Mutant of ergothioneine and application thereof

Abstract

The application discloses a mutant of ergothioneine and application thereof, relates to the technical field of genetic engineering and synthetic biology, and the mutant comprises a mutant ergothioneine synthase and a mutant cysteine sulfoxide cleaving enzyme; the amino acid sequence of the former is selected from any one of SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 and SEQ ID No: 8; and the amino acid sequence of the latter is selected from any one of SEQ ID No: 9, SEQ ID No: 10 and SEQ ID No: 11. The application obtains the mutant ergothioneine synthase and the mutant cysteine sulfoxide cleaving enzyme with high activity; the two mutants obtained through screening are used for producing ergothioneine, have the advantages of high acid production and short fermentation time, greatly save material cost, and are favorable for industrial production.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and synthetic biology, and in particular to a mutant for synthesizing ergothionein and its applications. Background Technology

[0002] Ergothioneine (ERG) is a natural amino acid with a very unique thiazoline ring structure, hailed as the "king of antioxidants," the "longevity vitamin," and the "guardian of cells." In recent years, it has received unprecedented attention in the fields of skincare products, health supplements, and medical research.

[0003] Ergothioneine's remarkable efficacy stems from its unique mechanism of action. Its core capability lies in its powerful antioxidant capacity, effectively neutralizing various reactive oxygen species (ROS) and reactive nitrogen species (RNS), and regenerating other antioxidants (such as vitamin C, vitamin E, and glutathione) to form a robust antioxidant network. Secondly, it specifically accumulates in mitochondria, directly protecting mitochondrial DNA and membrane structures from oxidative damage, ensuring stable cellular energy supply, thereby delaying cellular aging. Furthermore, it inhibits the activation of key inflammatory signaling pathways such as nuclear factor κB (NF-κB), thereby reducing the production of pro-inflammatory cytokines (such as TNF-α, IL-6, and IL-1β), helping to alleviate chronic inflammation at its root.

[0004] The human body cannot synthesize ergothioneine on its own and mainly obtains it through dietary supplements, including fungi, meat, and grains. However, naturally occurring ergothioneine is very limited and scarce, failing to meet the ever-expanding market demand. Currently, the main methods for synthesizing ergothioneine are chemical synthesis and biosynthesis. Chemical synthesis is mostly in the laboratory research stage and cannot be used for industrial production. Biosynthesis mainly relies on microbial fermentation. One method involves screening high-yielding original strains from nature for fermentation extraction. For example, CN120699781A discloses a high-yielding Cordyceps militaris strain and its application. Its mutant Cordyceps militaris strain achieved a maximum yield of 3.833 mg / g after the addition of precursor substances, but the fermentation time was relatively long, reaching 7 days, and the yield was average. CN120555527A discloses a method for simultaneously producing ergothioneine and enzyme preparations using Trichoderma reesei, with a maximum yield of 250 mg / L in shake flasks, which is a very low yield.

[0005] It is evident that the yield of strains produced through natural selection and mutation is very low and cannot meet the standards for industrial production. Therefore, relevant researchers attempted to construct ergothionein engineered bacteria using model strains. For example, CN112251392A disclosed a genetically engineered strain for producing ergothionein and its application. By integrating the ergothionein operator egtBCDE and sulfoxide synthase mutant into the Escherichia coli host MG1655, the yield reached a maximum of 2.9 g / L after continuous fermentation for 52 hours in a 5L fermenter. CN117844722A disclosed a genetically engineered bacterium and its use. Genes such as EgtB, EgtD, and EgtE were constructed in sodium-dependent Vibrio. After culturing in a 6L fermenter for 60 hours, the yield reached 4.55 g / L. CN120099041A disclosed a lipothermic yeast strain for synthesizing ergothionein and its application. The lipothermic yeast strain was modified as the original strain, and the key enzyme TrEGT1 was semi-rationally modified. After continuous fermentation in a 5L fermenter for 168 hours, the maximum yield reached 10.3 g / L.

[0006] Overall, fermentation through chassis cell modification using prokaryotic or eukaryotic strains has two main drawbacks. First, the fermentation yield is low, making it impossible to scale up to industrial production. Second, although some strains can achieve yields exceeding 10 g / L, the fermentation time is too long, consuming a large amount of material and human resources, which is not conducive to industrial production. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a mutant of synthetic ergothionein and its application, which increases yield and shortens fermentation time.

[0008] In order to achieve the objective of this invention, the following solution is proposed: First, the following explanation is provided: Ergothionein synthase is derived from non-alfalfa variant Fusarium ( Verticillium nonalfalfae) The sequence shown in SEQ ID No: 1 is the original gene sequence of ergothionein synthase, and the sequence shown in SEQ ID No: 2 is the original amino acid sequence of ergothionein synthase.

[0009] Cysteine ​​sulfoxide lyase is derived from Alternaria alternata ( Alternaria arborescens) The original gene sequence of cysteine ​​sulfoxide lyase shown in SEQ ID No: 3, and the original amino acid sequence of cysteine ​​sulfoxide lyase shown in SEQ ID No: 4.

[0010] In a first aspect, the present invention provides a mutant for synthesizing ergothioneine, comprising a mutant ergothioneine synthase and a mutant cysteine ​​sulfoxide lyase.

[0011] The amino acid sequence of the mutant ergothionein synthase is selected from any one of SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7, and SEQ ID No: 8.

[0012] (1) The sequence shown in SEQ ID No: 5 was obtained by replacing valine at position 117 with glycine, glutamic acid at position 141 with threonine, and glycine at position 188 with proline.

[0013] (2) The sequence shown in SEQ ID No: 6 was obtained by replacing valine at position 117 with glycine, phenylalanine at position 153 with tryptophan, and glutamic acid at position 237 with aspartic acid.

[0014] (3) The sequence shown in SEQ ID No: 7 was obtained by replacing the aspartic acid at position 512 of the amino acid sequence shown in SEQ ID No: 2 with glutamic acid, the valine at position 657 with leucine, and the lysine at position 724 with arginine.

[0015] (4) The sequence shown in SEQ ID No: 8 was obtained by replacing aspartic acid at position 512 with glutamic acid, proline at position 655 with glycine, and isoleucine at position 750 with methionine.

[0016] The amino acid sequence of the mutant cysteine ​​sulfoxide lyase is selected from any one of the sequences shown in SEQ ID No: 9, SEQ ID No: 10, and SEQ ID No: 11.

[0017] (1) The sequence shown in SEQ ID No: 9 was obtained by replacing alanine at position 183 with leucine, asparagine at position 258 with cysteine, and threonine at position 287 with serine. (2) The sequence shown in SEQ ID No: 10 was obtained by replacing serine at position 95 with cysteine, lysine at position 169 with arginine, and proline at position 246 with glycine. (3) The sequence shown in SEQ ID No: 11 was obtained by replacing isoleucine at position 73 with alanine, alanine at position 293 with leucine, and histidine at position 409 with lysine.

[0018] Secondly, the present invention provides a gene encoding the mutant ergothionein synthase.

[0019] Thirdly, the present invention provides a dual expression vector comprising a gene encoding the mutant ergothioneine synthase and a gene encoding the mutant cysteine ​​sulfoxide lyase.

[0020] Fourthly, the present invention provides an engineered bacterium, obtained by the following method: using *Escherichia coli* E. coli W3110 Using this initial strain, the genetic metabolic pathway of the strain was demonstrated by knocking out the gene for L-cysteine ​​desulfurization hydratase using CRISPR / Cas9 gene editing technology. tnaA ), genes for oxidoreductases ( yciW ), to reduce cysteine ​​degradation; in methionine adenosine transferase metK An enhancer was added to the front end of the gene, the enhancer sequence of which is shown in SEQ ID No: 12, to promote the conversion of L-methionine and obtain a chassis strain; the dual expression vector was then transferred into the chassis strain to obtain an engineered strain.

[0021] Fifthly, the present invention provides the application of the mutant for the synthesis of ergothioneine.

[0022] Sixthly, the present invention provides a method for synthesizing ergothioneine, comprising the following steps: (1) After culturing the engineered bacteria in 600 ml of LB medium in a shake flask, the culture was inoculated into a 100 L seed tank and cultured for 7 h until the OD value was reached. 600 ≥3, no bacteria detected under microscopy.

[0023] (2) 50L of seed culture was inoculated into a 1T fermenter. The initial culture medium was 300L, feed 1 was 400L, and feed 2 was 200L. The feed 1 formula was: glucose 800g / L, magnesium sulfate heptahydrate 20g / L, and antifoaming agent 3g / L. The feed 2 formula was: L-histidine 20g / L, L-methionine 40g / L, L-cysteine ​​hydrochloride 15.5g / L, and pyridoxal phosphate 0.3g / L. 5L of inducer was added, with the formula: IPTG 25g / L and L-arabinose 300g / L.

[0024] (3) Initial culture conditions: pH adjusted to 6.8-7.0, temperature 37℃, rotation speed 100 rpm, aeration rate 8 m³ / h 3 / h, dissolved oxygen controlled at 20%-40%, after dissolved oxygen rebound, start feeding one at a rate of 30ml / (L·h), when OD 600 At 25-30°C, add the inducing agent all at once and cool down to 30°C. Start feeding the second batch of feed at a rate of 40 ml / (L·h). After incubation for about 80 hours, remove the feed from the container.

[0025] Furthermore, in step (3), samples are taken every 5 hours, and the contents of ergothioneine, L-histidine, L-methionine, and L-cysteine ​​hydrochloride are determined by high performance liquid chromatography. L-cysteine ​​hydrochloride must not be detected, L-methionine ≤1g / L, and L-histidine ≤0.1g / L.

[0026] The beneficial effects of this invention are as follows: 1. This invention utilizes Fusarium oxysporum derived from non-alfalfa variants (… Verticillium nonalfalfae) Ergothioneine synthase and Alternaria alternata ( Alternaria arborescens) The cysteine ​​sulfoxide lyase was modified, and the corresponding mutants were screened to identify key residues affecting ergothioneine synthesis. Highly active mutant ergothioneine synthase and mutant cysteine ​​sulfoxide lyase were obtained. The two mutants obtained were applied to the production of ergothioneine using L-histidine, L-methionine, L-cysteine ​​hydrochloride, and PLP as feedstocks, showing good results.

[0027] 2. In the fermentation reaction of mutant ergothioneine synthase and mutant cysteine ​​sulfoxide lyase to prepare ergothioneine, it has good acid production efficiency. After continuous fermentation for 75 hours in a 1T fermenter, the highest yield reached 12.85 g / L. This is the metabolic engineered bacteria with the highest acid production in a short fermentation time known to date. It has the advantages of high acid production, short fermentation time, and great savings in material costs, which is conducive to industrial production. Attached Figure Description

[0028] Figure 1 Plasmid maps of mutant ergothioneine synthase (D512E, P655G, I750M) and mutant cysteine ​​sulfoxide lyase (S95C, K169R, P246G).

[0029] Figure 2 This is a high-performance liquid chromatography (HPLC) chromatogram of ergothionein.

[0030] Figure 3 Electrophoretic detection images of mutant ergothioneine synthase and mutant cysteine ​​sulfoxide lyase proteins.

[0031] Figure 4 Acid content and OD of 1T ergothionein fermentation strain 600 picture. Detailed Implementation

[0032] Example 1 The chassis strain construction is detailed below: (1) Primer design and synthesis With Escherichia coli E. coli W3110 Using the genome as a template, according to tnaAGene sequence homologous arm primers tnaA-UF / tnaA-UR and tnaA-DF / tnaA-DR were designed. PCR amplification was performed using these primers to obtain the tnaA-UP and tnaA-DOWN homologous arm fragments. Then, using the tnaA-UP and tnaA-DOWN homologous arm fragments as templates, PCR amplification was performed using tnaA-UR and tnaA-DF primers to obtain the tnaA-Del targeting fragment. Similarly, the following can be obtained... yciW The primers used for targeting the gene fragment yciW-Del are shown in Table 1.

[0033] Table 1 tnaA , yciW Primer sequence listing for upstream and downstream homologous arms of the gene according to metK Gene sequence design of upstream and downstream primers, with a Ptrc enhancer sequence (SEQ ID No: 12) added to the upstream primer, using metK-U-F1 / metK-UR as primers, and Escherichia coli. E. coli W3110 The metK-Ptrc fragment was obtained by PCR amplification using the genome as a template. Then, the knock-in fragment metK-Ptrc-Enter was obtained by amplification using metK-U-F2 / metK-UR primers and the metK-Ptrc fragment as a template. The primers used are shown in Table 2.

[0034] Table 2 metK Gene amplification primer sequence listing (2) Amplification of pEcsgRNA plasmid Using pEcsgRNA plasmid as a template, tnaA Taking the gene as an example, the SG-F1 (tnaA) and SG-R1 (tnaA) sequences were used as primers for whole-plasmid PCR amplification. The plasmid was then subjected to nucleic acid electrophoresis to confirm the correct fragment size. Dpn I was added for digestion, and the plasmid was transformed into DHα5. After plating and culturing, single colonies were selected and cultured on LB medium for 20 hours. The plasmid was then extracted, and its size was determined by nucleic acid electrophoresis. yciW , metK The gene repeats this process, which will not be repeated here. The primer sequences used in this process are shown in Table 3, and the PCR amplification systems used in all steps are shown in Table 4.

[0035] Table 3. Primer Sequence List for pEcsgRNA Plasmid Amplification Table 4 Record of 50ul PCR amplification system (3) Escherichia coli E. coli W3110 Preparation of competent cells E. coli was extracted from an ultra-low temperature freezer. E. coli W3110 One loopful of the bacterial strain was inoculated onto LB agar plates and incubated at 37°C for 16 hours. Single colonies were then selected and inoculated into 100 ml of LB medium (containing 20 mM MgSO4) and incubated at 37°C with a shaker at 180 rpm. After 3 hours of incubation, 1 ml of the bacterial suspension was taken every 20 minutes, and the OD was measured once using LB liquid medium as a control on a spectrophotometer. 600 Value, when OD 600 When the value reaches 0.4-0.5, remove from the ice bath for 30 min, centrifuge at 5000 rpm for 5 min at 4℃, discard the supernatant, add 40 ml of TFB1 pre-cooling solution and mix by pipetting, centrifuge at 5000 rpm for 5 min at 4℃, discard the supernatant, add 4 ml of TFB2 pre-cooling solution and mix by pipetting, then place in the ice bath for 15 min, then aliquot 100 μl into 1.5 ml centrifuge tubes and store in liquid nitrogen.

[0036] TFB1 solution preparation: 30mM potassium acetate, 10mM CaCl2, 50mM MnCl2, 100mM RbCl, 15% glycerol, 1M acetic acid to adjust pH to 5.8. Filter sterilize using a 0.22M filter and store at room temperature.

[0037] TFB2 solution preparation: 100 mM 3-morpholinopropanesulfonic acid (MOPS) or piperazine-N,N''-di(2-ethanesulfonic acid) (PIPES, pH 6.5), 75 mM CaCl2, 10 mM RbCl, 15% glycerol, and 1 M KOH to adjust the pH to 6.5. Filter sterilize using a 0.22 M filter and store at room temperature.

[0038] (4) Transform pEcCas plasmid into W3110 chemocompetent cells Take one W3110 chemocompetent cell and thaw it on ice for 5 min. Add 2 μL of pecCas plasmid to the W3110 cell, incubate on ice for 30 min, then heat shock for 1 min, incubate on ice for another 4 min, and add 1 ml of LB liquid medium. Incubate at 37°C in a shaker for 1 h, then spread 100 μL of the bacterial culture onto a kanamycin-resistant plate and incubate overnight.

[0039] (5) Preparation of W3110 / pEcCas electrocompetent cells Select a single colony of W3110 / pEcCas and inoculate it into 100 ml of LB medium (containing 60 mmol arabinose). Incubate at 37°C and 180 rpm in a constant temperature shaker for 5-6 hours. Every 20 minutes, take 1 mL of the bacterial culture and measure the OD value using a spectrophotometer, with LB liquid medium as a control. 600 Value, when OD 600 When the value reaches 0.8, remove from ice bath for 30 min, centrifuge at 4℃, 5000 rpm for 20 min, discard the supernatant, add 200 ml of pre-chilled Milli-Q ultrapure water and mix by pipetting, centrifuge at 4℃, 5000 rpm for 20 min, discard the supernatant, add 100 mL of pre-chilled Milli-Q ultrapure water containing 10% glycerol, gently shake to resuspend the cells, centrifuge at 4℃, 5000 rpm for 20 min, discard the supernatant, gently mix, then aliquot 100 μl into 1.5 ml centrifuge tubes and store in liquid nitrogen.

[0040] (6) Electroporation of pEcsgRNA plasmid and homologous fragment into W3110 / pEcCas electroporation competent cells Take one vial of W3110 / pecCas electroporation competent cells and thaw them on ice. Add homologous fragment: pecgRNA = 400 ng: 100 ng, mix gently, and incubate on ice for 5 min. Add the electroporated cells to an electroporation cuvette, gently shake to mix, and rapidly electroporate at 1.8 kV. Then quickly add LB medium (containing 60 mM arabinose) and incubate on a shaker for 2 h. First, plate 100 μL of the bacterial culture (K+ spe), then centrifuge, remove 900 μL of supernatant, and plate the remaining 100 μL again (K+ spe).

[0041] (7) Detecting gene knockout status Take a 1.5ml PE tube and add 0.5ml of K+Spe double antibody medium. Select 7 single colonies from each plate and place them into the PE tube. Incubate at 37℃ for approximately 4 hours. Prepare the relevant validation PCR amplification premix. After adding the bacterial culture, perform 30 cycles of amplification, annealing at 55℃ and extending for 60 seconds. Nucleic acid electrophoresis detection. tnaA , yciW Knockout status and metK Knock-in results. Selected knockout and knock-in strains were streaked onto K+Spe double antibiotic plates and incubated overnight.

[0042] (8) Elimination of pEcsgRNA plasmid Select suitable single colonies from the plate and inoculate them into 1.5 ml EP tubes. Add 1 ml of kanamycin + 100 mm rhamnose agar to each colony (or plate them onto kanamycin + 100 mm rhamnose agar, incubate overnight, and then transfer to liquid medium). Incubate at 37°C on a shaker for 1 hour, then remove and place in a 37°C incubator for 3 hours, and then incubate again at 37°C on a shaker for 1 hour. Prepare the relevant validation PCR amplification premix, perform PCR amplification, and detect the nucleic acid by electrophoresis. No bands are generated, indicating successful elimination.

[0043] (9) pEcCas plasmid elimination The eliminated strains were streaked onto 50 mm glucose + 50 mm sucrose agar plates and incubated overnight at 37°C. Twenty suitable single colonies were selected and inoculated into 1.5 ml EP tubes, each containing 0.5 ml of 50 mm glucose + 50 mm sucrose LB liquid medium, and incubated on a shaker for 4 hours. A premixed PCR amplification solution was prepared for validation, and PCR amplification and nucleic acid electrophoresis were performed. No band formation indicated successful elimination, thus successfully obtaining the chassis cells. E. coli W3110ΔtnaAΔyciW::Ptrc- metK .

[0044] Example 2 The mutant was validated by shake-flask fermentation, as detailed below: (1) Mutant ergothionein synthase and mutant cysteine ​​sulfoxide lyase Fusarium from non-alfalfa variants ( Verticillium nonalfalfae) Ergothioneine synthase EGT1 and Alternaria alternata ( Alternaria arborescens) Site-directed mutagenesis was performed on the cysteine ​​sulfoxide lyase EGT2 to obtain the following mutants: EGT1-1: V117G, E141T, G188P, amino acid sequence as shown in SEQ ID No: 5.

[0045] EGT1-2: V117G, F153W, E237D, amino acid sequence as shown in SEQ ID No: 6.

[0046] EGT1-3:D512E, V657L, K724R, amino acid sequence as shown in SEQ ID No: 7.

[0047] EGT1-4:D512E, P655G, I750M, amino acid sequence as shown in SEQ ID No: 8.

[0048] EGT2-1: A183L, N258C, T287S, amino acid sequence as shown in SEQ ID No: 9.

[0049] EGT2-2:S95C,K169R,P246G, amino acid sequence as shown in SEQ ID No: 10.

[0050] EGT2-3: I73A, A293L, H409K, amino acid sequence as shown in SEQ ID No: 11.

[0051] Based on the above amino acid sequence, the nucleotide sequence was optimized according to the codon preference of *E. coli*, and the two gene sequences were combined and constructed into the pETDuet-1 vector. The whole genome was then synthesized by Beijing Qingke Biotechnology. Figure 1 The plasmid maps of mutant ergothioneine synthase (D512E, P655G, I750M) and mutant cysteine ​​sulfoxide lyase (S95C, K169R, P246G) are shown, and the specific gene combinations on the vector are shown in Table 5.

[0052] Table 5. Record of the construction of mutant ergothionein synthase and mutant cysteine ​​sulfoxide lyase combination (2) Preparation E. coli W3110ΔtnaAΔyciW::Ptrc-metK competent cells E. coli E. coli W3110ΔtnaAΔyciW::Ptrc-metK Activate on LB solid agar plates, pick a single colony and inoculate into 50 mL of LB liquid agar, incubate at 30°C and 200 rpm until 0D. 600 After heating to 0.6 and incubating on ice for 30 min, centrifuge at 5000 rpm for 15 min at 4°C to collect bacterial cells. Wash the cells three times with pre-cooled 15% glycerol solution, resuspend the cells in 500 μL of 15% glycerol solution, and aliquot into sterile 1.5 mL EP tubes (80 μL per tube) to obtain *Escherichia coli*. E. coli W3110ΔtnaAΔyciW::Ptrc-metK Electrocompetent cells were stored at -80°C.

[0053] (3) Electrotransfer pETDuet-1 plasmid to E. coli W3110ΔtnaAΔyciW::Ptrc-metK competent cells Take 12 pieces E. coli W3110ΔtnaAΔyciW::Ptrc-metK Thaw the electrotransfer competent cells on ice, add 5 μL of the 12 plasmids from step 1, mix gently, and incubate on ice for 5 min. Add the electrotransfer cells to an electroporation cuvette, gently shake to mix, and rapidly electroporate at 1.8 kV. Then quickly add LB medium and incubate on a shaker for 1 h. First, take 100 μL of the bacterial culture for plating (ampicillin-resistant), then centrifuge, remove 900 μL of supernatant, and platch the remaining 100 μL (ampicillin-resistant).

[0054] (4) Screening of positive clones Single colonies were selected from 12 ampicillin-resistant plates, with 10 colonies selected from each plate. PCR amplification was performed using the T7 sequence, a commonly used primer for pET series vectors. Positive clones were screened, and positive clones were selected from the 10 strains selected from each plate for preservation. Each bacterial strain was preserved at -80℃ with 20% glycerol concentration.

[0055] (5) Verification by shake-flask fermentation Twelve glycerol bacteria were streaked onto LB ampicillin plates and incubated at 37°C for 16 hours. Three suitable single colonies from each plate were then inoculated into 50 ml of LB medium and cultured at 37°C with a shaker at 220 rpm until OD reached. 600 When the concentration reached approximately 0.8, 10 μL of IPTG and 5 mL of feed solution were added. The feed solution was prepared with 10 g / L L-histidine, 20 g / L L-methionine, and 10 g / L L-cysteine ​​hydrochloride. The ergothioneine content was determined by high-performance liquid chromatography (HPLC) every 6 hours, and the specific contents are shown in Table 6.

[0056] The high-performance liquid chromatography (HPLC) method for the determination of ergothionein is as follows: mobile phase: 30% acetonitrile; flow rate: 0.8 ml / min; detection wavelength: 254 nm; column temperature: 30℃; injection volume: 10 μL; chromatographic column: C18 column; dimensions: 150 mm * 4.6 mm * 5 μm; method: standard curve method, and the sample determination curve is shown below. Figure 2 As shown.

[0057] Table 6. Ergothioneine content determination in shake-flask fermentation (mg / L) The ergothioneine content of the final product from shake-flask fermentation showed that when the gene mutation combination was EGT1-4 / EGT2-2, the highest concentration reached 627.31 mg / L after 36 hours of induced fermentation, indicating good expression in shake-flasks. A portion of the bacterial culture was centrifuged, and the bacterial cells were subjected to ultrasonic disruption. Protein electrophoresis was used to detect the expression of the two enzymes. (Details are as follows...) Figure 3 As shown, ergothioneine synthase was expressed well, while cysteine ​​sulfoxide lyase was expressed only moderately due to its location in the second expression box. A portion of the bacteria was inoculated into LB liquid medium and cultured at 37°C for about 10 hours. The seed culture was then preserved with 20% glycerol and stored at -80°C.

[0058] Example 3 Fermentation verification of the 1T production line is as follows: (1) Activation of microbial strains Prepare LB agar plates with the following ingredients: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 20 g / L agar powder. After pouring the plates, allow them to cool and solidify. Take one EGT1-4 / EGT2-2 mutant strain from an ultra-low temperature freezer and streak or spread it on a clean area of ​​the plate. Invert the plate and place it in a 37°C incubator for 14-16 hours.

[0059] Prepare 800ml of LB liquid culture, and put 200ml into 1L Erlenmeyer flasks. Sterilize at 121℃ for 30min. After cooling, select relatively uniform single colonies from the plate and inoculate them into the Erlenmeyer flasks. Incubate at 37℃ and 220rpm for about 16-20h. If OD600≥2 and no contaminants are found under a microscope, the primary seed culture is qualified.

[0060] (2) Culture in a 100L seed tank Prepare 50L of seed culture medium with the following ingredients: 11.8g / L bone peptone, 23.6g / L yeast powder, 4g / L glycerol, 2.2g / L potassium dihydrogen phosphate, 9.4g / L dipotassium hydrogen phosphate, and 0.6ml / L antifoaming agent. After high-temperature sterilization, cool and allow to reach the desired pH. Pour the primary seed solution from step (1) of the inoculum activation process into the seed tank at a 1% inoculation rate. Initial process control: 37℃, rotation speed 100rpm, ventilation rate 2.5L / min, tank pressure 0.05Mpa, dissolved oxygen ≥35%, culture period 4h-7h, OD... 600 ≥3, after microscopic examination shows no contaminants, the container can be transferred.

[0061] (3) Cultivation in a 1T fermenter Prepare a 300L fermentation medium with the following ingredients: glucose 10g / L, magnesium sulfate heptahydrate 1.2g / L, potassium dihydrogen phosphate 13.3g / L, diammonium hydrogen phosphate 8g / L, citric acid monohydrate 1.7g / L, yeast extract 10g / L, calcium chloride dihydrate 0.2g / L, ferrous sulfate heptahydrate 30g, manganese sulfate monohydrate 1.5g, zinc sulfate pentahydrate 6.75g, copper sulfate pentahydrate 3g, sodium tetraborate decahydrate 0.69g, ammonium heptamolybdate 0.3g, hydrochloric acid 546g, biotin 0.105g, microbial B3 10.41g, microbial B1 10.41g, microbial B6 1.05g, microbial B5 10.41g, and defoamer 150g. Dissolve the ingredients and sterilize. Dissolve the glucose and magnesium sulfate heptahydrate in 5L of pure water and then separate them for further sterilization. After sterilization, inject the solutions into the large container using a syringe. The inducing agent is 25 g / L IPTG and 300 g / L arabinose. Prepare 5 L of the solution, dissolve it in pure water, and then filter it using a 0.2 μm filter.

[0062] 50L of seed culture was inoculated into a 1T fermenter for cultivation. The pH was adjusted to 6.8-7.0, the temperature was 37℃, the rotation speed was 100rpm, the aeration rate was 8m³ / h, and the dissolved oxygen was controlled at 20%-40%. When the dissolved oxygen rebounded after about 5 hours of fermentation, fed-batch feeding was started at a rate of 30ml / (L·h). In the early stage, the residual sugar was controlled at 0.5g / L-1g / L to accelerate growth. After induction, the residual sugar was controlled to be as close to 0 as possible. When the OD... 600 Between 25 and 30 degrees Celsius, the temperature was lowered to 36 degrees Celsius, and an inducing agent was added. The temperature was then lowered by 1 degree Celsius per hour until it reached 30 degrees Celsius. After induction, 10 L of feed 2 was added at once, followed by continuous feeding of feed 2 at a flow rate of 40 ml / (L·h). Samples were taken every 5 hours, and the contents of ergothioneine, L-histidine, L-methionine, and L-cysteine ​​hydrochloride were determined by high performance liquid chromatography. L-cysteine ​​hydrochloride was not detectable, L-methionine was ≤1 g / L, and L-histidine was ≤0.1 g / L.

[0063] If, after 40 hours of fermentation, OD values ​​are observed at 2-3 consecutive monitoring points... 600 If the growth rate stops or decreases, start adding feed 3 every 2 hours, for a total of 12L. If no growth occurs again after 60 hours, add another batch. When acid production stops or decreases after 75-80 hours of induction, stop adding feed 2 for 2-3 hours, stop adding glucose, wait for dissolved oxygen to rebound, and then remove the product from the tank. Denature at 80℃ for 0.5 hours, then cool to 60℃ before removing from the tank. The overall ergot thiocyanate acid production is as follows... Figure 4 As shown.

[0064] The total volume of feed 1 is 400L, feed 2 is 200L, and feed 3 is 12L. Feed 1 consists of: 800g / L glucose, 20g / L magnesium sulfate heptahydrate, and 3g / L defoamer. Feed 2 consists of: 20g / L L-histidine, 40g / L L-methionine, 15.5g / L L-cysteine ​​hydrochloride, and 0.3g / L pyridoxal phosphate. The supplementary ingredients are as follows: 1200g yeast extract, 2.4g calcium chloride dihydrate, 12g ferrous sulfate heptahydrate, 0.6g manganese sulfate monohydrate, 2.7g zinc sulfate heptahydrate, 1.2g copper sulfate pentahydrate, 0.276g sodium tetraborate decahydrate, 0.12g ammonium heptamolybdate, 218g hydrochloric acid, 0.042g biotin, 4.164g microbial B3, 4.164g microbial B1, 0.42g microbial B6, and 4.164g microbial B5.

[0065] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and are not intended to be unique or to limit the present invention. Those skilled in the art should understand that various changes or equivalent substitutions made to the present invention without departing from its scope are all within the scope of protection of the present invention.

Claims

1. A mutant strain that synthesizes ergothioneine, characterized in that, It includes mutant ergothioneine synthase and mutant cysteine ​​sulfoxide lyase. The amino acid sequence of mutant ergothioneine synthase is shown in SEQ ID No: 8, and the amino acid sequence of mutant cysteine ​​sulfoxide lyase is shown in SEQ ID No:

10.

2. A dual expression vector, characterized in that, It includes the gene encoding the mutant ergothionein synthase of claim 1 and the gene encoding the mutant cysteine ​​sulfoxide lyase of claim 1.

3. An engineered bacterium, characterized in that, Obtained via the following method: using Escherichia coli E. coli W3110 The initial strain was knocked out. tnaA Genes and yciW Genes, in methionine adenosine transferase metK Adding an enhancer to the gene terminus of the sequence shown in SEQ ID No: 12 enhances expression. metK Genes were extracted to obtain a chassis strain; the dual expression vector of claim 2 was then transferred into the chassis strain to obtain an engineered strain.

4. The application of the mutant of synthetic ergothioneine as described in claim 1, characterized in that, Used in the synthesis of ergothioneine.

5. A method for synthesizing ergothioneine, characterized in that, Includes the following steps: The seed culture containing the engineered bacteria described in claim 3 was inoculated into the fermenter. Initial culture conditions were: pH adjusted to 6.8-7.0, temperature 37℃, rotation speed 100 rpm, aeration rate 8 m³ / h, dissolved oxygen controlled at 20%-40%. After dissolved oxygen rebound, feedstock was started at a feed rate of 30 ml / (L·h). When OD... 600 At 25-30°C, add the inducing agent all at once and slowly cool down to 30°C. Start the second feeding process at a feeding rate of 40 ml / (L·h). After culturing for 80 hours, remove the container from the tank. The formula for supplementary feed 1 is: glucose 800g / L, magnesium sulfate heptahydrate 20g / L, and defoamer 3g / L; The formula for supplementary feed 2 is: L-histidine 20g / L, L-methionine 40g / L, L-cysteine ​​hydrochloride 15.5g / L, and pyridoxal phosphate 0.3g / L; The inducer formula is: IPTG 25g / L, L-arabinose 300g / L.

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