CN116970662A - Method for preparing ergothioneine by using engineering bacteria - Google Patents
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- SSISHJJTAXXQAX-ZETCQYMHSA-N L-ergothioneine Chemical compound C[N+](C)(C)[C@H](C([O-])=O)CC1=CNC(=S)N1 SSISHJJTAXXQAX-ZETCQYMHSA-N 0.000 title claims abstract description 68
- 229940093497 ergothioneine Drugs 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000894006 Bacteria Species 0.000 title claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 56
- 230000004151 fermentation Effects 0.000 claims abstract description 56
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 42
- 239000008103 glucose Substances 0.000 claims abstract description 42
- 150000001413 amino acids Chemical class 0.000 claims abstract description 29
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 239000000411 inducer Substances 0.000 claims abstract description 14
- 239000000758 substrate Substances 0.000 claims abstract description 13
- 230000006698 induction Effects 0.000 claims abstract description 11
- 239000006052 feed supplement Substances 0.000 claims abstract description 8
- 235000001014 amino acid Nutrition 0.000 claims description 25
- 101150040602 ERG4 gene Proteins 0.000 claims description 20
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical group CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 16
- 239000002609 medium Substances 0.000 claims description 16
- FRHBOQMZUOWXQL-UHFFFAOYSA-L ammonium ferric citrate Chemical compound [NH4+].[Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O FRHBOQMZUOWXQL-UHFFFAOYSA-L 0.000 claims description 13
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 13
- 230000001276 controlling effect Effects 0.000 claims description 13
- 229960004642 ferric ammonium citrate Drugs 0.000 claims description 13
- 239000004313 iron ammonium citrate Substances 0.000 claims description 13
- 235000000011 iron ammonium citrate Nutrition 0.000 claims description 13
- 229910052760 oxygen Inorganic materials 0.000 claims description 13
- 239000001301 oxygen Substances 0.000 claims description 13
- 239000013612 plasmid Substances 0.000 claims description 13
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 12
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 11
- 239000007788 liquid Substances 0.000 claims description 11
- 229930182817 methionine Natural products 0.000 claims description 11
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims description 10
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 10
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 10
- 235000018417 cysteine Nutrition 0.000 claims description 10
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 7
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 7
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 7
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 229960005322 streptomycin Drugs 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 238000009423 ventilation Methods 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 241001587143 Methylobacterium pseudosasicola Species 0.000 claims description 3
- 241000187480 Mycobacterium smegmatis Species 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- 230000035939 shock Effects 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims description 2
- 101150003509 tag gene Proteins 0.000 claims description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 abstract description 21
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 238000003786 synthesis reaction Methods 0.000 abstract description 4
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 230000001133 acceleration Effects 0.000 description 3
- 230000003078 antioxidant effect Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 101150067489 egtE gene Proteins 0.000 description 3
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- 101150028955 MBP gene Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 150000003462 sulfoxides Chemical class 0.000 description 2
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 241000589323 Methylobacterium Species 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 206010051246 Photodermatosis Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000036995 brain health Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000036996 cardiovascular health Effects 0.000 description 1
- 235000017471 coenzyme Q10 Nutrition 0.000 description 1
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 101150003448 egtB gene Proteins 0.000 description 1
- 101150005220 egtD gene Proteins 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 108091035539 telomere Proteins 0.000 description 1
- 210000003411 telomere Anatomy 0.000 description 1
- 102000055501 telomere Human genes 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- C12N9/10—Transferases (2.)
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- C12Y201/01—Methyltransferases (2.1.1)
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Abstract
The invention discloses a method for preparing ergothioneine by using engineering bacteria, belonging to the technical field of microbial fermentation. Inoculating seed solution of the ergothioneine recombinant strain into a fermentation culture medium, and feeding glucose in the culture process; the inducer and the feed supplement are added to start induction, and simultaneously, the substrate amino acid is added in a fed-batch mode until the fermentation is finished. The shake flask result shows that the effect of glucose as a carbon source is obviously better than that of glycerol; after 10g/L of substrate amino acid is directly added into a 5L fermentation tank at one time, the synthesis rate of the primary ergothioneine is higher than that of the fed-batch mode, but the yield of the ergothioneine finally obtained in the fed-batch mode is 3.1g/L, and the yield obtained in the one-time direct addition mode is 2.5g/L; 150g of each of the three amino acids was fed into a 30L fermenter, and after 44 hours of fermentation, the yield of ergothioneine was 3.2g/L, while 300g of each of the three amino acids was fed into the fermenter, and the yield of ergothioneine was 5.5g/L.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing ergothioneine by using engineering bacteria.
Background
Ergothioneine (Ergothioneine) is a naturally occurring amino acid derivative with a variety of biological functions. The ergothioneine has strong antioxidant activity, and clinical data show that the ability of the ergothioneine to remove free radicals is 14 times that of glutathione and 30 times that of coenzyme Q. Due to its specific antioxidant capacity, ergothioneine has been known as a highly latent anti-aging component of the skin kingdom, including anti-photoaging, anti-oxidative damage, anti-glycation, prevention of mitochondrial and telomere damage, and the like. Ergothioneine also protects brain health and improves cognitive function, and also has a role in eye protection, improving cardiovascular health and muscle repair.
The preparation method of the ergothioneine mainly comprises fungus mushroom extraction, chemical synthesis and synthetic biology, wherein the biosynthesis is an emerging industrialized preparation method of the ergothioneine, and has the advantages of low cost, less pollution, simple process and the like. The strain commonly used for biosynthesis of ergothioneine is Escherichia coli at present, and the strain has the advantages of easy gene editing, growth cycle end and the like because of the Escherichia coli, and the yield of ergothioneine reaches 4.34g/L after fermentation culture for 143h in a 2L tank in the paper of [ Chen Z, he Y, wu X, et al Toward more efficient ergothioneine production using the fungal ergothioneine biosynthetic pathway [ J ]. Microbial Cell Factories, 2022, 21 (1): 1-8) ]; [ Zhang L, tang J, feng M, et al Engineering Methyltransferase and Sulfoxide Synthase for High-Yield Production of Ergothioneine [ J ]. Journal of Agricultural and Food Chemistry, 2022, 71 (1): 671-679] in the 5L tank, the ergothioneine yield reached 5.4g/L after 94h of fermentation culture; in Chinese patent CN114854659A, the yield of ergothioneine reaches 7g/L after fermentation culture for 77 hours in a 2L tank; in Chinese patent CN116121161A, the yield of ergothioneine reaches 7.2g/L after fermentation culture for 60 hours in a 2L tank. The former two require relatively high ergothioneine yields through a long fermentation period, and the latter two require polygene editing of chassis strains, which is relatively complex and long in operation period.
The biosynthetic pathways of ergothioneine in different species vary, with two gene pathways of eukaryotes, three gene pathways of prokaryotes and five gene pathways, but in [ Kamide T, takuseawa S, tanaka N, et al High Production of Ergothioneine in Escherichia coli using the Sulfoxide Synthase from Methylobacterium strains [ J ]. Journal of agricultural and food chemistry, 2020, 68 (23): 6390-6394 ] articles, none of the three gene pathways of prokaryotes selected successfully achieved fed-batch culture in a fermenter.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a method for preparing ergothioneine by using engineering bacteria, which is used for promoting escherichia coli to improve the yield of ergothioneine.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for preparing ergothioneine by using engineering bacteria comprises inoculating seed solution of fermentation strain into fermentation medium, feeding glucose during fermentation, adding inducer to induce, adding feed supplement, and adding substrate amino acid in fed-batch manner to end of fermentation to obtain ergothioneine with dissolved oxygen not less than 30%; wherein, the ERG4 strain contains pCDP-EgtD-EgtE-MBP-EgtB plasmid.
The formula of the fermentation medium is as follows: glucose 30g/L, yeast powder 24g/L, peptone 12g/L, KH 2 PO 4 2g/L, naCl g/L, ferric ammonium citrate 0.06g/L, mgSO 4 ·7H 2 O 0.5g/L。
The inducer is isopropyl thiogalactoside.
The final concentration of the isopropyl thiogalactoside is 0.1mM.
The formula of the feed supplement comprises the following components: pyridoxine hydrochloride, na 2 S 2 O 3 Ferric ammonium citrate and CaCl 2 The final concentrations were 0.02g/L, 1.58g/L, 0.03g/L and 0.02g/L, respectively.
The substrate amino acids are histidine, methionine and cysteine.
The final concentration of histidine, methionine and cysteine is above 10 g/L.
In the fermentation process, the concentration of residual glucose in glucose is controlled to be below 5g/L, and the pH is controlled to be 6-8.
ERG4 cell OD 600 When the temperature reaches 25-30 ℃, adding an inducer isopropyl thiogalactoside to start induction, simultaneously adding a feed supplement, and reducing the temperature to 30 ℃ to ferment.
The method for preparing ergothioneine by using engineering bacteria comprises the following specific steps:
1) Selecting the EgtD and EgtE genes from Mycobacterium smegmati sources, the EgtB gene from Methylobacterium pseudosasicola sources and the MBP tag gene, designing primers, performing PCR cloning, constructing and obtaining pCDP-EgtD-EgtE-MBP-EgtB plasmids, and transferring the pCDP-EgtD-EgtE-MBP-EgtB plasmids into escherichia coli BL21 (DE 3) by a heat shock method to obtain engineering strain ERG4;
2) Selecting an engineering strain ERG4 monoclonal in a 5mL LB test tube containing streptomycin, placing in a shaking table at 37 ℃ for shake culture at 220rpm for 12 hours, transferring the bacterial liquid into a 1L triangular flask containing 200mL LB culture medium, placing in a shaking table at 37 ℃ for continuous culture at 220rpm for 12 hours, and obtaining a seed liquid of the engineering strain ERG4;
3) Inoculating the seed solution into fermentation culture medium according to 5% of inoculation amount, starting fermentation culture, controlling the temperature at 37deg.C, the rotation speed at 300r/min, pH at 6-8, ventilation rate at 2L/min, dissolved oxygen at 30% or above, and when initial glucose in culture medium is exhausted and dissolved oxygen rebounds, starting feeding glucose until the cell OD 600 Reaching 25 to 30; wherein, the formula of the fermentation medium is as follows: glucose 30g/L, yeast powder 24g/L, peptone 12g/L, KH 2 PO 4 2g/L, naCl g/L, lemonFerric ammonium acid 0.06g/L, mgSO 4 ·7H 2 O 0.5g/L；
4) Adding inducer isopropyl thiogalactoside with final concentration of 0.1mM to start induction, adding feed, cooling to 30deg.C, increasing rotation speed to 500-800r/min, continuously culturing for 2 hr, and adding amino acid to ferment to obtain ergothioneine;
wherein, the formula of the feed supplement comprises: pyridoxine hydrochloride, na 2 S 2 O 3 Ferric ammonium citrate and CaCl 2 Final concentrations were 0.02g/L, 1.58g/L, 0.03g/L and 0.02g/L, respectively; the amino acid consists of histidine, methionine and cysteine, and the final concentration of each amino acid is above 10-20 g/L;
the concentration of glucose residue in the fermentation process is controlled below 5g/L, and the pH is regulated by ammonia water and phosphoric acid and maintained at 6-8.
Compared with the prior art, the invention has the beneficial effects that:
the invention takes an engineering strain of escherichia coli BL21 (DE 3) named ERG4 as a fermentation strain, inoculates seed liquid of the fermentation strain into a fermentation culture medium, adds glucose in a flowing way in the fermentation process, adds a supplementary material when an inducer is added to start induction, and adds substrate amino acid in a flowing way to the end of fermentation to prepare the ergothioneine, wherein dissolved oxygen in the period is not less than 30 percent. The results show that the effect of glucose as a carbon source is obviously better than that of glycerol; although the synthesis rate of the primary ergothioneine is higher than that of the fed-batch mode after one-time direct addition, the yield of the ergothioneine obtained in the fed-batch mode is 3.1g/L, and the yield obtained in the one-time direct addition mode is 2.5g/L; 150g of each of the three amino acids was fed into a 30L fermenter, and after 44 hours of fermentation, the yield of ergothioneine was 3.2g/L, while 300g of each of the three amino acids was fed into the fermenter, and the yield of ergothioneine was 5.5g/L.
Drawings
FIG. 1 is a graph of ergothioneine yield in shake flasks for recombinant strains at different concentrations of glycerol and glucose;
FIG. 2 is a graph of ergothioneine yield of recombinant strains in a 5L fermenter with different addition of substrate amino acids;
FIG. 3 is a graph of ergothioneine yield of recombinant strains in a 5L fermenter under different medium conditions;
FIG. 4 is a graph of ergothioneine yield of recombinant strains in a 30L fermenter at different concentrations of substrate amino acids.
Detailed Description
The present invention will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present invention more apparent. Unless otherwise indicated, all technical means used in the following examples are conventional means well known to those skilled in the art.
In the following examples, HPLC analysis conditions were used: the chromatographic column is Shimadzu Shim-pack GIST C18-AQ column (5 μm, 4.6X1250 mm), the mobile phase A phase is ultrapure water, and the mobile phase B phase is acetonitrile; the sample is eluted with the equality degree of 95% A phase and 5% B phase, the flow rate is 0.6mL/min, the sample injection amount is 10 mu L, and the detection wavelength is 257mn.
Example 1
1. The method comprises the steps of selecting an EgtD (SEQ ID NO. 1) and an EgtE gene (SEQ ID NO. 2) from Mycobacterium smegmati sources, selecting an EgtB gene (SEQ ID NO. 3) from Methylobacterium pseudosasicola sources, designing related primers (Table 1), and carrying out PCR cloning and related vector construction, wherein the specific steps are as follows:
1) First, egtD, egtE, egtB and a carrier containing MBP genes are used as templates, primers EgtD-F/EgtD-R, egtE-F/EgtE-R, egtB-MF/EgtB-R and MBP-F/EgtB-MR are respectively utilized, and EgtD, egtE, M-EgtB and MBP gene fragments are obtained through PCR amplification of high-fidelity enzymes; and then, the M-EgtB gene fragment and the MBP gene fragment are taken as templates together, and the MBP-EgtB is obtained by utilizing the primer MBP-F/EgtB-R to carry out overlapping PCR amplification.
2) The gene fragments EgtD, egtE and MBP-EgtB obtained by the PCR are respectively connected to NdeI and XhoI of an expression vector pCDP2 by using T4 ligase, and are transformed into escherichia coli DH5 alpha by a heat shock method to obtain pCDP-EgtD, pCDP-EgtE and pCDP-MBP-EgtB.
3) Taking pCDP-EgtD as a plasmid donor, carrying out double digestion at NheI and SalI sites, taking pCDP-EgtE as a gene donor, carrying out double digestion at avrII and SalI sites, and connecting an EgtE gene fragment obtained by double digestion to a linearized pCDP-EgtD plasmid by using T4 ligase according to the principle of homotail enzyme ligation to obtain plasmid pCDP-EgtD-EgtE; and then taking pCDP-EgtD-EgtE as a plasmid donor, continuing to carry out double digestion at NheI and SalI sites, taking pCDP-MBP-EgtB as a gene donor, carrying out double digestion at AvrII and SalI sites, and connecting the MBP-EgtB gene fragment obtained by double digestion to a linearized pCDP-EgtD-EgtE plasmid to obtain the plasmid pCDP-EgtD-EgtE-MBP-EgtB.
2. The pCDP-EgtD-EgtE-MBP-EgtB plasmid obtained in the above is transferred into escherichia coli BL21 (DE 3) to obtain a recombinant strain producing ergothioneine, which is named ERG4.
TABLE 1 primers related thereto
Example 2
Respectively picking up recombinant strain ERG4 monoclonal in 5mL LB test tubes containing streptomycin, placing in a shaking table at 37 ℃ and shake culturing at 220rpm for 12h to obtain seed solution of the ergothioneine recombinant strain; the seed solution was then transferred to a fermentation medium (yeast powder 24g/L, peptone 12g/L, KH) containing 25mL at an inoculum size of 2% 2 PO 4 4 g/L、Na 2 HPO 4 ·12H 2 O8 g/L, naCl 3g/L, ferric ammonium citrate 0.06g/L, mgSO 4 ·7H 2 O0.5 g/L) and adding carbon sources (1% glycerol, 2% glycerol, 3% glycerol, 1% glucose, 2% glucose and 3% glucose) with different concentrations respectively, adding 50mg/L of streptomycin, placing the mixture in a shaking table at 37 ℃ for shaking culture at 220rpm for 2-3 hours, adding an inducer isopropyl thiogalactoside (IPTG) with the final concentration of 0.1mM for starting induction, adding three substrate amino acids (histidine, methionine and cysteine) simultaneously, placing the mixture in a shaking table at 30 ℃ for fermenting culture at 220rpm for 24 hours, and detecting and analyzing the ergothioneine yield by HPLC.
As a result, as shown in FIG. 1, glucose was significantly better than glycerol in effect as a carbon source.
Example 3
Picking recombinant strain ERG4 monoclonal in 5mL LB test tube containing streptomycinPlacing in a shaking table at 37 ℃ for shake culture at 220rpm for 12 hours, transferring the bacterial liquid into a 1L triangular flask containing 200mL of LB culture medium, placing in a shaking table at 37 ℃ for continuous culture at 220rpm for 12 hours, and obtaining seed liquid of recombinant strain ERG4; inoculating the seed solution to 2L fermentation medium (glucose 30g/L, yeast powder 24g/L, peptone 12g/L, KH) 2 PO 4 2g/L, naCl g/L, ferric ammonium citrate 0.06g/L, mgSO 4 ·7H 2 O0.5 g/L), starting fermentation culture, controlling the rotating speed at 37 ℃ and 300r/min, controlling the pH value at 6-8, controlling the ventilation rate at about 2L/min, controlling the dissolved oxygen to be more than 30%, starting feeding glucose when the initial glucose in the culture medium is exhausted and the dissolved oxygen rebounds, and controlling the flow acceleration of glucose through the sugar consumption speed of the thallus (the residual glucose concentration of the glucose in the whole fermentation process is controlled below 5 g/L). The strain is cultured for about 4-6 hours, and the OD of the thallus is obtained 600 When 25-30 times, adding inducer isopropyl thiogalactoside (IPTG) (final concentration 0.1 mM) to start induction, and adding feed (pyridoxine hydrochloride, na) 2 S 2 O 3 Ferric ammonium citrate and CaCl 2 The final concentration is 0.02g/L, 1.58g/L, 0.03g/L and 0.02g/L respectively, the temperature is reduced to 30 ℃, the rotating speed is increased to 500-800r/min, the culture is continued for about 2 hours, the addition of substrate amino acids (histidine, methionine and cysteine) is started, the addition mode (1) directly adds 20g of each of three amino acids at one time, and the culture is continued for 96 hours; (2) 20g of each of the three amino acids is added in a fed-batch mode, the culture is continued for 96 hours, the pH value of the whole fermentation process is regulated by ammonia water and phosphoric acid, and the ergothioneine yield is detected and analyzed by HPLC.
As a result, as shown in FIG. 2, although the synthesis rate of the primary ergothioneine was higher than that of the fed-batch form after one-time direct addition, the yield of ergothioneine finally obtained in the fed-batch form was 3.1g/L, and the yield obtained in the one-time direct addition form was 2.5g/L.
Example 4
Selecting recombinant strain ERG4 monoclonal in 5mL LB test tube containing streptomycin, placing in a shaking table at 37deg.C, shake culturing at 220rpm for about 12 hr, transferring bacterial liquid into 1L triangular flask containing 200mL LB culture medium, placing in a shaking table at 37deg.CAfter the culture is continued for 12 hours at 220rpm, seed liquid of the recombinant strain ERG4 is obtained; inoculating the seed solution into 5L triple fermenters containing 2L of different culture mediums (R culture medium (glucose 10g/L, KH) of Zhang Shan, etc 2 PO 4 13.3g/L，(NH 4 ) 2 HPO 4 4.0 g/L，MgSO 4 ·7H 2 O1.2 g/L, citric acid 1.7 g/L, trace elements 5mL: EDTA 8.4mg/L, coCl 2 ·6H 2 O 2.5mg/L，MnCl 2 ·4H 2 O 15.0mg/L，CuCl 2 ·2H 2 O 1.5mg/L，H 3 BO 33.0mg/L，Na 2 MoO 4 ·2H 2 O 2.5mg/L，Zn(CH 3 COO) 2 ·2H 2 EGT11 medium (glucose 10g/L, yeast extract 0.5g/L, (NH) 100mg/L, ferric (III) citrate 4.5mg/L, and Wu Heyun etc 4 ) 2 SO 4 5g/L，K 2 HPO 4 7g/L，MgSO 4 ·7H 2 O2 g/L, sodium citrate 2g/L, methionine 2g/L, feSO 4 ·7H 2 O 100mg/L，MnSO 4 ·7H 2 The fermentation culture is started by O20 mg/L, VB1, VB3, VB5 and VB12 of 3mg/L, VH 5mg/L and VB6 mg/L, the temperature is 37 ℃, the rotating speed is 300r/min, the pH is controlled to be 6-8, the ventilation rate is controlled to be about 2L/min, the dissolved oxygen is controlled to be more than 30 percent, when the initial glucose in the culture medium is exhausted, the dissolved oxygen rebounds, the glucose starts to be fed, and the glucose flow acceleration is controlled through the thallus glucose consumption speed (the residual glucose concentration of the glucose in the whole fermentation process is controlled to be less than 5 g/L). The strain is cultured for about 4-8h, and the OD of the thallus is obtained 600 When 25-30 times, adding inducer isopropyl thiogalactoside (IPTG) (final concentration 0.1 mM) to start induction, and adding feed (pyridoxine hydrochloride, na) 2 S 2 O 3 Ferric ammonium citrate and CaCl 2 Final concentrations of 0.02g/L, 1.58g/L, 0.03g/L and 0.02g/L respectively), the temperature is reduced to 30 ℃, the rotating speed is increased to 500-800r/min, after continuous culture for about 2 hours, the three substrate amino acids (histidine, methionine and cysteine) are added in a fed-batch mode until the fermentation is finished (the pH value of the whole fermentation process is regulated by ammonia water and phosphoric acid), HAnd the PLC detection is used for analyzing the ergothioneine yield.
As a result, as shown in FIG. 3, zhang Shan et al, the yield of ergothioneine obtained by fermentation in R medium was about 2.4g/L, wu Heyun et al, and the yield of ergothioneine obtained by fermentation in EGT11 medium was about 2.6g/L, and the strains grew slower and the yield of ergothioneine was lower during fermentation in R medium and EGT11 medium than in the fermentation medium used in the present invention (example 3).
Example 5
Selecting recombinant strain ERG4 monoclonal in 5mL LB test tube containing streptomycin, placing in a shaking table at 37 ℃ and shaking and culturing at 220rpm for about 12h, transferring the bacterial liquid into a 1L triangular flask containing 200mL LB culture medium, placing in a shaking table at 37 ℃ and continuously culturing at 220rpm for 12h to obtain seed liquid of recombinant strain ERG4; the seed solution was inoculated to a medium containing 15L of fermentation medium (glucose 30g/L, yeast powder 24g/L, peptone 12g/L, KH) 2 PO 4 2g/L, naCl g/L, ferric ammonium citrate 0.06g/L, mgSO 4 ·7H 2 O0.5 g/L), starting fermentation culture, controlling the temperature at 37 ℃ and the rotating speed at 300r/min, controlling the pH value at 6-8, controlling the ventilation rate at about 2L/min, controlling the dissolved oxygen at more than 30%, starting feeding glucose when the initial glucose in the culture medium is exhausted and the dissolved oxygen rebounds, and controlling the glucose feeding acceleration through the thallus glucose consumption speed (controlling the glucose concentration in the whole fermentation process to be less than 5 g/L). The strain is cultured for about 4 hours, and the OD of the thallus is that of the strain 600 When 25-30 times, adding inducer isopropyl thiogalactoside (IPTG) (final concentration 0.1 mM) to start induction, and adding feed (pyridoxine hydrochloride, na) 2 S 2 O 3 Ferric ammonium citrate and CaCl 2 The final concentration is 0.02g/L, 1.58g/L, 0.03g/L and 0.02g/L respectively), the temperature is reduced to 30 ℃, the rotating speed is increased to 500-800r/min, after the continuous culture is carried out for about 2 hours, three substrate amino acids (histidine, methionine and cysteine) are added, and (1) 150g of each of the three amino acids are added (the final concentration is 10 g/L); (2) 300g of each of the three amino acids was fed (final concentration: 20 g/L), pH of the whole fermentation process was adjusted by ammonia water and phosphoric acid, and the ergothioneine yield was analyzed by HPLC detection.
As a result, as shown in FIG. 4, 150g of each of the three amino acids was fed during fermentation, and the yield of ergothioneine was about 3.2g/L, while 300g of each of the three amino acids was fed, and the yield of ergothioneine was about 5.5g/L.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for preparing ergothioneine by using engineering bacteria is characterized in that: inoculating a seed solution of a fermentation strain into a fermentation culture medium by taking an engineering strain of escherichia coli BL21 (DE 3) named ERG4 as the fermentation strain, adding glucose in a fed-batch manner in the fermentation process, adding an inducer for induction, adding a feed supplement at the same time, and adding substrate amino acid in a fed-batch manner until the fermentation is finished, so as to prepare ergothioneine, wherein the dissolved oxygen is not less than 30%; wherein, the ERG4 strain contains pCDP-EgtD-EgtE-MBP-EgtB plasmid.
2. The method for preparing ergothioneine by engineering bacteria according to claim 1, wherein the fermentation medium comprises the following formula: glucose 30g/L, yeast powder 24g/L, peptone 12g/L, KH 2 PO 4 2g/L, naCl g/L, ferric ammonium citrate 0.06g/L, mgSO 4 ·7H 2 O 0.5g/L。
3. The method for preparing ergothioneine by engineering bacteria according to claim 1, wherein the inducer is isopropyl thiogalactoside.
4. The method for producing ergothioneine using engineering bacteria according to claim 1 or 3, wherein the final concentration of isopropyl thiogalactoside is 0.1mM.
5. The method for preparing ergothioneine by engineering bacteria according to claim 1The method is characterized in that the feed formula comprises the following steps: pyridoxine hydrochloride, na 2 S 2 O 3 Ferric ammonium citrate and CaCl 2 The final concentrations were 0.02g/L, 1.58g/L, 0.03g/L and 0.02g/L, respectively.
6. The method for preparing ergothioneine by engineering bacteria according to claim 1, wherein the substrate amino acids are histidine, methionine and cysteine.
7. The method for preparing ergothioneine by engineering bacteria according to claim 1 or 6, wherein the final concentration of histidine, methionine and cysteine is above 10 g/L.
8. The method for preparing ergothioneine by engineering bacteria according to claim 1, wherein the concentration of residual glucose in the fermentation process is controlled to be below 5g/L, and the pH is controlled to be 6-8.
9. The method for preparing ergothioneine by engineering bacteria according to claim 1, wherein ERG4 bacteria OD 600 When the temperature reaches 25-30 ℃, adding an inducer isopropyl thiogalactoside to start induction, simultaneously adding a feed supplement, and reducing the temperature to 30 ℃ to ferment.
10. The method for preparing ergothioneine by engineering bacteria according to claim 1, which is characterized by comprising the following specific steps:
1) Selecting the EgtD and EgtE genes from Mycobacterium smegmati sources, the EgtB gene from Methylobacterium pseudosasicola sources and the MBP tag gene, designing primers, performing PCR cloning, constructing and obtaining pCDP-EgtD-EgtE-MBP-EgtB plasmids, and transferring the pCDP-EgtD-EgtE-MBP-EgtB plasmids into escherichia coli BL21 (DE 3) by a heat shock method to obtain engineering strain ERG4;
2) Selecting an engineering strain ERG4 monoclonal in a 5mL LB test tube containing streptomycin, placing in a shaking table at 37 ℃ for shake culture at 220rpm for 12 hours, transferring the bacterial liquid into a 1L triangular flask containing 200mL LB culture medium, placing in a shaking table at 37 ℃ for continuous culture at 220rpm for 12 hours, and obtaining a seed liquid of the engineering strain ERG4;
3) Inoculating the seed solution into fermentation culture medium according to 5% of inoculation amount, starting fermentation culture, controlling the temperature at 37deg.C, the rotation speed at 300r/min, pH at 6-8, ventilation rate at 2L/min, dissolved oxygen at 30% or above, and when initial glucose in culture medium is exhausted and dissolved oxygen rebounds, starting feeding glucose until the cell OD 600 Reaching 25 to 30; wherein, the formula of the fermentation medium is as follows: glucose 30g/L, yeast powder 24g/L, peptone 12g/L, KH 2 PO 4 2g/L, naCl g/L, ferric ammonium citrate 0.06g/L, mgSO 4 ·7H 2 O 0.5g/L；
4) Adding inducer isopropyl thiogalactoside with final concentration of 0.1mM to start induction, adding feed, cooling to 30deg.C, increasing rotation speed to 500-800r/min, continuously culturing for 2 hr, and adding amino acid to ferment to obtain ergothioneine;
wherein, the formula of the feed supplement comprises: pyridoxine hydrochloride, na 2 S 2 O 3 Ferric ammonium citrate and CaCl 2 Final concentrations were 0.02g/L, 1.58g/L, 0.03g/L and 0.02g/L, respectively; the amino acid consists of histidine, methionine and cysteine, and the final concentration of each amino acid is above 10-20 g/L;
the concentration of glucose residue in the fermentation process is controlled below 5g/L, and the pH is regulated by ammonia water and phosphoric acid and maintained at 6-8.
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| CN114854659A (en) * | 2022-05-27 | 2022-08-05 | 深圳中科欣扬生物科技有限公司 | Ergothioneine production process and application thereof |
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| KAMIDE T ET AL.: "High Production of Ergothioneine in Escherichia coli using the Sulfoxide Synthase from Methylobacterium strains", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》, vol. 68, no. 23, pages 6391 * |
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