CN109554325B - Escherichia coli engineering bacterium capable of highly producing tyrosine and application thereof - Google Patents
Escherichia coli engineering bacterium capable of highly producing tyrosine and application thereof Download PDFInfo
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- CN109554325B CN109554325B CN201910019537.5A CN201910019537A CN109554325B CN 109554325 B CN109554325 B CN 109554325B CN 201910019537 A CN201910019537 A CN 201910019537A CN 109554325 B CN109554325 B CN 109554325B
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 title claims abstract description 74
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 title claims abstract description 67
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 65
- 241000894006 Bacteria Species 0.000 title claims abstract description 31
- 238000000855 fermentation Methods 0.000 claims abstract description 57
- 230000004151 fermentation Effects 0.000 claims abstract description 57
- PJWIPEXIFFQAQZ-PUFIMZNGSA-N 7-phospho-2-dehydro-3-deoxy-D-arabino-heptonic acid Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@H](O)CC(=O)C(O)=O PJWIPEXIFFQAQZ-PUFIMZNGSA-N 0.000 claims abstract description 10
- 230000006698 induction Effects 0.000 claims abstract description 10
- 108010000898 Chorismate mutase Proteins 0.000 claims abstract description 5
- 108010035004 Prephenate Dehydrogenase Proteins 0.000 claims abstract description 5
- 239000008186 active pharmaceutical agent Substances 0.000 claims abstract description 5
- 239000002773 nucleotide Substances 0.000 claims description 32
- 125000003729 nucleotide group Chemical group 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 19
- 238000004519 manufacturing process Methods 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 10
- -1 aromatic amino acid Chemical class 0.000 claims description 2
- 101150010999 aroP gene Proteins 0.000 abstract description 18
- 101150049689 tyrP gene Proteins 0.000 abstract description 17
- 230000000813 microbial effect Effects 0.000 abstract description 6
- 108050005273 Amino acid transporters Proteins 0.000 abstract description 4
- 102000034263 Amino acid transporters Human genes 0.000 abstract description 4
- 101100002724 Thermus thermophilus aroH gene Proteins 0.000 abstract description 4
- 101150076125 aroG gene Proteins 0.000 abstract description 4
- 125000003118 aryl group Chemical group 0.000 abstract description 4
- 101150083154 tyrA gene Proteins 0.000 abstract description 4
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- 238000010353 genetic engineering Methods 0.000 abstract description 3
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- 238000000137 annealing Methods 0.000 description 4
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- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
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- FTVWIRXFELQLPI-ZDUSSCGKSA-N (S)-naringenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 FTVWIRXFELQLPI-ZDUSSCGKSA-N 0.000 description 2
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- 230000015572 biosynthetic process Effects 0.000 description 2
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- KTVIXTQDYHMGHF-UHFFFAOYSA-L cobalt(2+) sulfate Chemical compound [Co+2].[O-]S([O-])(=O)=O KTVIXTQDYHMGHF-UHFFFAOYSA-L 0.000 description 2
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 2
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
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- 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 2
- 239000011684 sodium molybdate Substances 0.000 description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
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- 239000011691 vitamin B1 Substances 0.000 description 2
- 229910001868 water Inorganic materials 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 239000011686 zinc sulphate Substances 0.000 description 2
- NJWJQMLMBXIOJW-UHFFFAOYSA-N (2S)-3',5,7-trihydroxyflavanone Natural products OC1=CC=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 NJWJQMLMBXIOJW-UHFFFAOYSA-N 0.000 description 1
- VOZDNCFKGOLECZ-BTVCFUMJSA-N 2-hydroxypropanoic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound CC(O)C(O)=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O VOZDNCFKGOLECZ-BTVCFUMJSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- RTIXKCRFFJGDFG-UHFFFAOYSA-N Chrysin Natural products C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=CC=C1 RTIXKCRFFJGDFG-UHFFFAOYSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 1
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 1
- 238000012220 PCR site-directed mutagenesis Methods 0.000 description 1
- 102000015731 Peptide Hormones Human genes 0.000 description 1
- 108010038988 Peptide Hormones Proteins 0.000 description 1
- 201000011252 Phenylketonuria Diseases 0.000 description 1
- FGUBFGWYEYFGRK-HNNXBMFYSA-N Pinocembrin Natural products Cc1cc(C)c2C(=O)C[C@H](Oc2c1)c3ccccc3 FGUBFGWYEYFGRK-HNNXBMFYSA-N 0.000 description 1
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 description 1
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- 229910000329 aluminium sulfate Inorganic materials 0.000 description 1
- DIZPMCHEQGEION-UHFFFAOYSA-H aluminium sulfate (anhydrous) Chemical compound [Al+3].[Al+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O DIZPMCHEQGEION-UHFFFAOYSA-H 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 230000004186 co-expression Effects 0.000 description 1
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
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- WGEYAGZBLYNDFV-UHFFFAOYSA-N naringenin Natural products C1(=O)C2=C(O)C=C(O)C=C2OC(C1)C1=CC=C(CC1)O WGEYAGZBLYNDFV-UHFFFAOYSA-N 0.000 description 1
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- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
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- 239000000813 peptide hormone Substances 0.000 description 1
- URFCJEUYXNAHFI-ZDUSSCGKSA-N pinocembrin Chemical compound C1([C@@H]2CC(=O)C3=C(O)C=C(C=C3O2)O)=CC=CC=C1 URFCJEUYXNAHFI-ZDUSSCGKSA-N 0.000 description 1
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- 239000001632 sodium acetate Substances 0.000 description 1
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- 238000003786 synthesis reaction Methods 0.000 description 1
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- 150000003667 tyrosine derivatives Chemical class 0.000 description 1
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- C07K—PEPTIDES
- 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
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
- C12P13/225—Tyrosine; 3,4-Dihydroxyphenylalanine
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Abstract
The invention discloses an escherichia coli engineering bacterium capable of highly producing tyrosine and application thereofIt belongs to the technical field of genetic engineering and microbial engineering. The present invention expresses a gene aroG encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (DS) in Escherichia coli simultaneously by knocking out aroP encoding an aromatic amino acid transporter and/or tyrP encoding a tyrosine-specific transporter in Escherichia colifbrAnd/or tyrA gene encoding chorismate mutase and prephenate dehydrogenase (CM-PD)fbrGreatly improving the tyrosine yield of the escherichia coli; the escherichia coli engineering bacteria are used for induction fermentation for 46 hours, so that the tyrosine content in the fermentation liquor can reach 44.5g/L, and is improved by 57 percent compared with escherichia coli HGXP.
Description
Technical Field
The invention relates to an escherichia coli engineering bacterium capable of highly producing tyrosine and application thereof, belonging to the technical field of genetic engineering and microbial engineering.
Background
Tyrosine (L-tyrosine, Tyr) is a conditionally essential amino acid of human body, is often used as a nutritional supplement for phenylketonuria patients, a raw material for preparing pharmaceutical and chemical products such as polypeptide hormones, antibiotics, L-dopa and the like, and a precursor for synthesizing high-value-added tyrosine derivatives such as resveratrol, naringenin, pinocembrin and the like, and is widely applied to industries such as food, feed, medicine, chemical industry and the like.
Currently, tyrosine is mainly prepared industrially by an enzymatic method, which has the advantages of short period, strong selectivity, high conversion rate and simple separation and purification steps, but the disadvantages of low natural enzymatic activity, poor stability and the like limit the application of the enzymatic method. Compared with an enzyme method, the microbial fermentation method can realize the head-on synthesis of tyrosine by using a biomass raw material, so that the production cost of the tyrosine is reduced, and meanwhile, the microbial fermentation method has obvious advantages in the aspect of stability.
Therefore, the method for producing tyrosine by using the microbial fermentation method to replace the enzyme method has very wide development prospect.
However, existing Tyrosine-producing bacteria, such as e.coli DPD4193(Olson, m.m., Templeton, l.j., Suh, w., Youderian, p., sariaslland, f.s., gateby, a.a., Dyk, t.k.v.,2007.Production of type from floor or glucose exposed by crude genes, tophyll et al, e.g., soybean, 0.59g/L and 2.65g/L, which undoubtedly greatly hinders the industrialization process of producing tyrosine by microbial fermentation.
Therefore, there is an urgent need to find a method for increasing the tyrosine production of tyrosine-producing bacteria.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to obtain the tyrosine production strain with improved tyrosine yield.
[ solution ]
In order to solve the problems, the invention provides an escherichia coli engineering bacterium capable of highly producing tyrosine, wherein a gene aroP coding an aromatic amino acid transporter and/or a gene tyrP coding a tyrosine specific transporter are knocked out.
In one embodiment of the present invention, the engineered Escherichia coli expresses a gene aroG encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (DS) based on the deletion of aroP encoding an aromatic amino acid transporter and/or tyrP encoding a tyrosine-specific transporterfbrAnd/or tyrA gene encoding chorismate mutase and prephenate dehydrogenase (CM-PD)fbr。
In one embodiment of the invention, the aroP has the nucleotide sequence shown in SEQ ID NO. 1.
In one embodiment of the invention, the tyrP nucleotide sequence is as shown in SEQ ID NO. 2.
In one embodiment of the present invention, the aroGfbrThe nucleotide sequence of (A) is shown in SEQ ID NO. 3.
In one embodiment of the invention, the tyrAfbrThe nucleotide sequence of (A) is shown in SEQ ID NO. 4.
In one embodiment of the present invention, the aroGfbrAnd/or tyrAfbrIs expressed by assembling the plasmid with a heat-induced expression vector pAP-B03 in an Escherichia coli engineering bacterium.
In one embodiment of the invention, the engineered Escherichia coli expresses a gene aroG encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (DS) based on the knockout of the aroP gene encoding the aromatic amino acid transporterfbrAnd tyrA gene encoding chorismate mutase and prephenate dehydrogenase (CM-PD)fbr。
The invention also provides a method for producing tyrosine, which comprises the step of inoculating the escherichia coli engineering bacterium capable of producing tyrosine with high yield into a fermentation culture medium for fermentation by using the escherichia coli engineering bacterium capable of producing tyrosine with high yield.
In one embodiment of the invention, the fermentation is to inoculate the above engineering bacterium of escherichia coli capable of producing tyrosine with high yield into a fermentation medium and culture the engineering bacterium to OD600After 23-27 deg.C, the induction is carried out while controlling the temperature at 36-40 deg.C.
In one embodiment of the invention, the fermentation is to inoculate the above engineering bacterium of escherichia coli capable of producing tyrosine with high yield into a fermentation medium and culture the engineering bacterium to OD600After 23-27 ℃, induction was performed while controlling the temperature at 38 ℃.
In one embodiment of the invention, the components of the fermentation medium comprise glucose 35g/L, (NH)4)2SO45g/L、KH2PO43g/L、MgSO4·7H2O3 g/L, NaCl1g/L, sodium citrate 1.5g/L, CaCl g2·2H2O 0.015g/L、CaCO312g/L、FeSO4·7H2O0.1125 g/L, vitamin B10.075g/L, peptone 4g/L, yeast powder 2g/L, kanamycin sulfate 0.04g/L and trace element nutrient solution (TES)1.5 mL/L; the microelement nutrient solution (TES) contains Al2(SO4)3·18H2O 2.0g/L、CoSO4·7H2O 0.75g/L、CuSO4·5H2O 2.5g/L、H3BO30.5g/L、MnSO4·H2O 24g/L、Na2MoO4·2H2O 3.0g/L、NiSO4·6H2O 2.5g/L、ZnSO4·7H2O 15g/L。
The invention also provides the application of the escherichia coli engineering bacterium capable of highly producing tyrosine or the method in preparation of tyrosine.
[ advantageous effects ]
(1) The present invention expresses a gene aroG encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (DS) in Escherichia coli simultaneously by knocking out aroP encoding an aromatic amino acid transporter and/or tyrP encoding a tyrosine-specific transporter in Escherichia colifbrAnd/or tyrA gene encoding chorismate mutase and prephenate dehydrogenase (CM-PD)fbrGreatly improving the tyrosine yield of the escherichia coli; the escherichia coli engineering bacteria are used for induction fermentation for 46 hours, so that the tyrosine content in the fermentation liquor can reach 44.5g/L, and is improved by 57 percent compared with escherichia coli HGXP;
(2) the strain of the invention uses a heat-induced expression vector pAP-aroGfbr-tyrAfbrCompared with other engineering strains needing to be added with expensive inducers, the strain can produce tyrosine by thermally inducing and expressing target genes, has the advantages of economy, saving and difficult contamination, and is more suitable for industrial large-scale production.
Drawings
FIG. 1: influence of temperature on the yield of Escherichia coli engineering bacteria HGXP, HGPP or HGEP tyrosine.
FIG. 2: and (3) a supplemented batch fermentation curve of escherichia coli HGPP.
FIG. 3: fed batch fermentation curve of Escherichia coli HGEP.
Detailed Description
Escherichia coli BL21(DE3), Escherichia coli JM109, and Escherichia coli WSH-Z06(pAP-B03) mentioned in the following examples were purchased from China Center for Type Culture Collection (CCTCC), wherein the preservation number of Escherichia coli WSH-Z06(pAP-B03) is CCTCC No: m2010009.
The pAP-B03 plasmid referred to in the following examples is described in the literature "Zhou, H., Liao, X., Wang, T., Du, G., Chen, J.,2010.Enhanced L-phenylalkane biosynthesis by co-expression of PheAfbrand aroFwtBioresource technology.101(11): 4151-; the pCas and p-Target plasmids referred to in the following examples are described in the literature "Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S.,2015.Multigene editing in the Escherichia coli genome via the CRISPR-cassette 9system, applied and Environmental microbiology.81(7): 2504 ″; the recombinant plasmid pCDF-aroG referred to in the following examplesfbr-tyrAfbrDescribed in "Wu, J., Zhou, T., Du, G., Zhou, J., Chen, J.,2014. modulation optimization of biotechnology pathways for de novosynthesis of (2S) -naringenin in Escherichia coli. plos one.9(7): 1-9".
The media involved in the following examples are as follows:
seed medium (LB medium): 5g/L of yeast powder and 10g/L, NaCl 10g/L of peptone.
Fermentation medium: glucose 35g/L, (NH)4)2SO45g/L、KH2PO43g/L、MgSO4·7H2O3 g/L, NaCl1g/L and sodium citrate 1.5g/L, CaCl2·2H2O 0.015g/L、CaCO312g/L、FeSO4·7H2O0.1125 g/L, vitamin B10.075g/L, peptone 4g/L, yeast powder 2g/L, kanamycin sulfate 0.04g/L, trace element nutrient solution (TES)1.5mL/L, pH 6.8 +/-0.1;
wherein, the TES solution: al (Al)2(SO4)3·18H2O 2.0g/L、CoSO4·7H2O 0.75g/L、CuSO4·5H2O2.5g/L、H3BO30.5g/L、MnSO4·H2O 24g/L、Na2MoO4·2H2O 3.0g/L、NiSO4·6H2O 2.5g/L、ZnSO4·7H2O 15g/L。
The detection methods referred to in the following examples are as follows:
the tyrosine content detection method comprises the following steps:
HPLC detection is carried out by adopting an Agilent C18(250 multiplied by 4.6mm) chromatographic column, and the mobile phase comprises 0.1mol/L sodium acetate (glacial acetic acid is used for adjusting the pH value to 4.0) and methanol, the volume percentage is 90 percent and 10 percent, the flow rate is 1.0mL/min, the wavelength of an ultraviolet detector is 280nm, the column temperature is 30 ℃, and the sample injection amount is 10 mu L.
The DCW detection method comprises the following steps:
determining the concentration of thallus in fermentation culture in a fermentation tank, adding 6mol/L HCl to dissolve tyrosine in fermentation liquor before constant volume, centrifuging the treated fermentation liquor at 12000 r/min for 10min, and discarding supernatant; washing the thallus precipitate with deionized water for 2 times, drying the wet thallus obtained after centrifugation at 105 ℃ to constant weight, and calculating the Dry Cell Weight (DCW); after the fermentation broth was diluted to an appropriate range, its absorbance (OD) at 600nm was measured using a model 722s spectrophotometer600) Obtaining OD600Relation with DCW, wherein DCW (g/L) ═ 0.364 XOD600。
The detection method of the glucose content comprises the following steps:
centrifuging 1mL fermentation liquid at 12000 r/min for 5min, diluting the supernatant by a certain multiple, and measuring the glucose concentration by using a glucose-lactate biosensing analyzer (Shenzhen Sielman science and technology Co., Ltd.).
Substrate conversion calculation method:
substrate conversion (mol/mol) ═ total moles of product (mol)/total moles of glucose consumed (mol).
The production intensity calculation method comprises the following steps:
production intensity (g/L/h) ═ tyrosine yield (g/L)/total fermentation time (h).
Example 1: construction of Escherichia coli starting Strain HGX
Escherichia coli WSH-Z06(pAP-B03) was passed through serial passages to eliminate pAP-B03 plasmid carried by itself, and Escherichia coli HGX without plasmid was obtained as the starting strain.
Example 2: construction of Escherichia coli aroP single gene knockout bacterium
The method comprises the following specific steps:
(1) transferring the pCas plasmid into an original strain HGX to construct a strain HGX (pCas);
(2) designing specific primers according to 500bp gene sequences of the upstream and downstream aroP, and amplifying the upstream and downstream genes by using Escherichia coli BL21(DE3) genomes as templates, wherein the amplification primers are as follows:
aroP-up-F (the nucleotide sequence is shown as SEQ ID NO. 5): 5-CCACTTGCCGAAGTCAATTGGTC-3;
aroP-up-R (the nucleotide sequence is shown as SEQ ID NO. 6):
5-CGGGTGAGGGCGTAGAGAGAGAAACCTCGTGCGGTGGTTG-3；
aroP-down-F (the nucleotide sequence is shown as SEQ ID NO. 7):
5-CAACCACCGCACGAGGTTTCTCTCTCTACGCCCTCACCCG-3；
aroP-down-R (the nucleotide sequence is shown as SEQ ID NO. 8):
5-ACGAACGTGAGTATTTGCGTGAG-3；
the PCR conditions were as follows: denaturation at 94 deg.C for 2 min; denaturation at 98 ℃ for 30 s; annealing at 60 ℃ for 30s, and extending at 72 ℃ for 30 s;
(3) connecting the upstream and downstream genes obtained in the step (2) by using a fusion PCR technology to obtain an aroP gene upstream and downstream homologous arm;
(4) designing primers, and constructing an aroP-pTarget plasmid by using a PCR (polymerase chain reaction) site-directed mutagenesis technology by taking a p-Target plasmid as a template, wherein the primers are as follows:
aroP-sgRNA-F (nucleotide sequence shown in SEQ ID NO. 9):
5-CCTCCGTAATACAGTCCGCAGTTTTAGAGCTAGAAATAGCAAG-3；
aroP-sgRNA-R (the nucleotide sequence is shown as SEQ ID NO. 10):
5-ACTAGTATTATACCTAGGACTGAG-3；
(5) designing primers, and verifying the aroP-pTarget plasmid obtained in (4) by PCR, wherein the primers are as follows:
aroP-sgRNA-v-F (nucleotide sequence shown in SEQ ID NO. 11):
5-GACCTACACCGAACTGAGATACCTA-3；
aroP-sgRNA-v-R (the nucleotide sequence is shown in SEQ ID NO. 12):
5-TGCGGACTGTATTACGGAGG-3；
(6) and (3) electrically transforming HGX (pCas) competent cells by using the upstream and downstream homology arms of the aroP gene obtained in the step (3) and the aroP-pTarget plasmid obtained in the step (5) to obtain Escherichia coli aroP single gene knock-out bacteria HGX delta aroP.
Example 3: construction of Escherichia coli tyrP single gene knockout bacterium
The method comprises the following specific steps:
(1) transferring the pCas plasmid into an original strain HGX to construct a strain HGX (pCas);
(2) designing specific primers according to tyrP upstream and downstream 500bp gene sequences, and amplifying upstream and downstream genes by taking an escherichia coli BL21(DE3) genome as a template, wherein the primers are as follows:
tyrP-up-F (nucleotide sequence shown as SEQ ID NO. 13): 5-GCGAAGGTCTGTATTTTATCGAC-3;
tyrP-up-R (the nucleotide sequence is shown as SEQ ID NO. 14):
5-AGGAATTTGAGGCTATCTGAGCTTTCTTCTGTCCTGACGA-3；
tyrP-down-F (nucleotide sequence shown in SEQ ID NO. 15):
5-TCGTCAGGACAGAAGAAAGCTCAGATAGCCTCAAATTCCT-3；
tyrP-down-R (the nucleotide sequence is shown as SEQ ID NO. 16): 5-AAGAGATGACGCGCTTTATG-3;
the PCR conditions were as follows: denaturation at 94 deg.C for 2 min; denaturation at 98 ℃ for 30 s; annealing at 60 ℃ for 30s, and extending at 72 ℃ for 30 s;
(3) connecting the upstream and downstream genes obtained in the step (2) by using a fusion PCR technology to obtain upstream and downstream homology arms of the tyrP gene;
(4) designing primers, and constructing tyrP-pTarget plasmid by using a PCR site-directed mutagenesis technology by taking a p-Target plasmid as a template, wherein the primers are as follows:
tyrP-sgRNA-F (nucleotide sequence shown in SEQ ID NO. 17):
5-GGAGGTGTACCAGCATGTTCGTTTTAGAGCTAGAAATAGCAAG-3；
tyrP-sgRNA-R (the nucleotide sequence is shown in SEQ ID NO. 18):
5-ACTAGTATTATACCTAGGACTGAG-3；
(5) designing primers, and verifying the tyrP-pTargett plasmid obtained in (4) by PCR, wherein the primers are as follows:
tyrP-sgRNA-v-F (nucleotide sequence shown in SEQ ID NO. 19):
5-GACCTACACCGAACTGAGATACCTA-3；
tyrP-sgRNA-v-R (nucleotide sequence shown in SEQ ID NO. 20):
5-GAACATGCTGGTACACCTCC-3；
(6) and (3) electrically transforming HGX (pCas) competent cells by the upstream and downstream homology arms of the tyrP gene obtained in the step (3) and the tyrP-pTarget plasmid obtained in the step (5) to obtain escherichia coli tyrP single-gene knock-out sterilized HGX delta tyrP.
Example 4: construction of Escherichia coli aroP/tyrP double-gene knockout bacterium
The method comprises the following specific steps:
on the basis of the Escherichia coli single gene knock-out HGX. DELTA. aroP obtained in example 2, the tyrP gene was further knocked out by the method of example 3 to construct Escherichia coli aroP/tyrP double gene knock-out HGX. DELTA. aroP/tyrP.
Example 5: heat-inducible expression vector pAP-aroGfbr-tyrAfbrConstruction of
The elements cI857, pR, pL and pCDF-aroG were expressed thermally on plasmid pAP-B03 using Gibson assembly techniquefbr-tyrAfbraroG offbr、tyrAfbrAssembling the genes to construct a heat-induced expression vector pAP-aroGfbr-tyrAfbr。
The method comprises the following specific steps:
(1) the plasmid pAP-B03 was digested with Bgl II and Nco I, and the target fragment was recovered by gel recovery kit to obtain a 4545bp linearized vector containing the Kan resistance gene, p15A replicon, cI857 and pR;
(2) designing a primer with a homology arm, and carrying out PCR by using a pAP-B03 plasmid as a template to obtain a fragment pL, wherein the amplification primers are as follows:
pL-up (the nucleotide sequence is shown as SEQ ID NO. 21):
5-CGTCGCGGGTAAGAGGTTTATTATGGTTGCTGAATTG-3；
pL-down (the nucleotide sequence is shown as SEQ ID NO. 22):
5-TTCAGCAACCATAGTGTTGCCTTTTTGTTATCAATAA-3；
the PCR conditions were as follows: denaturation at 94 deg.C for 2 min; denaturation at 98 ℃ for 30 s; annealing at 55 deg.C for 30s, and extending at 72 deg.C for 1 min;
(3) primers with homology arms were designed as pCDF-aroGfbr-tyrAfbrPlasmid as template, PCR to obtain fragment aroGfbr、tyrAfbrWherein, the amplification primers are as follows:
aroG-up (the nucleotide sequence is shown as SEQ ID NO. 23):
5-GGCAAACCAAGACAGCTAAAATGAATTATCAGAACGACGATTTAC-3；
aroG-down (the nucleotide sequence is shown as SEQ ID NO. 24):
5-ATAATAAACCTCTTACCCGCGACGCGCTTTTACTGCA-3；
tyrA-up (nucleotide sequence shown in SEQ ID NO. 25):
5-GCAACACTATGGTTGCTGAATTGACCGCATTAC-3；
tyrA-down (nucleotide sequence shown in SEQ ID NO. 26):
5-TCCAGATAGAACATCTCTTCCTTACTGGCGATTGTCATTCGCCTGA-3；
the PCR conditions were as follows: denaturation at 94 deg.C for 2 min; denaturation at 98 ℃ for 30 s; annealing at 55 ℃ for 30s and extension at 72 ℃ for 70 s.
(4) The 4 fragments thus recovered were diluted by an appropriate factor to prepare a solution containing 1. mu.L of each of the 4 fragments, 5. mu.L of AssemblyMaster Mix (2X) and H2O1 muL, placing the prepared system at 50 ℃ for reaction for 60min by using a Gibson assembly kit to obtain a heat-induced expression vector pAP-aroGfbr-tyrAfbr；
(5) Thermally inducing expression vector pAP-aroGfbr-tyrAfbrEscherichia coli JM109 was transformed, and the correct heat-inducible expression vector pAP-aroG was obtained by colony PCR verificationfbr-tyrAfbrWherein, the primers for PCR verification are as follows:
pL-up (the nucleotide sequence is shown as SEQ ID NO. 27):
5-CGTCGCGGGTAAGAGGTTTATTATGGTTGCTGAATTG-3；
pL-down (the nucleotide sequence is shown in SEQ ID NO. 28):
5-TTCAGCAACCATAGTGTTGCCTTTTTGTTATCAATAA-3。
example 6: construction of recombinant escherichia coli and shake flask fermentation
The method comprises the following specific steps:
(1) the heat-inducible expression vector pAP-aroG obtained in example 5 was usedfbr-tyrAfbrRespectively transforming an original strain HGX, a single-gene knockout bacterium HGX delta aroP, a single-gene knockout bacterium HGX delta tyrP and a double-gene knockout bacterium HGX delta aroP/tyrP to obtain recombinant escherichia coli HGXP, HGPP, HGEP and HGRP;
(2) streaking the recombinant escherichia coli HGXP, HGPP, HGEP and HGRP obtained in the step (1) on an LB (LB) plate (obtained by adding 20g/L of agar into an LB culture medium), and culturing for 10-14 h at 37 ℃ to obtain a single colony;
(3) adding 50mL of LB culture medium into a 500mL shake flask;
(4) picking the single colony obtained in the step (2) to an LB culture medium, and culturing at 37 ℃ at 200r/min for 10-14 h to obtain a seed solution;
(5) adding 25mL of fermentation medium into a 250mL shake flask;
(6) inoculating the recombinant Escherichia coli HGXP, HGPP, HGEP and HGRP obtained in the step (4) into a fermentation medium in an inoculation amount of 10% (v/v), and culturing at 33 ℃ to OD600And (3) heating to 38 ℃, and continuing fermentation culture for 48 hours to obtain fermentation liquor, wherein the temperature is 1.5-2.0 ℃.
Detecting the tyrosine content in the fermentation liquor, wherein the detection result is as follows: the tyrosine content in the fermentation liquid obtained by fermenting the recombinant Escherichia coli HGXP is 3.14 g/L; the tyrosine content in the fermentation liquor obtained by fermenting the recombinant escherichia coli HGPP and HGEP is respectively 3.74g/L and 3.45g/L, which are obviously improved compared with the HGXP; the tyrosine content in the fermentation liquor obtained by the fermentation of the recombinant Escherichia coli HGRP is 2.53g/L, which is obviously reduced compared with HGXP because the growth of the recombinant Escherichia coli HGRP is inhibited.
Example 7: influence of temperature on tyrosine of recombinant Escherichia coli HGPP and HGEP
The method comprises the following specific steps:
(1) inoculating the single colony of the recombinant escherichia coli HGXP, HGPP and HGEP obtained in the embodiment 6 into a 500mL triangular flask filled with 50mL of seed culture medium for culture, and culturing at 37 ℃ at 200r/min for 10-14 h to obtain seed liquid;
(2) adding 25mL of fermentation medium into a 250mL shake flask;
(3) inoculating the seed liquid obtained in (1) into a fermentation medium in an inoculation amount of 10% (v/v), and performing fermentation culture at 33 ℃ until OD is reached600And after the fermentation time is 1.5-2.0 ℃, respectively controlling the temperature to be 33, 36, 38 and 40 ℃ and continuing to induce and ferment for 48 hours to obtain fermentation liquor.
The tyrosine content in the fermentation liquid is detected, and the detection result is shown in figure 1.
As can be seen from FIG. 1, the tyrosine production of the HGXP, HGPP and HGEP strains under the induction condition of 38 ℃ was increased by 96.7%, 77.4% and 92.6% respectively as compared with the culture condition of 33 ℃, 17%, 19% and 18% respectively as compared with the induction condition of 36 ℃ and 8%, 9% and 12% respectively as compared with the induction condition of 40 ℃.
The result shows that the induction temperature of 38 ℃ can improve the tyrosine production capability of the tyrosine genetic engineering bacteria.
Example 8: fermentation of recombinant Escherichia coli HGXP, HGPP and HGEP in tank
The method comprises the following specific steps:
(1) adding 50mL of seed culture medium into a 500mL shake flask;
(2) inoculating the single colonies of the recombinant escherichia coli HGXP, HGPP and HGEP obtained in the embodiment 6 into a seed culture medium, and culturing at 37 ℃ for 12-14 h to obtain a seed solution;
(3) adding 1.1L of fermentation medium into 3-L fermentation tank;
(4) inoculating the seed liquid obtained in the step (2) into a fermentation culture medium in an inoculation amount of 10% (v/v), controlling the initial rotation speed to be 400r/min, and culturing at 33 ℃ for 16h to OD600After 23-27 ℃, heating to 38 ℃, and continuously culturing for 48h to obtain fermentation liquor; in the whole fermentation process, the pH is controlled to be 6.7-6.9 by feeding 20% ammonia water, the rotation speed or ventilation is gradually increased when the DO is reduced to 20% so as to maintain the DO to be above 20%, when the glucose concentration in the culture medium is exhausted, a feeding program is started to feed 700g/L glucose for fed-batch fermentation, and the glucose concentration is maintained to be below 10 g/L.
And (3) detecting the tyrosine content, the glucose content and the variation of the colibacillus thallus concentration in the fermentation liquid in the whole fermentation process (the detection results of the fermentation liquid obtained by HGPP and HGEP fermentation are shown in figures 2-3), and calculating the substrate conversion rate and the production intensity.
The tyrosine yield of the starting strain HGXP on a 3-L fermentation tank is 28.3g/L at most; as shown in FIGS. 2-3, although HGPP was not as grown as well as HGEP at the shake flask level, the maximum DCW of 2 strains was similar at the fermenter level due to more abundant dissolved oxygen, 26.65g/L and 25.96g/L, respectively; as shown in FIGS. 2-3, in the tyrosine production aspect, the tyrosine yield of the HGPP strain fermented for 46h reaches 44.5g/L, the tyrosine yield is improved by 57% compared with the original strain, the substrate conversion rate is 0.179, the production intensity is 0.967g/L/h, the HGEP strain fermented for 46h can produce 35.1g/L of L-tyrosine, the L-tyrosine yield is improved by 24% compared with the original strain, the substrate conversion rate is 0.143, and the production intensity is 0.763 g/L/h.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that 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.
Sequence listing
<110> university of south of the Yangtze river
<120> Escherichia coli engineering bacterium capable of highly producing tyrosine and application thereof
<160>28
<170>PatentIn version 3.3
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atgaattatc agaacgacga tttacgcatc aaagaaatca aagagttact tcctcctgtc 60
gcattgctgg aaaaattccc cgctactgaa aatgccgcga atacggttgc ccatgcccga 120
aaagcgatcc ataagatcct gaaaggtaat gatgatcgcc tgttggttgt gattggccca 180
tgctcaattc atgatcctgt cgcggcaaaa gagtatgcca ctcgcttgct ggcgctgcgt 240
gaagagctga aagatgagct ggaaatcgta atgcgcgtct attttgaaaa gccgcgtacc 300
acggtgggct ggaaagggct gattaacgat ccgcatatgg ataatagctt ccagatcaac 360
gacggtctgc gtatagcccg taaattgctg cttgatatta acgacagcgg tctgccagcg 420
gcaggtgagt ttctcaatat gatcacccca caatatctcg ctgacctgat gagctggggc 480
gcaattggcg cacgtaccac cgaatcgcag gtgcaccgcg aactggcatc agggctttct 540
tgtccggtcg gcttcaaaaa tggcaccgac ggtacgatta aagtggctat cgatgccatt 600
aatgccgccg gtgcgccgca ctgcttcctg tccgtaacga aatgggggca ttcggcgatt 660
gtgaatacca gcggtaacgg cgattgccat atcattctgc gcggcggtaa agagcctaac 720
tacagcgcga agcacgttgc tgaagtgaaa gaagggctga acaaagcagg cctgccagca 780
caggtgatga tcgatttcag ccatgctaac tcgtccaaac aattcaaaaa gcagatggat 840
gtttgtgctg acgtttgcca gcagattgcc ggtggcgaaa aggccattat tggcgtgatg 900
gtggaaagcc atctggtgga aggcaatcag agcctcgaga gcggggagcc gctggcctac 960
ggtaagagca tcaccgatgc ctgcatcggc tgggaagata ccgatgctct gttacgtcaa 1020
ctggcgaatg cagtaaaagc gcgtcgcggg taa 1053
<210>4
<211>1122
<212>DNA
<213> Artificial sequence
<400>4
atggttgctg aattgaccgc attacgcgat caaattgatg aagtcgataa agcgctgctg 60
aatttattag cgaagcgtct ggaactggtt gctgaagtgg gcgaggtgaa aagccgcttt 120
ggactgccta tttatgttcc ggagcgcgag gcatctattt tggcctcgcg tcgtgcagag 180
gcggaagctc tgggtgtacc gccagatctg attgaggatg ttttgcgtcg ggtgatgcgt 240
gaatcttact ccagtgaaaa cgacaaagga tttaaaacac tttgtccgtc actgcgtccg 300
gtggttatcg tcggcggtgg cggtcagatg ggacgcctgt tcgagaagat gctgaccctc 360
tcgggttatc aggtgcggat tctggagcaa catgactggg atcgagcggc tgatattgtt 420
gccgatgccg gaatggtgat tgttagtgtg ccaatccacg ttactgagca agttattggc 480
aaattaccgc ctttaccgaa agattgtatt ctggtcgatc tggcatcagt gaaaaatggg 540
ccattacagg ccatgctggt ggcgcatgat ggtccggtgc tggggctaca cccgatgttc 600
ggtccggaca gcggtagcct ggcaaagcaa gttgtggtct ggtgtgatgg acgtaaaccg 660
gaagcatacc aatggtttct ggagcaaatt caggtctggg gcgctcggct gcatcgtatt 720
agcgccgtcg agcacgatca gaatatggcg tttattcagg cactgcgcca ctttgctact 780
tttgcttacg ggctgcacct ggcagaagaa aatgttcagc ttgagcaact tctggcgctc 840
tcttcgccga tttaccgcct tgagctggcg atggtcgggc gactgtttgc tcaggatccg 900
cagctttatg ccgacatcat tatgtcgtca gagcgtaatc tggcgttaat caaacgttac 960
tataagcgtt tcggcgaggc gattgagttg ctggagcagg gcgataagca ggcgtttatt 1020
gacagtttcc gcaaggtgga gcactggttc ggcgattacg tacagcgttt tcagagtgaa 1080
agccgcgtgt tattgcgtca ggcgaatgac aatcgccagt aa 1122
<210>5
<211>23
<212>DNA
<213> Artificial sequence
<400>5
ccacttgccg aagtcaattg gtc 23
<210>6
<211>40
<212>DNA
<213> Artificial sequence
<400>6
cgggtgaggg cgtagagaga gaaacctcgt gcggtggttg 40
<210>7
<211>40
<212>DNA
<213> Artificial sequence
<400>7
caaccaccgc acgaggtttc tctctctacg ccctcacccg 40
<210>8
<211>23
<212>DNA
<213> Artificial sequence
<400>8
acgaacgtga gtatttgcgt gag 23
<210>9
<211>43
<212>DNA
<213> Artificial sequence
<400>9
cctccgtaat acagtccgca gttttagagc tagaaatagc aag 43
<210>10
<211>24
<212>DNA
<213> Artificial sequence
<400>10
actagtatta tacctaggac tgag 24
<210>11
<211>25
<212>DNA
<213> Artificial sequence
<400>11
gacctacacc gaactgagat accta 25
<210>12
<211>20
<212>DNA
<213> Artificial sequence
<400>12
<210>13
<211>23
<212>DNA
<213> Artificial sequence
<400>13
gcgaaggtct gtattttatc gac 23
<210>14
<211>40
<212>DNA
<213> Artificial sequence
<400>14
aggaatttga ggctatctga gctttcttct gtcctgacga 40
<210>15
<211>40
<212>DNA
<213> Artificial sequence
<400>15
tcgtcaggac agaagaaagc tcagatagcc tcaaattcct 40
<210>16
<211>20
<212>DNA
<213> Artificial sequence
<400>16
<210>17
<211>43
<212>DNA
<213> Artificial sequence
<400>17
ggaggtgtac cagcatgttc gttttagagc tagaaatagc aag 43
<210>18
<211>24
<212>DNA
<213> Artificial sequence
<400>18
actagtatta tacctaggac tgag 24
<210>19
<211>25
<212>DNA
<213> Artificial sequence
<400>19
gacctacacc gaactgagat accta 25
<210>20
<211>20
<212>DNA
<213> Artificial sequence
<400>20
<210>21
<211>37
<212>DNA
<213> Artificial sequence
<400>21
cgtcgcgggt aagaggttta ttatggttgc tgaattg 37
<210>22
<211>37
<212>DNA
<213> Artificial sequence
<400>22
ttcagcaacc atagtgttgc ctttttgtta tcaataa 37
<210>23
<211>45
<212>DNA
<213> Artificial sequence
<400>23
ggcaaaccaa gacagctaaa atgaattatc agaacgacga tttac 45
<210>24
<211>37
<212>DNA
<213> Artificial sequence
<400>24
ataataaacc tcttacccgc gacgcgcttt tactgca 37
<210>25
<211>33
<212>DNA
<213> Artificial sequence
<400>25
gcaacactat ggttgctgaa ttgaccgcat tac 33
<210>26
<211>46
<212>DNA
<213> Artificial sequence
<400>26
tccagataga acatctcttc cttactggcg attgtcattc gcctga 46
<210>27
<211>37
<212>DNA
<213> Artificial sequence
<400>27
cgtcgcgggt aagaggttta ttatggttgc tgaattg 37
<210>28
<211>37
<212>DNA
<213> Artificial sequence
<400>28
ttcagcaacc atagtgttgc ctttttgtta tcaataa 37
Claims (8)
1. An engineering bacterium of colibacillus capable of producing tyrosine with high yield, which is characterized in thatThe Escherichia coli engineering bacteria knock out genes encoding aromatic amino acid transportersaroPOr a gene encoding a tyrosine-specific transportertyrPOn the basis of (a), a gene encoding 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (DS) is expressedaroG fbr And genes encoding chorismate mutase and prephenate dehydrogenase (CM-PD)tyrA fbr 。
2. The engineered Escherichia coli with high tyrosine yield of claim 1, wherein the engineered Escherichia coli with high tyrosine yield is obtained by culturingaroPThe nucleotide sequence of (A) is shown in SEQ ID NO. 1.
3. The engineered Escherichia coli having high tyrosine productivity according to claim 1 or 2, wherein the engineered Escherichia coli has a high tyrosine productivitytyrPThe nucleotide sequence of (A) is shown in SEQ ID NO. 2.
4. The engineered Escherichia coli having high tyrosine productivity according to claim 3, wherein the engineered Escherichia coli has a high tyrosine productivityaroG fbr The nucleotide sequence of (A) is shown in SEQ ID NO. 3.
5. The engineered Escherichia coli with high tyrosine yield of claim 4, wherein the engineered Escherichia coli with high tyrosine yield is obtained by culturingtyrA fbr The nucleotide sequence of (A) is shown in SEQ ID NO. 4.
6. A method for producing tyrosine, which is characterized in that the method comprises the step of inoculating the engineering bacterium of Escherichia coli capable of highly producing tyrosine according to any one of claims 1 to 5 into a fermentation culture medium for fermentation.
7. The method for producing tyrosine according to claim 6, wherein the fermentation is carried out by inoculating the engineered Escherichia coli strain capable of producing tyrosine with high yield according to any one of claims 1-5 into a fermentation medium and culturing to OD600After the induction at the temperature of 36-40 ℃ is carried out after the induction of 23-27 ℃.
8. Use of the engineered bacterium of Escherichia coli capable of highly producing tyrosine according to any one of claims 1 to 5 or the method according to claim 6 or 7 for preparing tyrosine.
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| WO2022082362A1 (en) * | 2020-10-19 | 2022-04-28 | 陈振暐 | Non-pathogenic bacterial gene expression system and transformant for metabolizing tyrosine, use thereof for preparing composition for reducing urinary toxins, and method for metabolizing tyrosine using same |
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Application publication date: 20190402 Assignee: ZHEJIANG LYUCHUANG BIOTECHNOLOGY Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2023980042310 Denomination of invention: An engineered Escherichia coli strain with high tyrosine production and its application Granted publication date: 20201103 License type: Common License Record date: 20230922 |
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Application publication date: 20190402 Assignee: SHAANXI MICROBE BIOTECHNOLOGY Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2023980052098 Denomination of invention: An engineered Escherichia coli strain with high tyrosine production and its application Granted publication date: 20201103 License type: Common License Record date: 20231214 |