, remove the supernatant, add about 500ul of 0.9% NaCl to resuspend the cells, and spread on SD-Ura solid medium.

[0065] The method of transforming Aspergillus niger by using PEG-mediated protoplasts is as follows: the filtered mycelium is transferred to the protoplast hydrolysate, the system of which is 1% cellulase, 1% snail enzyme, 0.5% lytic enzyme, 1× sodium phosphate buffer, and 0.8M sodium chloride. The filtered mycelium is added to 20mL of enzyme solution and hydrolyzed at 120rpm at 30°C. After the hydrolysis is completed, the protoplasts are collected by filtration with 4 layers of Milipore, and then the bottle of the hydrolysate and Milipore are rinsed with 20mL of 0.8M sodium chloride. The protoplasts are centrifuged at 900g for 10min at 4°C, and the supernatant is discarded. 5mL of STC buffer is added to resuspend the protoplasts, and after uniform dispersion, 25mL of STC buffer is added to resuspend, and then the protoplasts are collected by centrifugation at 900g for 10min at 4°C. This step is repeated once. The protoplasts are resuspended with 600μL of STC buffer. Add 100 μL of knockout plasmid and expression plasmid to 160 μL protoplasts, and add PEG solution, gently blow with a sharpened pipette to mix evenly, and place on ice for 30 minutes. After adding 1.5 mL of PEG solution, cover the centrifuge tube cap and turn it upside down to mix it thoroughly, and place it at room temperature for 25 minutes. During this period, pour the lower plate, that is, hypertonic CD culture medium (containing 2% agar). Finally, pour the above transformation mixture into a 15 mL sterile centrifuge tube containing 6 mL soft agar hypertonic CD culture medium (containing 0.5% agar) at 30°C to 45°C and 3 mL of STC buffer, and mix it upside down. Finally, the mixture (upper plate, containing 0.5% hypertonic CD medium) was evenly poured onto the sucrose CD hypertonic solid medium (lower plate, containing 2% hypertonic CD medium), and cultured at 30°C for 6 days. During this period, the upper medium (containing 0.5% hypertonic CD medium) solidified. Transformants emerging from the transformation plate were selected and placed in new ordinary CD medium (containing 2% agar), and cultured in a 30°C incubator for 4 days. The transformant genes were extracted and identified by PCR. The primer sequences for PCR identification are shown in Table 1. The schematic diagram of gene integration is shown in Figure 2 .

[0066] The correct transformant was identified as OE1-N1N2, and the above-mentioned strain was subjected to shake flask fermentation culture, and the specific steps were as follows: the transformant OE1-N1N2 was ground with a grinding rod and then accessed into liquid CD (containing 0.05% agar) and cultured to a certain amount, and then accessed the fermentation medium with a 10% (v:v) inoculum amount, and culture supernatant was obtained after 7d. Ergothioneine production was detected using high performance liquid chromatography, and the peak diagram was as shown in FIG. Figure 3 As shown, (I) is the HPLC detection result of ergothioneine standard sample, (II) is the standard curve of ergothioneine, (III) is the HPLC detection of fermentation sample, and (IV) is the HPLC detection peak diagram of Aspergillus niger fermentation sample of precursor transformation engineering. Figure 4 The constructed engineered bacteria OE1-N1N2 produced ergothioneine at a production rate of 191.7 mg / L.

[0067] Example 2 Screening of fungal-derived ergothioneine synthesis pathway genes

[0068] The enzymes of ergothioneine synthesis pathway from different fungi, including Tregt1 (XP_006968620) and Tregt2 (XP_006968735) from Trichoderma reesei, Cp egt2 (CCE33140.1) from Claviceps ergotii, and Sp egt1 (NP_596639.2) from Schizosaccharomyces pombe, were connected with the linearized Yep352 vector by one-step seamless cloning or TAR cloning method to obtain vectors pYep352-N1T2, pYep352-N1C2, pYep352-T1N2, pYep352-S1N2, and pYep352-T1T2, respectively. According to Example 1, these plasmids were transformed into Aspergillus niger and the Aspergillus niger engineered bacteria OE2-N1T2, OE3-N1C2, OE4-T1N2, OE5-S1N2, and OE6-T1T2 were identified. According to Example 1, the above-mentioned transformants were fermented in shake flasks and the yield of ergothioneine in the fermentation broth was detected. The amplification primers and identification primers are shown in Table 1. The peak diagram obtained by high performance liquid chromatography detection of the fermentation broth is shown in Figure 3 (IV), the fermentation yield of each transformant was calculated as shown in Figure 4 When Trichoderma reesei-derived Tregt1 and Neurospora crassa-derived Nc egt2 were simultaneously overexpressed in the engineered Aspergillus niger OE4-T1N2, the ergothioneine production was the highest, reaching 419.3 mg / L, which was about 118.7% higher than that of the basic engineered strain OE1-N1N2.

[0069] Example 3 Optimization of ergothioneine precursor amino acid metabolic pathway

[0070] Based on the OE4-T1N2 engineered bacteria obtained in Example 2, the cystathionine γ-lyase mecB (FungiDB ID: An16g08720), cysteine ​​synthetase cysB (FungiDB ID: An02g10750), and Cys1 (FungiDB ID: An12g09880) genes in the cysteine ​​synthesis pathway were overexpressed, the histidine synthesis pathway gene His1 (FungiDBID: An17g01640), and histidinol dehydrogenase His2 (FungiDB ID: An14g07210) were overexpressed, and the SAM synthesis pathway gene methionine adenosyltransferase SAM1 (FungiDB ID: An02g10660) were overexpressed, and the corresponding primers were used to amplify the above gene fragments. The amplification primers are shown in Table 1. According to the method of vector construction and Aspergillus niger transformation and identification in Example 1, the engineered strain OE7-T1N2-cys was obtained by simultaneously overexpressing the cysteine ​​synthesis pathway genes mecB, cysB, and Cys1; the engineered strain OE8-T1N2-his was obtained by simultaneously overexpressing the histidine synthesis pathway genes His1 and His2; the engineered strain OE9-T1N2-sam was obtained by overexpressing the SAM synthesis pathway gene SAM1; the engineered strain OE10-T1N2-CSH was obtained by simultaneously overexpressing the cysteine, histidine, and SAM synthesis pathway genes. According to Example 1, the above-mentioned transformants were fermented in shake flasks, and all the cells after 7 days of growth in the fermentation medium were collected and weighed using a vacuum filtration pump. The cells were ground using liquid nitrogen, and 0.1 g of the ground cells were dissolved in 1 ml of water to obtain a cell crushing liquid. The ergothioneine yield in the fermentation liquid and the cell crushing liquid was detected using HPLC, and the intracellular cysteine ​​content of OE7-T1N2-cys was detected using a cysteine ​​detection kit. The ergothioneine yield is shown in Figure 5 , intracellular cysteine ​​content Figure 6 . The engineered bacteria OE4-T1N2 has a stable ergothioneine production, with an extracellular secretion of 398.6 mg / L, an intracellular production of 419.7 mg / L, and a total ergothioneine production of 818.3 mg / L. The modified engineered strains OE7-T1N2-cys, OE8-T1N2-his, OE9-T1N2-sam, and OE10-T1N2-CSH have different degrees of inhibition on the synthesis of ergothioneine and a slight promotion effect on the growth of bacteria; among them, the engineered strain OE7-T1N2-cys has an increased level of intracellular cysteine ​​synthesis in the fermentation medium after overexpressing three genes in the cysteine ​​synthesis pathway.

[0071] Example 2 is a preferred implementation mode of the present invention, but the implementation mode of the present invention is not limited to the above-mentioned example. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for constructing an engineered Aspergillus niger producing ergothioneine, characterized in that, At least one of the following improvements was made based on the starting strain: (a) expressing fungal genes of ergothioneine synthesis pathway: the genes include Ncegt1 and Ncegt2 from Neurospora crassa, Tregt1 and Tregt2 from Trichoderma reesei, Cp egt2 from Claviceps ergotii or Sp egt1 from Schizosaccharomyces pombe; (b) strengthening the genes of the ergothioneine precursor cysteine ​​synthesis pathway branch; (c) strengthening the ergothioneine precursor histidine synthesis pathway branch gene; (d) strengthening the branch genes of the synthesis pathway of ergothioneine precursor S-adenosylmethionine; (e) Simultaneously strengthening the genes for the synthesis pathway of the three precursor amino acids of ergothioneine: cysteine, histidine, and S-adenosylmethionine.

2. according to the construction method of a kind of thioneine-producing engineering Aspergillus niger of claim 1, it is characterized in that: The precursor synthesis pathway branch gene is at least one of the genes cysB, mecB, Cys1, His1, His2, and SAM1.

3. according to the construction method of a kind of thioneine-producing engineering Aspergillus niger of claim 2, it is characterized in that: The sequences of cysB, mecB, Cys1, His1, His2, and SAM1 are shown in SEQ ID NOs: 7 to 12.

4. A method for constructing an engineering Aspergillus niger producing ergothioneine according to claim 1, characterized in that: The NCBI database sequence accession number of the amino acid sequence of gene Nc egt1 is XP_956324, the NCBI database sequence accession number of the amino acid sequence of gene Nc egt2 is XP_001728131, the NCBI database sequence accession number of the amino acid sequence of gene Tr egt1 is XP_006968620, the NCBI database sequence accession number of the amino acid sequence of gene Tr egt2 is XP_006968735, the NCBI database sequence accession number of the amino acid sequence of gene Cp egt2 is CCE33140.1, and the NCBI database sequence accession number of the amino acid sequence of gene Spegt1 is NP_596639.

2.

5. according to the construction method of a kind of thioneine-producing engineering Aspergillus niger of claim 2, it is characterized in that: The FungiDB database sequence accession number of the amino acid sequence of the protein encoded by gene mecB is An16g08720, the FungiDB database sequence accession number of the amino acid sequence of the protein encoded by gene cysB is An02g10750, the FungiDB database sequence accession number of the amino acid sequence of the protein encoded by gene Cys1 is An12g09880, the FungiDB database sequence accession number of the amino acid sequence of the protein encoded by gene His1 is An17g01640, the FungiDB database sequence accession number of the amino acid sequence of the protein encoded by gene His2 is An14g07210, and the FungiDB database sequence accession number of the amino acid sequence of the protein encoded by gene SAM1 is An02g10660.

6. A method for constructing an engineering Aspergillus niger producing ergothioneine according to claim 1, characterized in that: The starting strain is Aspergillus niger SH-2.

7. A method for constructing an engineering Aspergillus niger producing ergothioneine according to claim 1, characterized in that: In a kind of Aspergillus niger, the gene Nc egt1 and the gene Nc egt2 are expressed to synthesize ergothioneine; in a kind of Aspergillus niger, the gene Nc egt1 and the gene Tr egt2 are expressed to synthesize ergothioneine; in a kind of Aspergillus niger, the gene Nc egt1 and the gene Cpegt2 are expressed to synthesize ergothioneine; in a kind of Aspergillus niger, the gene Tr egt1 and the gene Tr egt2 are expressed to synthesize ergothioneine; in a kind of Aspergillus niger, the gene Nc egt1 and the gene Nc egt2 are expressed to synthesize ergothioneine, and the synthesis level of ergothioneine is greatly improved.

8. A method for constructing an engineering Aspergillus niger producing ergothioneine according to claim 1, characterized in that: The order of influence of egt1 on ergothioneine synthesis is Tr egt1>Nc egt1>Sp egt1.

9. A method for constructing an engineering Aspergillus niger producing ergothioneine according to claim 1, characterized in that: The order of influence of egt2 on ergothioneine synthesis is Nc egt2>Tr egt2>Cp egt2.

10. A method for constructing an engineering Aspergillus niger producing ergothioneine according to claim 1, characterized in that: At the same time, overexpression of genes cysB, mecB, and cys1 increased the accumulation of intracellular cysteine.

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