An engineered bacterium producing ergothioneine and its construction method and application

Abstract

The invention discloses an engineering bacterium producing ergothioneine and a construction method and application thereof. The engineering bacterium construction method is to use Escherichia coli as a starting strain, and to introduce a constitutive promoter PrrnD and a new gold Mycobacterium Mycobacterium neoaurum Ergothioneine biosynthesis gene cluster egtABCDE Integrate into the genome of the starting strain; further convert the ATP of the resulting strain into phosphoribosyl kinase HisG The 271st glutamic acid of gene is mutated into lysine, and the engineering bacteria of producing thioneine is obtained. With glucose as main raw material, the engineering bacteria of the present invention are utilized to carry out fermentation culture, and thioneine can be produced. The engineering bacteria fermentation synthesis of thioneine of the present invention has the advantages of simple process and high production efficiency, can avoid the use of a large amount of organic solvents and antibiotics, and is suitable for industrialized production.

Description

Technical Field

[0001] The invention belongs to the field of synthetic biology, and specifically relates to an engineering bacterium producing ergothioneine, a construction method and application thereof. Background Art

[0002] Ergothioneine is a thiol compound with the chemical name 2-thioimidazole amino acid. Its left-handed structure has good physiological activity. Ergothioneine has two isomeric forms, thiol and thiol, and exists mainly in the form of thiol in aqueous solution. Ergothioneine was originally isolated from the ergot fungus of rye in the Poaceae family. Microorganisms such as mycobacteria, methylbacteria, cyanobacteria and mushrooms can naturally synthesize ergothioneine. Ergothioneine has been shown to exist in almost all human cells and tissues, usually at millimolar levels in the brain, bone marrow, lens and cornea of ​​the eye, and red blood cells, but the human body cannot synthesize ergothioneine by itself and can usually only be ingested and supplemented from food.

[0003] Ergothioneine plays an important role in human cells and tissues, which is mainly reflected in the following aspects: ① Ergothioneine has excellent antioxidant properties and stability, and can remove cellular reactive oxygen, resist oxidative damage and maintain redox balance; ② Ergothioneine has the potential to prevent and treat diseases such as cancer, cardiovascular disease, cognitive dysfunction caused by oxidative stress; ③ Ergothioneine also has the effect of improving sleep disorders, improving intestinal health, and resisting UV damage and photoaging.

[0004] Current thioneine production methods mainly include mushroom extraction, chemical synthesis and biosynthesis. As CN118271245 A discloses a method for extracting thioneine from Pleurotus citrinum, first Pleurotus citrinum is subjected to liquid culture, mycelium is collected, and after physical ultrasonic shearing treatment, an active composition containing thioneine is obtained by water extraction, but in addition to thioneine, the composition also contains protein, polypeptide, polysaccharide, etc.; CN 118271245 A discloses a chemical synthesis method for preparing thioneine using dimethylhistidine as raw material, and the obtained product has high chiral purity, but organic solvents such as toluene, chloroform, carbon tetrabromide, tetrahydrofuran, acetonitrile, etc. need to be used in the synthesis process; CN 117802015 A discloses a method for producing thioneine by fermentation of genetically engineered bacteria, and genetically engineered bacteria are cultured in a culture medium for a period of time, and thioneine is produced by inducing IPTG, and the batch fed-batch fermentation yield reaches 4063.15mg / L.

[0005] With the deepening of efficacy research, thioneine has been widely used in the fields of cosmetics, functional foods, and market demand continues to emerge. The synthesis of thioneine by fermentation of engineered bacteria has the advantages of simple process and high production efficiency, and can avoid the use of a large amount of organic solvents, which is expected to become the mainstream method for producing thioneine. However, the thioneine output level of the current process is low, which poses certain challenges to industrial production. Therefore, it is of great significance to improve the ability of engineered bacteria to synthesize thioneine and make it suitable for industrial production. Summary of the invention

[0006] In order to solve the problems in the prior art, the present invention provides an engineering bacterium for producing ergothioneine and a construction method and application thereof.

[0007] The technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for constructing an engineering bacterium that produces ergothioneine, comprising:

[0009] 1) Using Escherichia coli as the starting strain, the constitutive promoter PrrnD and the ergothioneine biosynthesis gene cluster egtABCDE from Mycolicibacterium neoaurum were integrated into the genome of the starting strain;

[0010] 2) Mutating the 271st glutamic acid of the ATP phosphoribosylkinase HisG gene of the strain obtained in step 1) into lysine to obtain the engineered bacteria producing ergothioneine.

[0011] The step 1) is: performing Escherichia coli codon optimization on the ergothioneine synthesis gene cluster MnegtABCDE from Mycolicibacterium neoaurum, adding a constitutive promoter PrrnD in front of the optimized sequence to obtain a MnegtABCDE gene cluster fragment with the constitutive promoter PrrnD, and integrating the fragment into the motA site of the Escherichia coli BL21 (DE3) genome.

[0012] According to a preferred embodiment of the present invention, the codon-optimized MnegtABCDE gene sequence of Escherichia coli is shown as SEQ ID NO.1; the constitutive promoter PrrnD sequence is shown as SEQ ID NO.2.

[0013] In a second aspect, the present invention provides an engineered bacterium producing ergothioneine constructed by the method.

[0014] In a third aspect, the present invention provides a method for the biosynthesis of ergothioneine, which uses glucose as a main raw material and utilizes the engineered bacteria for fermentation culture to produce ergothioneine.

[0015] Compared with the prior art, the present invention uses Escherichia coli BL21 (DE3) as a host, and integrates the constitutive promoter PrrnD and the ergothioneine synthesis gene cluster egtABCDE (codon optimized by Escherichia coli) from Mycolicibacterium neoaurum into the motA site of the genome through gene editing technology, so that the host cell has the ability to synthesize ergothioneine using glucose. The constitutive promoter PrrnD used in the present invention is the promoter of Escherichia coli ribosomal RNA, which has high expression intensity and is conducive to the heterologous expression of the ergothioneine synthesis gene cluster. The gene cluster integration site motA selected by the present invention is the flagella gene (non-essential gene) of Escherichia coli, and the destruction of the gene will not affect the growth metabolism of the strain, and also reduces the consumption of non-essential energy. Further, the 271st glutamic acid of the ATP phosphoribosyltransferase HisG of the host cell is mutated to lysine through target gene point mutation, the feedback inhibition of L-histidine is released, the accumulation of the precursor substance L-histidine is increased, and the fermentation yield of ergothioneine is greatly increased.

[0016] The engineered bacteria for producing ergothioneine proposed by the present invention has good genetic stability, and no antibiotics and inducers need to be added during the fermentation process, thereby reducing material costs and better ensuring the application safety of the finished product in cosmetics and food. The invention has significant advantages over the disclosed ergothioneine engineered bacteria.

[0017] The ergothioneine finished product prepared by fermentation of the ergothioneine-producing engineering bacteria provided by the present invention has excellent properties, a content of more than 99%, and has the prospect of large-scale industrial production and practical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the synthesis pathway of ergothioneine in engineered bacteria.

[0019] Figure 2 Schematic diagram for modifying HisG to relieve feedback inhibition by L-histidine.

[0020] Figure 3 Be the ergothioneine finished product obtained by embodiment. DETAILED DESCRIPTION

[0021] The overall construction idea of ​​the engineering bacteria producing ergothioneine of the present invention is: the constitutive promoter PrrnD and the ergothioneine synthesis gene cluster MnegtABCDE (codon-optimized by Escherichia coli) from Mycolicibacterium neoaurum are integrated into the motA site of the genome of the starting strain Escherichia coli BL21 (DE3), and the ergothioneine synthesis pathway (such as Figure 1As shown in the figure), the recombinant strain BL21 (DE3) ΔmotA::MnegtABCDE was obtained and named as the engineered strain EGT01. Furthermore, using EGT01 as the host cell, the 271st glutamic acid of ATP phosphoribosyltransferase hisG was mutated to lysine by target gene point mutation technology to relieve the feedback inhibition of L-histidine (as shown in the figure). Figure 2 As shown), the recombinant strain EGT01 hisG E271K , named as engineered bacteria EGT02.

[0022] The present invention is further described and illustrated below in conjunction with specific embodiments. The embodiments are merely exemplary of the present disclosure and do not limit the scope.

[0023] Several common reaction systems and reaction conditions are involved in each embodiment, and the systems and conditions are described as follows:

[0024] PCR reaction system (DNA fragment, whole plasmid amplification): 5× PrimeSTAR buffer 10 μL, dNTP 4 μL, upstream primer and downstream primer 1 μL each, DNA template (genomic or plasmid DNA containing the target gene) 0.5 μL, HS PrimeStar DNA polymerase 0.5 μL, and fill with ddH2O to a total volume of 50 μL.

[0025] PCR reaction conditions (DNA fragment, whole plasmid amplification): 95℃ pre-denaturation for 5min; 95℃ denaturation for 30sec; 55℃ annealing for 30sec; 72℃ extension (extension time calculated as 1kb / min), 32 cycles; 72℃ extension for 10min; 4℃ storage.

[0026] PCR reaction system (colony PCR verification): 2× Rapid Taq Master Mix 10μL, upstream primer and downstream primer 0.4μL each, ddH2O 9.2μL, appropriate amount of DNA template, total volume 20μL.

[0027] PCR reaction conditions (colony PCR verification): pre-denaturation at 95°C for 10 min; denaturation at 95°C for 15 sec; annealing at 58°C for 15 sec; extension at 72°C (extension time calculated at 4 kb / min), 28 cycles; extension at 72°C for 2 min; storage at 4°C.

[0028] Recombination reaction system: 2×GenRec Assembly Master Mix 10 μL, linearized vector 0.03 pmol, target gene fragment 0.09 pmol, ddH2O filled to a total volume of 20 μL.

[0029] Recombination reaction conditions: Place the prepared reaction solution in a 50°C water bath for 50 min.

[0030] Example 1 Construction of engineered bacteria EGT01

[0031] The ergothioneine synthesis gene cluster egtABCDE (GenBank accession number: NZ_CP074376.1, location = 1102768..1104015, 1104012..1105292, 1105285..1105971, 1105968..1106906, 1106903..1107985) from Mycolicibacterium neoaurum was codon optimized in Escherichia coli, and the optimized sequence is shown in SEQ ID NO.1; a constitutive promoter PrrnD was added in front of the sequence (the PrrnD sequence is shown in SEQ ID NO.2), synthesized by a chemical method, and the synthesized gene was used as a template. The primers egt-F and egt-R in Table 1 were used for PCR amplification to obtain the MnegtABCDE gene cluster fragment with the constitutive promoter PrrnD.

[0032] Table 1

[0033]

[0034] Using the E. coli BL21 (DE3) genome as a template, the primers motA-UF / motA-UR and motA-DF / motA-DR were designed according to the sequence of the motA gene (GenBank accession number: NZ_CP053602.1, location=1923468..1924355), and PCR amplification was performed to obtain the gene fragments motA-U and motA-D. Then, using the gene fragments motA-U, motA-D, and MnegtABCDE as templates, and motA-UF and motA-DR as primers, fusion PCR amplification was performed to obtain the MnegtABCDE gene cluster expression cassette. The primer sequences used in this process are shown in Table 2.

[0035] Table 2

[0036]

[0037] The pTarget plasmid was used as a template, and the whole plasmid was amplified by PCR using sgmotA-F (motA site sgRNA primer) and pTarget-R as primers. The obtained PCR product was digested, purified and ligated, and transformed into Escherichia coli JM109 competent cells by chemical transformation. The plates were coated with LB solid medium containing 30 mg / L spectinomycin and cultured at 37°C to obtain single colonies. Colony PCR verification was performed using primers testsg-motA-F and pTarget-R to verify that the correct strain contained the pTargetF-sgmotA plasmid targeting the motA gene. The primer sequences used in this process are shown in Table 3.

[0038] Table 3

[0039]

[0040] Escherichia coli BL21 (DE3) was activated on LB solid medium plates and inoculated into LB liquid medium for overnight culture. The seed solution was transferred to 25 mL LB liquid medium at 1% and cultured at 30°C and 200 rpm until OD 600 =0.5, ice bath for 30 min; 4°C, 4000 rpm centrifugation for 10 min to collect the bacteria, wash the bacteria three times with pre-cooled 10% glycerol solution; add 300 μL 10% glycerol solution to resuspend the bacteria, and distribute them into sterile 1.5 mL EP tubes, 80 μL / tube, to prepare Escherichia coli BL21 (DE3) electroporation competent cells.

[0041] The plasmid pCas carrying the transporter gene was electrotransformed into the electrocompetent cells of Escherichia coli BL21 (DE3), coated on LB solid medium plates containing 30 mg / L kanamycin, and cultured at 37°C to obtain a single colony BL21 (DE3) / pCas. The electrocompetent cells of Escherichia coli BL21 (DE3) / pCas were prepared, and the MnegtABCDE gene cluster expression cassette and pTargetF-sgmotA plasmid were simultaneously electrotransformed into the competent cells, coated on LB solid medium plates containing 30 mg / L kanamycin and spectinomycin, and cultured at 30°C to obtain a single colony. The primers motA-UF and motA-DR in Table 2 were used for colony PCR verification and sequencing verification, and the correct strain was verified to be Escherichia coli BL21 (DE3) ΔmotA::MnegtABCDE / pCas / pTargetF-sgmotA.

[0042] E. coli BL21 (DE3) ΔmotA::MnegtABCDE / pCas / pTargetF-sgmotA was inoculated into LB liquid medium containing kanamycin, IPTG with a final concentration of 0.5 mM was added, and cultured at 30°C and 200 rpm for 2 h, and then coated with LB solid medium plates containing 30 mg / L kanamycin and cultured at 30°C. Single colonies without spectinomycin resistance were screened to obtain E. coli BL21 (DE3) ΔmotA::MnegtABCDE / pCas without the pTargetF-sgmotA plasmid.

[0043] Escherichia coli BL21 (DE3) ΔmotA::MnegtABCDE / pCas was inoculated into non-antibiotic LB liquid medium, cultured at 42°C and 200 rpm for 2 h, and then coated onto non-antibiotic LB solid medium plates and cultured at 42°C. Single colonies without kanamycin resistance were screened to obtain Escherichia coli BL21 (DE3) ΔmotA::MnegtABCDE with the pCas plasmid removed, which was named engineered bacteria EGT01.

[0044] Example 2 Construction of engineered bacteria EGT02

[0045] Using the E. coli BL21 (DE3) genome as a template, primers hisG-F / hisG-R were designed according to the sequence of hisG gene (GenBank accession number: NZ_CP053602.1, location=1987293..1988192), and PCR amplification was performed to obtain the gene fragment hisG. Using the vector pET-24a(+) as a template, primers pET-F and pET-R were used to perform PCR amplification to obtain the linearized vector pET-24a(+). The primer sequences used in this process are shown in Table 4.

[0046] Table 4

[0047]

[0048] The hisG gene was connected to the linearized vector pET-24a(+) by recombination reaction and sequenced to obtain the recombinant vector pET-24a(+)-hisG. The primers hisGE271K-F and hisGE271K-R in Table 5 were used for whole plasmid PCR to obtain the recombinant vector pET-24a(+)-hisG E271K After digesting the template plasmid, transform E. coli and extract the plasmid for sequencing verification. E271K As a template, PCR amplification was performed using primers hisG-F / hisG-R in Table 4 to obtain the gene fragment hisG E271K .

[0049] Table 5

[0050]

[0051] Using the pTarget plasmid as a template, sghisG-F (sgRNA primer for hisG site) and pTarget-R in Table 3 as primers, the whole plasmid was amplified by PCR. The resulting PCR product was digested, purified and ligated, and transformed into Escherichia coli JM109 competent cells by chemical transformation. The cells were coated with LB solid medium plates containing 30 mg / L spectinomycin and cultured at 37°C to obtain single colonies. Colony PCR verification was performed using primers testsg-hisG-F and pTarget-R in Table 3 to verify that the correct strain contained the pTargetF-sghisG plasmid targeting the hisG gene. The primer sequences used in this process are shown in Table 6.

[0052] Table 6

[0053]

[0054] The engineered bacteria EGT01 / pCas electroporation competent cells were prepared according to the method in Example 1, and hisG E271K The gene fragment and pTargetF-sghisG plasmid were electroporated into competent cells at the same time, spread on LB solid medium plates containing 30 mg / L kanamycin and spectinomycin, and cultured at 30°C to obtain single colonies. Primers hisG-F and hisG-R were used for colony PCR verification and sequencing verification, and the correct strain was E. coli EGT01-hisG E271K / pCas / pTargetF-sghisG.

[0055] E. coli EGT01-hisG E271K / pCas / pTargetF-sghisG was inoculated with LB liquid medium containing kanamycin, and IPTG with a final concentration of 0.5 mM was added. The culture was shaken at 30°C and 200 rpm for 2 h, and then coated with LB solid medium plates containing 30 mg / L kanamycin and cultured at 30°C. Single colonies without spectinomycin resistance were screened to obtain E. coli EGT01-hisG without pTargetF-sghisG plasmid. E271K / pCas.

[0056] E. coli EGT01-hisG E271K / pCas was inoculated with non-antibiotic LB liquid medium, cultured at 42°C and 200 rpm for 2 h, and then coated with non-antibiotic LB solid medium plates and cultured at 42°C. Single colonies without kanamycin resistance were screened to obtain E. coli EGT01-hisG without pCas plasmid. E271K , named as engineered bacteria EGT02.

[0057] Examples 3-5 and Comparative Examples 1-3 are fermentation tests of related strains, wherein the culture medium formula involved is as follows:

[0058] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L.

[0059] LB solid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, agar 15 g / L.

[0060] Ergothioneine seed culture medium: glucose 20~25g / L, yeast extract 4~6g / L, peptone 2~4g / L, dipotassium hydrogen phosphate 6~8g / L, magnesium sulfate heptahydrate 1~2g / L, ferrous sulfate heptahydrate 40~60mg / L, manganese sulfate monohydrate 10~20mg / L, vitamin B1 2~5mg / L.

[0061] Ergothioneine fermentation medium: glucose 20~25g / L, yeast extract 1~5g / L, ammonium sulfate 5~7g / L, dipotassium hydrogen phosphate 8~10g / L, citric acid monohydrate 2~4g / L, magnesium sulfate heptahydrate 1~2g / L, ferrous sulfate heptahydrate 150~200mg / L, manganese sulfate monohydrate 10~20mg / L, vitamin B1 2~5mg / L, methionine 2~4g / L, trace elements 1~2mL / L; trace element composition is: sodium molybdate dihydrate 2.5g / L, nickel sulfate hexahydrate 2.5g / L, calcium chloride dihydrate 2g / L, copper sulfate pentahydrate 0.75g / L, aluminum sulfate 18hydrate 2.25g / L, cobalt chloride hexahydrate 2.5g / L, zinc chloride 0.5g / L, boric acid 3g / L.

[0062] Ergothioneine feed medium 1: 600~800g / L glucose, 8~10g / L yeast extract.

[0063] Ergothioneine-supplemented medium II: 10~15g / L methionine, 6~8g / L cysteine.

[0064] The materials used in preparing the culture medium were all commercially available analytical grade reagents.

[0065] The high performance liquid chromatography detection method of thioneine adopted in each embodiment of the present invention and comparative example is:

[0066] (1) Instrument: Shimadzu LC-2050 high performance liquid chromatograph;

[0067] (2) Chromatographic column: Thermo Fisher Scientific Hypersil ODS-2, 250×4.6 mm, 5 μm;

[0068] (3) Method parameters: The mobile phase was 5% (v / v) methanol in water, the flow rate was 1 mL / min, the UV detector wavelength was 257 nm, the column temperature was 35 °C, the injection volume was 5 μL, and the detection time was 15 min.

[0069] (4) Method for establishing a standard curve: Accurately weigh 0.0500g of thioneine standard sample (commercially available 99% thioneine), dissolve it in ultrapure water, dilute to 50mL, and prepare a 1g / L standard sample stock solution. Take 1mL, 2mL, 3mL, 4mL, and 5mL of the stock solution respectively, dilute to 10mL with ultrapure water, and prepare standard sample solutions with concentrations of 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, and 0.5g / L. Filter with a 0.22μm aqueous needle filter membrane, perform HPLC detection, and draw a standard curve with concentration as the horizontal axis and peak area as the vertical axis, and calculate the standard curve equation.

[0070] (5) Finished product content detection: Take an appropriate amount of ergothioneine reference substance, accurately weigh 0.01g (accurate to 0.00001g), place it in a 50mL volumetric flask, add ultrapure water to dissolve and dilute to the scale, shake well, filter with a 0.22μm filter membrane to prepare a reference solution. Take an appropriate amount of ergothioneine sample, accurately weigh 0.01g (accurate to 0.00001g), place it in a 50mL volumetric flask, add ultrapure water to dissolve and dilute to the scale, shake well, filter with a 0.22μm filter membrane to prepare a sample solution, and prepare two copies in parallel. Take the reference solution and the sample solution, inject them according to the chromatographic conditions respectively, record the chromatogram, and calculate the content of ergothioneine in the sample by the external standard one-point method.

[0071] The content of ergothioneine in the sample = (As×mr×Pr) / (Ar×ms)×100%

[0072] Where As is the peak area of ​​ergothioneine in the sample solution, Ar is the peak area of ​​ergothioneine in the reference solution, ms is the sample weight (g) of the ergothioneine sample, mr is the sample weight (g) of the ergothioneine reference, and Pr is the content of the ergothioneine reference. The arithmetic mean of the results of two parallel determinations is used as the reported value, and the absolute difference between the two parallel determination results is not greater than 2% of the arithmetic mean.

[0073] Example 3 Shake flask fermentation of engineered bacteria EGT02

[0074] The engineered bacteria EGT02 was streaked and activated on an LB solid medium plate, cultured at 37°C for 12 hours, inoculated with 10 mL of seed culture medium, cultured at 37°C and 220 rpm for 12 hours, inoculated with 50 mL of fermentation medium at a 5% inoculum, and cultured at 37°C and 220 rpm for 48 hours. The titer of ergothioneine in the fermentation broth was 0.74 g / L by high performance liquid chromatography (HPLC).

[0075] Comparative Example 1 Shake flask fermentation of engineered bacteria EGT01

[0076] The shake flask fermentation experiment was carried out using the engineered bacteria EGT01 according to the conditions of Example 3. The ergothioneine titer in the shake flask fermentation broth after fermentation for 48 hours was 0.19 g / L as determined by high performance liquid chromatography (HPLC).

[0077] Example 4 5L tank fermentation of engineered bacteria EGT02

[0078] The engineered bacteria EGT02 was streaked and activated on the LB solid culture medium plate, cultured at 37°C for 12 hours, inoculated with 10 mL of seed culture medium, and cultured at 37°C and 220 rpm for 12 hours to obtain the first-level seed solution. 100 mL of seed culture medium was inoculated at a 2% inoculation rate, and cultured at 37°C and 220 rpm for 5 hours to obtain the second-level seed solution. 2 L of fermentation medium was inoculated at an 8% inoculation rate, the culture temperature was 37°C, the initial speed was 250 rpm, the ventilation volume was 2 L / min (1 vvm), and the pH was controlled at 7.0 ± 0.05 with 25% ~ 28% ammonia water. After inoculation, the dissolved oxygen was controlled at 30% by the dissolved oxygen-stirring linkage. Samples were taken every 2 hours to detect the OD 600 and glucose. When the glucose concentration in the initial culture medium is lower than 1g / L, feed medium 1 is added to maintain the glucose concentration in the fermentation broth at 1-2g / L. When the OD value reaches 10, feed medium 2 is added at a rate of 20mL / h. The fermentation is completed after 48h, and the titer of ergothioneine in the fermentation broth is 6.22g / L by high performance liquid chromatography (HPLC).

[0079] Comparative Example 2 5L tank fermentation of engineered bacteria EGT01

[0080] A 5 L tank fermentation experiment was carried out using the engineered bacteria EGT01 according to the conditions of Example 4. The ergothioneine titer in the shake flask fermentation broth after 48 h of fermentation was 2.49 g / L as determined by high performance liquid chromatography (HPLC).

[0081] Example 5 50L tank fermentation of engineered bacteria EGT02

[0082] The engineered bacteria EGT02 was streaked and activated on the LB solid culture medium plate, cultured at 37°C for 12 hours, inoculated with 10 mL of seed culture medium, and cultured at 37°C and 220 rpm for 12 hours to obtain the first-level seed solution. 400 mL of seed culture medium was inoculated at 2% inoculation volume, cultured at 37°C and 220 rpm for 5 hours to obtain the second-level seed solution. 20 L of fermentation medium was inoculated at 8% inoculation volume, the culture temperature was 37°C, the initial speed was 200 rpm, the ventilation volume was 1.2 m³ / h (1 vvm), and the pH was controlled at 7.0±0.05 with 25%~28% ammonia water. After inoculation, the dissolved oxygen was controlled at 30% by the dissolved oxygen-stirring association. Samples were taken every 2 hours to detect the OD 600 When the glucose concentration in the initial culture medium is lower than 1g / L, feed medium 1 is added to maintain the glucose concentration in the fermentation broth at 1~2g / L. 600 When the value reached 10, feed medium 2 was added at a rate of 200 mL / h. The fermentation was continued until 48 h, and the titer of ergothioneine in the fermentation broth was 6.80 g / L by high performance liquid chromatography (HPLC).

[0083] Comparative Example 3 50L tank fermentation of engineered bacteria EGT01

[0084] A 50 L tank fermentation experiment was carried out using the engineered bacteria EGT01 according to the conditions of Example 5. The ergothioneine titer in the shake flask fermentation broth after 48 h of fermentation was 2.91 g / L as determined by high performance liquid chromatography (HPLC).

[0085] Example 6 Separation and purification of ergothioneine in fermentation broth

[0086] Embodiment 5 thioneine fermentation liquid is filtered through 50nm ceramic membrane, pressure control is 0.45~0.55MPa, temperature control is <40 ℃, adding pure water top wash to yield is 98.2%, removes the cells and insoluble solids in the fermentation liquid, and collects the clear liquid.The ceramic membrane clear liquid is ultrafiltered by 5000D ultrafiltration membrane, pressure control is 0.45~0.55MPa, temperature control is <40 ℃, adding pure water top wash to yield is 98.5%, removes the macromolecular protein and sugar in the feed liquid, and collects the clear liquid.The ultrafiltration membrane clear liquid is decolorized by LX-98 decolorizing resin, and top wash with purified water to yield is 99.3%, and decolorizing liquid and top wash liquid are collected.Decolorizing liquid and top wash liquid are vacuum concentrated, and temperature control is at 65 ℃, and it is 202g / L to be concentrated to thioneine content.5 times of volume of anhydrous ethanol are added to the concentrated solution, and crystallization is stirred at room temperature for 16h. The crystal solution was filtered, the wet crystals were washed 3 times with anhydrous ethanol, and the wet crystals were placed at 65 ° C and dried for 12 h to obtain the ergothioneine finished product (attached Figure 3 ). The content of ergothioneine in the finished product was 99.55% as determined by high performance liquid chromatography (HPLC).

[0087] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for constructing an engineering bacterium that produces ergothioneine, characterized in that, include: 1) Using Escherichia coli as the starting strain, the constitutive promoter PrrnD and Mycobacterium neogoldenum were introduced Mycolicibacterium neoaurum Ergothioneine biosynthesis gene cluster egtABCDE Integrated into the genome of the starting strain; The step 1) is: Mycobacterium neogoldensis Mycolicibacterium neoaurum Ergothioneine biosynthesis gene cluster MnegtABCDE The E. coli codon was optimized, and the constitutive promoter PrrnD was added in front of the optimized sequence to obtain a gene with the constitutive promoter PrrnD. MnegtABCDE The gene cluster fragment was integrated into the motA site of the Escherichia coli BL21 (DE3) genome; the GenBank accession number of the motA gene is: NZ_CP053602.1; motA The sgRNA primer of the site is shown in SEQ ID NO.9; E. coli codon optimized MnegtABCDE The gene sequence is shown in SEQ ID NO.1; the constitutive promoter PrrnD sequence is shown in SEQ ID NO.2; 2) mutating the 271st glutamic acid of the ATP phosphoribosyl kinase HisG gene of the strain obtained in step 1) to lysine to obtain the engineered bacteria producing ergothioneine; The step 2) includes: 2-1) Using the E. coli BL21 (DE3) genome as a template, PCR amplification was performed to obtain hisG gene; hisG The G en Bank accession number of the gene is: NZ_CP053602.1, and the primers used for PCR amplification are the primers shown in SEQ ID NO.12 hisG -F and the primer shown in SEQ ID NO.13 his -R; 2-2) Through recombination reaction his The gene was connected to the linearized vector pET-24a(+) to obtain the recombinant vector pET-24a(+)- hisG ; Use the primer shown in SEQ ID NO.16 his E271K-F and primers shown in SEQ ID NO.17 his E271K-R was subjected to full plasmid PCR to obtain the recombinant vector pET-24a(+)- his E271K ; 2-3) Using the recombinant vector pET-24a(+)- hisG E271K As template, PCR amplification was performed to obtain hisG E271K Gene fragments; 2-4) Using pTarget plasmid as template, construct a target his pTargetF-sg hisG Plasmids; 2-5) Electroporation of the plasmid pCas carrying the transporter gene into competent cells of the engineered bacteria EGT01 / pCas obtained from step 1. his E271K Gene fragment and pTargetF-sg his The plasmids were simultaneously electroporated into competent cells to obtain E. coli EGT01- his E271K / pCas / pTargetF-sg his ; 2-6) Escherichia coli EGT01- hisG E271K / pCas / pTargetF-sg his Screening and culture were performed to obtain the pTargetF-sg his Plasmids and pCas plasmids in E. coli EGT01- hisG E271K , which is the engineered bacteria that produces ergothioneine.

2. the engineering bacteria of the production of ergothioneine constructed by the method described in claim 1.

3. A biosynthetic method for ergothioneine, characterized in that, Glucose is used as the main raw material, and the engineered bacteria described in claim 2 are used for fermentation culture to produce ergothioneine.

4. The method according to claim 3, characterized in that include: Inoculating the activated engineered bacteria according to claim 2 into a seed culture medium for cultivation to obtain a seed solution; Inoculate the seed liquid into the fermentation medium and take samples regularly to detect OD 600 When the glucose concentration in the culture medium is lower than 1g / L, feed medium 1 is added to maintain the glucose concentration in the fermentation broth at 1~2g / L. 600 When the value reaches 10, feed medium 2 is started to be added at a flow rate of 200 mL / h; and fermentation is continued until the end of fermentation, wherein the feed medium 2 is composed of: 10-15 g / L methionine and 6-8 g / L cysteine.

5. The method according to claim 4, characterized in that The culture temperature in the fermentation medium was 37°C, the pH was controlled at 7.0±0.05 with 25%~28% ammonia water, and the dissolved oxygen was controlled at 30%; The seed culture medium is composed of: 20-25 g / L glucose, 4-6 g / L yeast extract, 2-4 g / L peptone, 6-8 g / L dipotassium hydrogen phosphate, 1-2 g / L magnesium sulfate heptahydrate, 40-60 mg / L ferrous sulfate heptahydrate, 10-20 mg / L manganese sulfate monohydrate, and 2-5 mg / L vitamin B1; The fermentation medium is composed of: 20-25 g / L glucose, 1-5 g / L yeast extract, 5-7 g / L ammonium sulfate, 8-10 g / L dipotassium hydrogen phosphate, 2-4 g / L citric acid monohydrate, 1-2 g / L magnesium sulfate heptahydrate, 150-200 mg / L ferrous sulfate heptahydrate, 10-20 mg / L manganese sulfate monohydrate, 2-5 mg / L vitamin B1, 2-4 g / L methionine, and 1-2 mL / L trace elements; wherein the trace elements include: 2.5 g / L sodium molybdate dihydrate, 2.5 g / L nickel sulfate hexahydrate, 2 g / L calcium chloride dihydrate, 0.75 g / L copper sulfate pentahydrate, 2.25 g / L aluminum sulfate 18hydrate, 2.5 g / L cobalt chloride hexahydrate, 0.5 g / L zinc chloride, and 3 g / L boric acid; The feed medium comprises: 600-800 g / L glucose and 8-10 g / L yeast extract.

6. The method according to claim 3, characterized in that The method further comprises a post-processing step after the fermentation is completed. The post-processing step comprises: filtering, decolorizing and concentrating the fermentation liquid obtained by the fermentation, adding 3 to 5 times the volume of anhydrous ethanol to the concentrated liquid, stirring and crystallizing at room temperature, suction filtering the crystallization liquid, washing the wet crystals with anhydrous ethanol and drying to constant weight to obtain ergothioneine.

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