CN106834128A - One plant of genetic engineering bacterium and its construction method and application that beta Alanine is produced using glucose fermentation - Google Patents
One plant of genetic engineering bacterium and its construction method and application that beta Alanine is produced using glucose fermentation Download PDFInfo
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- CN106834128A CN106834128A CN201710196096.7A CN201710196096A CN106834128A CN 106834128 A CN106834128 A CN 106834128A CN 201710196096 A CN201710196096 A CN 201710196096A CN 106834128 A CN106834128 A CN 106834128A
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- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 title claims abstract description 70
- 238000000855 fermentation Methods 0.000 title claims abstract description 44
- 230000004151 fermentation Effects 0.000 title claims abstract description 40
- 229940000635 beta-alanine Drugs 0.000 title claims abstract description 35
- 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 title claims abstract description 29
- 239000008103 glucose Substances 0.000 title claims abstract description 29
- 241000894006 Bacteria Species 0.000 title claims abstract description 24
- 238000010353 genetic engineering Methods 0.000 title claims abstract description 20
- 238000010276 construction Methods 0.000 title claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 239000013612 plasmid Substances 0.000 claims abstract description 20
- 241000588724 Escherichia coli Species 0.000 claims abstract description 18
- CKLJMWTZIZZHCS-UWTATZPHSA-N D-aspartic acid Chemical compound OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 claims abstract description 14
- 230000001580 bacterial effect Effects 0.000 claims abstract description 13
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229960005261 aspartic acid Drugs 0.000 claims abstract description 12
- 101150087106 pncB gene Proteins 0.000 claims abstract description 11
- 108700040046 Nicotinate phosphoribosyltransferases Proteins 0.000 claims abstract description 10
- 230000014509 gene expression Effects 0.000 claims abstract description 9
- 239000013613 expression plasmid Substances 0.000 claims abstract description 8
- 108020003285 Isocitrate lyase Proteins 0.000 claims abstract description 5
- 108020004687 Malate Synthase Proteins 0.000 claims abstract description 5
- 239000012634 fragment Substances 0.000 claims description 26
- 239000002773 nucleotide Substances 0.000 claims description 21
- 125000003729 nucleotide group Chemical group 0.000 claims description 21
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- XZNUGFQTQHRASN-XQENGBIVSA-N apramycin Chemical compound O([C@H]1O[C@@H]2[C@H](O)[C@@H]([C@H](O[C@H]2C[C@H]1N)O[C@@H]1[C@@H]([C@@H](O)[C@H](N)[C@@H](CO)O1)O)NC)[C@@H]1[C@@H](N)C[C@@H](N)[C@H](O)[C@H]1O XZNUGFQTQHRASN-XQENGBIVSA-N 0.000 claims description 11
- 229950006334 apramycin Drugs 0.000 claims description 11
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 9
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 8
- 238000012408 PCR amplification Methods 0.000 claims description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- 230000035939 shock Effects 0.000 claims description 5
- 108010046276 FLP recombinase Proteins 0.000 claims description 4
- 230000029087 digestion Effects 0.000 claims description 4
- 238000003209 gene knockout Methods 0.000 claims description 4
- 229960003512 nicotinic acid Drugs 0.000 claims description 4
- 235000001968 nicotinic acid Nutrition 0.000 claims description 4
- 239000011664 nicotinic acid Substances 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 claims description 3
- 108010091086 Recombinases Proteins 0.000 claims description 3
- 102000018120 Recombinases Human genes 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 239000001569 carbon dioxide Substances 0.000 claims description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 2
- 241000432767 Asparagus setaceus Species 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 2
- 229910021592 Copper(II) chloride Inorganic materials 0.000 claims description 2
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 2
- 239000007836 KH2PO4 Substances 0.000 claims description 2
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 2
- 229910000329 aluminium sulfate Inorganic materials 0.000 claims description 2
- 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 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 2
- 229910052564 epsomite Inorganic materials 0.000 claims description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- -1 phosphoribosyl Chemical group 0.000 claims description 2
- 239000011684 sodium molybdate Substances 0.000 claims description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 229910009112 xH2O Inorganic materials 0.000 claims description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 2
- 239000011686 zinc sulphate Substances 0.000 claims description 2
- 108090000992 Transferases Proteins 0.000 claims 1
- 238000010564 aerobic fermentation Methods 0.000 claims 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims 1
- 229960005091 chloramphenicol Drugs 0.000 claims 1
- 239000002994 raw material Substances 0.000 abstract description 4
- 108090000489 Carboxy-Lyases Proteins 0.000 abstract description 3
- 239000002028 Biomass Substances 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 abstract 2
- 102000004031 Carboxy-Lyases Human genes 0.000 abstract 2
- 230000000694 effects Effects 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 8
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 6
- 230000000692 anti-sense effect Effects 0.000 description 5
- 230000005611 electricity Effects 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 102000000780 Nicotinate phosphoribosyltransferase Human genes 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000001530 fumaric acid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 3
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000002425 crystallisation Methods 0.000 description 2
- 230000008025 crystallization Effects 0.000 description 2
- HHLFWLYXYJOTON-UHFFFAOYSA-N glyoxylic acid Chemical compound OC(=O)C=O HHLFWLYXYJOTON-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 108010011619 6-Phytase Proteins 0.000 description 1
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 1
- 241000432824 Asparagus densiflorus Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- 101001076781 Fructilactobacillus sanfranciscensis (strain ATCC 27651 / DSM 20451 / JCM 5668 / CCUG 30143 / KCTC 3205 / NCIMB 702811 / NRRL B-3934 / L-12) Ribose-5-phosphate isomerase A Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- AGSPXMVUFBBBMO-UHFFFAOYSA-N beta-aminopropionitrile Chemical compound NCCC#N AGSPXMVUFBBBMO-UHFFFAOYSA-N 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 101150036810 eco gene Proteins 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1077—Pentosyltransferases (2.4.2)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- 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
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
- C12Y203/03009—Malate synthase (2.3.3.9)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02011—Nicotinate phosphoribosyltransferase (2.4.2.11)
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- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01011—Aspartate 1-decarboxylase (4.1.1.11)
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- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/03—Oxo-acid-lyases (4.1.3)
- C12Y401/03001—Isocitrate lyase (4.1.3.1)
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- C12Y—ENZYMES
- C12Y403/00—Carbon-nitrogen lyases (4.3)
- C12Y403/01—Ammonia-lyases (4.3.1)
- C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
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Abstract
The invention discloses the genetic engineering bacterium that a plant is produced beta Alanine using glucose fermentation, knockout preserving number is CGMCC NO：The encoding gene aceBA of malate synthetase and isocitrate lyase, causes it to inactivate in 2301 bacterial strains, and the gene that L Aspartases and L aspartic acid α decarboxylases are separately encoded is inserted on the position of aceBA genes, obtains Escherichia coli AL12；Nicotinic acid phosphoribosyl transferase gene pncB is cloned on expression plasmid, recombinant plasmid is obtained, by recombinant plasmid transformed Escherichia coli AL12, that is, the genetic engineering bacterium AL13 that glucose fermentation produces beta Alanine is utilized.The present invention realizes the composing type high-activity expression of L Aspartases and L aspartic acid α decarboxylases, and uses renewable biomass resources glucose that the route of beta Alanine is prepared for fermenting raw materials completely, route green, environmental protection.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to one plant of gene that Beta-alanine is produced using glucose fermentation
Engineering bacteria and its construction method and application.
Background technology
Beta-alanine, also known as 3- alanines, white prismatic crystallization or the crystallization of Orthogonal Double centrum are unique in nature
The β type amino acid of presence.The physiological function of Beta-alanine is mainly the intermediate product of metabolism, using extremely wide.Medical discovery,
In mammalian nervous system, it can be used as the neurotransmission person in brain.Can be used as synthetic pantothenic acid and auxiliary in terms of medicine
The important precursor of enzyme.In terms of chemical industry, the catalyst that can be chemically reacted as some.Beta-alanine has a wide range of applications
Field and good market prospects.
The production of current Beta-alanine mainly uses chemical synthesis and enzyme transforming process.Chemical synthesis is mainly logical
Cross acrylonitrile method, BAPN method and succinimide say that solution synthesizes Beta-alanine, these method raw materials, intermediate and
Accessory substance is poisonous, and pollution environment is extremely serious.The country has also been studied enzyme transforming process at present, is with L-Aspartic acid mainly
Raw material, is synthesized using biological enzyme, and L-Aspartic acid is prepared by fumaric acid at present, and fumaric acid mainly uses chemical method
Prepare, therefore from complete period analysis, the preparation process of Beta-alanine is complicated, the cycle long still relies on fossil resource.And glucose
From reproducible biomass resource, its abundance, screening or structure obtain one plant and can directly utilize glucose fermentation
The production bacterial strain for preparing Beta-alanine has great importance, and not yet has relevant report at present.
The content of the invention
The technical problem to be solved in the present invention is to provide one plant of genetic engineering that Beta-alanine is produced using glucose fermentation
Bacterium.
The technical problem also to be solved of the invention is to provide the construction method of said gene engineering bacteria.
The technical problem finally to be solved of the invention is to provide the application of said gene engineering bacteria.
In order to solve the above technical problems, the present invention is adopted the following technical scheme that：
One plant of genetic engineering bacterium that Beta-alanine is produced using glucose fermentation, knockout preserving number is CGMCC NO：2301 bacterium
The encoding gene aceBA of malate synthetase and isocitrate lyase, causes it to inactivate in strain, and by L-Aspartic acid enzyme and L-
The gene that aspartic acid-α-decarboxylase is separately encoded is inserted on the position of aceBA genes, obtains Escherichia coli AL12, institute
The GenBank registration numbers for stating the encoding gene aceBA of malate synthetase and isocitrate lyase are EU889415.1；
Nicotinic acid phosphoribosyl transferase gene pncB is cloned on expression plasmid, recombinant plasmid is obtained, by the restructuring matter
Grain conversion Escherichia coli AL12, that is, be utilized the genetic engineering bacterium AL13 that glucose fermentation produces Beta-alanine.Described large intestine
Bacillus CGMCC NO：2301 is one plant of genetic engineering bacterium of high-yield fumaric acid, and the specifying information of the bacterial strain is in Application No.
Disclosed in 200810019216.7 patent.
Nicotinic acid phosphoribosyl transferase is the rate-limiting enzyme of NAD (H) synthesis system, and overexpression pncB can improve NAD (H)
Total amount NADH/NAD suitable with maintenance+, and promote cells use glucose growth metabolism.By knock out malate synthetase and
The encoding gene aceBA of isocitrate lyase, can block glyoxalic acid circulation branch road path, and strengthen TCA circulating paths.By
The coexpression of L-Aspartic acid enzyme and L-Aspartic acid-α-decarboxylase so that thalline directly can produce β-the third using glucose fermentation
Propylhomoserin.
Wherein, the nucleotide sequence such as SEQ ID NO of the L-Aspartic acid enzyme gene AspC：Shown in 1, L-Aspartic acid
The GenBank registration numbers of enzyme gene are X03629.1.
Wherein, the nucleotide sequence of L-Aspartic acid-α-decarboxylase gene panD such as SEQ ID NO：Shown in 2, L- asparagus ferns
The GenBank registration numbers of propylhomoserin-α-decarboxylase gene are NC_003450.3.
Wherein, the nucleotide sequence of nicotinic acid phosphoribosyl transferase gene pncB such as SEQ ID NO：Shown in 3, nicotinic acid turns phosphorus
The EcoGene registration numbers of sour ribokinase gene pncB are EG10742.
Wherein, described expression plasmid is pTrc99a.
Above-mentioned utilization glucose fermentation produces the construction method of the genetic engineering bacterium of Beta-alanine, comprises the following steps：
(1) with SEQ ID NO：4 and SEQ ID NO：Nucleotides sequence shown in 5 is classified as primer, and plasmid pIJ773 is template,
PCR amplifications obtain linear fragment 1；
With SEQ ID NO：6 and SEQ ID NO：Nucleotides sequence shown in 7 is classified as primer, SEQ ID NO：Core shown in 1
Nucleotide sequence is template, and PCR amplifications obtain linear fragment 2；
With SEQ ID NO：8 and SEQ ID NO：Nucleotides sequence shown in 9 is classified as primer, SEQ ID NO：Core shown in 2
Nucleotide sequence is template, and PCR amplifications obtain linear fragment 3；
With SEQ ID NO：4 and SEQ ID NO：Nucleotides sequence shown in 9 is classified as primer, linear fragment 1, linear fragment 2
With linear fragment 3 for template amplification obtains gene knockout fragment；
(2) by pKD46 plasmids conversion CGMCC NO：2301 bacterial strains, its expression λ recombinase is induced using L-arabinose,
The bacterial strain is prepared into competence again；
(3) in the competence for obtaining gene knockout fragment step of converting (2) in step (1), it is coated with the flat of apramycin
Screen selects positive recombinant；
(4) pCP20 is transformed into the positive recombinant that step (3) is obtained, 42 DEG C of heat shocks make its expression FLP recombinase,
Flat board using non-resistant flat board and containing apramycin resistance carry out it is double choose, can be grown on non-resistant flat board, but can not
The bacterial strain grown in apramycin resistant panel is Escherichia coli AL12；
(5) by SEQ ID NO：The nucleotide sequence of pncB genes shown in 3 be cloned into pTrc99a plasmids Nco I and
Hind III digestion sites, obtain pTrc99a-pncB recombinant plasmids, and recombinant plasmid transformed Escherichia coli AL12 is obtained final product
To the genetic engineering bacterium Escherichia coli AL13 that Beta-alanine is produced using glucose fermentation.
Above-mentioned utilization glucose fermentation produces genetic engineering bacterium the applying in fermentation prepares Beta-alanine of Beta-alanine
Within protection scope of the present invention.
Wherein, seed liquor incubation is as follows：
(S1) it is transferred in LB culture mediums from cryopreservation tube for 1~2% by volume fraction, 10~12h of aerobic culture；
(S2) it is 1~2% to be transferred in the LB culture mediums of seed fermentation tank by volume fraction；
(S3) thalline OD is treated600It is by volume 5~10% inoculation fermentation culture mediums, the fermented and cultured during to 2.5~4
The formula of base is：JSP culture mediums, nicotinic acid 0.1mM；Citric acid 3.0g/L；Na2HPO4·7H2O 3.00g/L；KH2PO4 8.00g/
L；(NH4)2HPO420.00g/L；NH4Cl 10g/L；(NH4)2SO45g/L；MgSO4·7H2O 1.00g/L；CaCl2·2H2O
10.0mg/L；ZnSO4·7H2O 0.5mg/L；CuCl2·2H2O 0.25mg/L；MnSO4·H2O 2.5mg/L；CoCl2·6H2O
1.75mg/L；H3BO30.12mg/L；Al2(SO4)3·xH2O 1.77mg/L；Na2MoO4·2H2O 0.5mg/L；Fe(III)
Citrate 16.1mg/L, solvent is water, and it is 8.0 to adjust pH with ammoniacal liquor after sterilizing, and wherein glucose is divided into 3 times after individually sterilizing
Add.
In step (S1) and (S2), cultivation temperature is 35~37 DEG C.
In step (S3), using two benches fermentation pattern, as thalline OD600For less than 20 when, logical oxygen carries out aerobic hair
Ferment, dissolved oxygen is 5~40%；As thalline OD600Changing logical carbon dioxide during to more than 20 carries out anaerobic fermentation.
Wherein, temperature is 30~32 DEG C in two benches fermentation process, and incubation pH ammoniacal liquor is adjusted to 7.8~8.1.
Beneficial effect：
The present invention innovatively instead of method of original Beta-alanine using enzymatic conversion, solve production Beta-alanine week
Phase problem long, there is provided a kind of breeding objective clearly, efficiently, the strong engineered strain of synthesis Beta-alanine ability and use completely
Glucose prepares the route of Beta-alanine for fermenting raw materials, route green, environmental protection.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, without should also without limitation on sheet described in detail in claims
Invention.
Embodiment 1：
This example demonstrates that using aceBA in overlapping pcr and homologous recombination technique knockout parental E. coli JM125
Gene (SEQ ID NO：3) L-Aspartic acid enzyme gene AspC (SEQ ID NO are inserted while：And L-Aspartic acid-α 1)-de-
Decarboxylase gene panD (SEQ ID NO：, and the apramycin resistant strain process that is eliminated 2).
(1) LB culture mediums are utilized, in culture Escherichia coli JM125 to OD under 37 DEG C, aerobic conditions600=0.4~0.6, system
It is standby to turn competence into electricity；
(2) recombinant plasmid electricity is transferred to the Escherichia coli CGMCC NO of competence：2301.Electric shock condition is：200 Ω, 25 μ
F, shock voltage 2.3kv, shock by electricity 4~5ms of the time.The rapid SOC culture mediums by thalline addition precooling 1mL after electric shock, 150r/
The LB culture medium flat plates with ampicillin (Amp) are coated after min, 30 DEG C of culture 1h filter out positive transformant CGMCC
NO：2301(pKD46)；
(3) L-arabinose of 10mM is added in LB culture mediums, λ restructuring is given expression in inducing plasmid pKD46 at 30 DEG C
Enzyme, is made electricity and turns competence；
(4) with both sides with the apramycin resistance gene (pIJ773) in FRT sites, L-Aspartic acid enzyme gene
(GenBank：X03629.1) and L-Aspartic acid-α-decarboxylase gene is template design primer F1, R1, F2, R2 and F3, R3,
Particular sequence is：
F1(SEQ ID NO：4)：
CCTTCGTTCACAGTGGGGAAGTTTTCGGATCCATGACGAGGAGCTGCACGTGTAGGCTGGAGCTGCTTC
GAAG
R1(SEQ ID NO：5)：ATTCCGGGGATCCGTCGACTACAAACTCTTGTAATGGCGGCGF2(SEQ ID
NO：6)：TAAGGCCCCTAGGCAGCTGATGTTTGAGAACATTACCGCCGCR2(SEQ ID NO：7)：
TGCGGCGTGAACGCCTTATCCGGCCTACAGTCAGCAACGGTTGTTGTTGCCGGGCTTCATTGTTTTTAA
TGCTTACAGCA
F3(SEQ ID NO：8)：ATGGGTCGCGGATCCGAATTCATGCTGCGCACCATCCTC
R3(SEQ ID NO：9)：
CTCGAGTGCGGCCGCAAGCTTCTAAATGCTTCTCGACGTCAAAAGC
(5) with F1, R1, F2, R2 and F3, R3 amplify apramycin resistance gene (linear fragment 1), L- asparagus fern ammonia respectively
Phytase gene (linear fragment 2) and L-Aspartic acid-α-decarboxylase gene, then carried as primer amplifies two ends with F1 and R3
The DNA of aceBA DNA homolog arms knocks out fragment；
(6) electricity turns the Escherichia coli CGMCCNO of artificial synthesized linear DNA fragment to induced expression λ recombinases：2301
(pKD46) competence, and coat the LB flat screens with apramycin and select positive recombinant, and carried out PCR identifications；
(7) positive recombinant be prepared into after competence pour into can induced expression FLP recombinases plasmid pCP20, in 42 DEG C
Apramycin resistance can be eliminated after heat shock expression FLP recombinases.Using a pair of plates, parallel point sample is carried out, can be in nonreactive
Grown on mild-natured plate, but the bacterium colony that can not be grown in resistant panel has as knocked out the bacterial strain of resistance, is named as AL12 (△
aceBA-aspC-30-panD)。
Embodiment 2
This example demonstrates that building the expression plasmid of overexpression nicotinic acid phosphoribosyl transferase, bacterial strain is improved in anaerobism bar
Coenzyme NAD under part+Consumption and regeneration rate, maintain co-factor balance, obtain the process of strains A L13.
1st, the expression plasmid of overexpression nicotinic acid phosphoribosyl transferase is built, its process includes：
(1) primer of engineer and synthesis with Nco I and Hind III digestions site：
Sense primer (SEQ ID NO：10)：5’-CGCCATGGATGACACAATTCGCTTCTCCTG-3’
Anti-sense primer (SEQ ID NO：11)：5’-CCCAAGCTTCACTTGTCCACCCGTAAATGG-3’
(2) with e. coli k12 as template, PCR amplification, reaction condition be 94 DEG C, 45 seconds, 54 DEG C, 45 seconds, 72 DEG C,
1.2min, totally 30 circulations.After the pncB genes that purifying is amplified, expression plasmid pTrc99a uses Nco I and Hind III respectively
Double digestion, connection obtain recombinant plasmid pTrc99a-pncB.
The 2nd, plasmid pTrc99a-pncB is imported in embodiment 1 AL12 (the △ aceBA- for eliminating apramycin resistant strain
AspC-30-panD) competence.The positive transformant of acquisition is new structure strains A L13 of the invention.
Embodiment 3
This example demonstrates that new the recombination bacillus coli AL12, AL13 for building and starting strain CGMCC NO：2301 fermentations
Produce the contrast of Beta-alanine ability.
1st, accessed in triangular flask from cryopreservation tube by 1~2% (v/v) inoculum concentration using LB culture mediums, aerobic culture 10~
12h, is further seeded to seed fermentation tank (culture medium is also LB) by 1~2% (v/v) inoculum concentration, and thalline is treated after 4~6h of culture
OD600To between 2.5~4, by 5~10% inoculation fermentation culture mediums (JSP culture mediums, glucose is added in batches for carbon source)；
2nd, seed culture process temperature control is not required to adjust pH at 35~37 DEG C, in culture, and dissolved oxygen is controlled 5~40%.
Fermentation process uses two benches fermentation pattern, as thalline OD600Changing logical carbon dioxide during to 20 or so carries out anaerobic fermentation,
At 30~32 DEG C, incubation pH is controlled 7.8~8.1 fermentation process temperature control with ammoniacal liquor.
1 is the results are shown in Table after three anaerobic fermentation 48h of bacterial strain.
The starting strain of table 1 and two plants of recombinant bacterium fermentation and acid situations
SEQUENCE LISTING
<110>Nanjing University of Technology
<120>One plant of genetic engineering bacterium and its construction method and application that Beta-alanine is produced using glucose fermentation
<130> 20151116002
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 1191
<212> DNA
<213>The nucleotide sequence of L-Aspartic acid enzyme gene AspC
<400> 1
atgtttgaga acattaccgc cgctcctgcc gacccgattc tgggcctggc cgatctgttt 60
cgtgccgatg aacgtcccgg caaaattaac ctcgggattg gtgtctataa agatgagacg 120
ggcaaaaccc cggtactgac cagcgtgaaa aaggctgaac agtatctgct cgaaaatgaa 180
accaccaaaa attacctcgg cattgacggc atccctgaat ttggtcgctg cactcaggaa 240
ctgctgtttg gtaaaggtag cgccctgatc aatgacaaac gtgctcgcac ggcacagact 300
ccggggggca ctggcgcact acgcgtggct gccgatttcc tggcaaaaaa taccagcgtt 360
aagcgtgtgt gggtgagcaa cccaagctgg ccgaaccata agagcgtctt taactctgca 420
ggtctggaag ttcgtgaata cgcttattat gatgcggaaa atcacactct tgacttcgat 480
gcactgatta acagcctgaa tgaagctcag gctggcgacg tagtgctgtt ccatggctgc 540
tgccataacc caaccggtat cgaccctacg ctggaacaat ggcaaacact ggcacaactc 600
tccgttgaga aaggctggtt accgctgttt gacttcgctt accagggttt tgcccgtggt 660
ctggaagaag atgctgaagg actgcgcgct ttcgcggcta tgcataaaga gctgattgtt 720
gccagttcct actctaaaaa ctttggcctg tacaacgagc gtgttggcgc ttgtactctg 780
gttgctgccg acagtgaaac cgttgatcgc gcattcagcc aaatgaaagc ggcgattcgc 840
gctaactact ctaacccacc agcacacggc gcttctgttg ttgccaccat cctgagcaac 900
gatgcgttac gtgcgatttg ggaacaagag ctgactgata tgcgccagcg tattcagcgt 960
atgcgtcagt tgttcgtcaa tacgctgcag gaaaaaggcg caaaccgcga cttcagcttt 1020
atcatcaaac agaacggcat gttctccttc agtggcctga caaaagaaca agtgctgcgt 1080
ctgcgcgaag agtttggcgt atatgcggtt gcttctggtc gcgtaaatgt ggccgggatg 1140
acaccagata acatggctcc gctgtgcgaa gcgattgtgg cagtgctgta a 1191
<210> 2
<211> 411
<212> DNA
<213>The nucleotide sequence of L-Aspartic acid-α-decarboxylase gene panD
<400> 2
atgctgcgcaccatcctcggaagtaagattcaccgagccactgtcactcaagctgatcta 60
gattatgttggctctgtaaccatcgacgccgacctggttcacgccgccggattgatcgaa 120
ggcgaaaaagttgccatcgtagacatcaccaacggcgctcgtctggaaacttatgtcatt 180
gtgggcgacgccggaacgggcaatatttgcatcaatggtgccgctgcacaccttattaat 240
cctggcgatcttgtgatcatcatgagctaccttcaggcaactgatgcggaagccaaggcg 300
tatgagccaaagattgtgcacgtggacgccgacaaccgcatcgttgcgctcggcaacgat 360
cttgcggaagcactacctggatccgggcttttgacgtcgagaagcatttag 411
<210> 3
<211> 1203
<212> DNA
<213>The nucleotide sequence of nicotinic acid phosphoribosyl transferase gene pncB
<400> 3
atgacacaat tcgcttctcc tgttctgcac tcgttgctgg atacagatgc ttataagttg 60
catatgcagc aagccgtgtt tcatcactat tacgatgtgc atgtcgcggc ggagtttcgt 120
tgccgaggtg acgatctgct gggtatttat gccgatgcta ttcgtgaaca ggttcaggcg 180
atgcagcacc tgcgcctgca ggatgatgaa tatcagtggc tttctgccct gcctttcttt 240
aaggccgact atcttaactg gttacgcgag ttccgcttta acccggaaca agtcaccgtg 300
tccaacgata atggcaagct ggatattcgt ttaagcggcc cgtggcgtga agtcatcctc 360
tgggaagttc ctttgctggc ggttatcagt gaaatggtac atcgctatcg ctcaccgcag 420
gccgacgttg cgcaagccct cgacacgctg gaaagcaaat tagtcgactt ctcggcgtta 480
accgccggtc ttgatatgtc gcgcttccat ctgatggatt ttggcacccg tcgccgtttt 540
tctcgcgaag tacaagaaac catcgttaag cgtctgcaac aggaatcctg gtttgtgggc 600
accagcaact acgatctggc gcgtcggctt tccctcacgc cgatgggaac acaggcacac 660
gaatggttcc aggcacatca gcaaatcagc ccggatctag ccaacagcca gcgagctgca 720
cttgctgcct ggctggaaga gtatcccgac caacttggca ttgcattaac cgactgcatc 780
actatggatg ctttcctgcg tgatttcggt gtcgagttcg ctagtcggta tcagggcctg 840
cgtcatgact ctggcgaccc ggttgaatgg ggtgaaaaag ccattgcaca ttatgaaaag 900
ctgggaattg atccacagag taaaacgctg gttttctctg acaatctgga tttacgcaaa 960
gcggttgagc tataccgcca cttctcttcc cgcgtgcaat taagttttgg tattgggact 1020
cgcctgacct gcgatatccc ccaggtaaaa cccctgaata ttgtcattaa gttggtagag 1080
tgtaacggta aaccggtggc gaaactttct gacagccctg gcaaaactat ctgccatgat 1140
aaagcgtttg ttcgggcgct gcgcaaagcg ttcgaccttc cgcatattaa aaaagccagt 1200
taa 1203
<210> 4
<211> 73
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of linear fragment 1
<400> 4
ccttcgttca cagtggggaa gttttcggat ccatgacgag gagctgcacg tgtaggctgg 60
agctgcttcg aag 73
<210> 5
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of linear fragment 1
<400> 5
attccgggga tccgtcgact acaaactctt gtaatggcgg cg 42
<210> 6
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of linear fragment 2
<400> 6
taaggcccct aggcagctga tgtttgagaa cattaccgcc gc 42
<210> 7
<211> 80
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of linear fragment 2
<400> 7
tgcggcgtga acgccttatc cggcctacag tcagcaacgg ttgttgttgc cgggcttcat 60
tgtttttaat gcttacagca 80
<210> 8
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of linear fragment 3
<400> 8
atgggtcgcg gatccgaatt catgctgcgc accatcctc 39
<210> 9
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of linear fragment 3
<400> 9
Ctcgcgtgcg gccgcaagct tctaaatgct tctcgacgtc aaaagc 46
<210> 10
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the sense primer of nicotinic acid phosphoribosyl transferase
<400> 10
cgccatggat gacacaattc gcttctcctg 30
<210> 11
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223>Expand the anti-sense primer of nicotinic acid phosphoribosyl transferase gene
<400> 11
cccaagcttc acttgtccac ccgtaaatgg 30
Claims (10)
1. one plant utilizes the genetic engineering bacterium that glucose fermentation produces Beta-alanine, it is characterised in that knockout preserving number is CGMCC
NO：The encoding gene aceBA of malate synthetase and isocitrate lyase, causes it to inactivate in 2301 bacterial strains, and by L- asparagus ferns
The gene of the coding of the Gene A spC and L-Aspartic acid-α-decarboxylase PanD of propylhomoserin enzyme coding is inserted into aceBA genes jointly
Position on, obtain Escherichia coli AL12；
Nicotinic acid phosphoribosyl transferase gene pncB is cloned on expression plasmid, recombinant plasmid is obtained, the recombinant plasmid is turned
Change Escherichia coli AL12, that is, be utilized the genetic engineering bacterium AL13 that glucose fermentation produces Beta-alanine.
2. utilization glucose fermentation according to claim 1 produces the genetic engineering bacterium of Beta-alanine, it is characterised in that described
Encode the gene order such as SEQ ID NO of L-Aspartic acid enzyme：Shown in 1, the L-Aspartic acid-α-decarboxylase gene sequence of coding
Such as SEQ ID NO：Shown in 2.
3. utilization glucose fermentation according to claim 1 produces the genetic engineering bacterium of Beta-alanine, it is characterised in that nicotinic acid
The nucleotide sequence of phosphoribosyl transferase gene pncB such as SEQ ID NO：Shown in 3.
4. utilization glucose fermentation according to claim 1 produces the genetic engineering bacterium of Beta-alanine, it is characterised in that described
Expression plasmid be pTrc99a.
5. the utilization glucose fermentation described in claim 1 produces the construction method of the genetic engineering bacterium of Beta-alanine, and its feature exists
In comprising the following steps：
(1) with SEQ ID NO：4 and SEQ ID NO：Nucleotides sequence shown in 5 is classified as primer, and plasmid pIJ773 is template, PCR
Amplification obtains linear fragment 1；
With SEQ ID NO：6 and SEQ ID NO：Nucleotides sequence shown in 7 is classified as primer, SEQ ID NO：Nucleotides shown in 1
Sequence is template, and PCR amplifications obtain linear fragment 2；
With SEQ ID NO：8 and SEQ ID NO：Nucleotides sequence shown in 9 is classified as primer, SEQ ID NO：Nucleotides shown in 2
Sequence is template, and PCR amplifications obtain linear fragment 3；
With SEQ ID NO：4 and SEQ ID NO：Nucleotides sequence shown in 9 is classified as primer, linear fragment 1, linear fragment 2 and line
Property fragment 3 obtains gene knockout fragment for template amplification；
(2) by pKD46 plasmids conversion CGMCC NO：2301 bacterial strains, its expression λ recombinase is induced using L-arabinose, then will
The bacterial strain is prepared into competence；
(3) in the competence for obtaining gene knockout fragment step of converting (2) in step (1), it is coated with the flat screen of apramycin
Select positive recombinant；
(4) pCP20 is transformed into the positive recombinant that step (3) is obtained, 42 DEG C of heat shocks make its expression FLP recombinase, utilize
Non-resistant flat board and the flat board containing apramycin resistance carry out it is double choose, can grow on non-resistant flat board, but can not pacify
The bacterial strain grown on general chloramphenicol resistance flat board is Escherichia coli AL12；
(5) by SEQ ID NO：The nucleotide sequence of pncB genes shown in 3 is cloned into the Nco I and Hind of pTrc99a plasmids
III digestion site, obtains pTrc99a-pncB recombinant plasmids, by recombinant plasmid transformed Escherichia coli AL12, that is, obtains profit
The genetic engineering bacterium Escherichia coli AL13 of Beta-alanine is produced with glucose fermentation.
6. the utilization glucose fermentation described in claim 1 produces the genetic engineering bacterium of Beta-alanine in fermentation prepares Beta-alanine
Application.
7. application according to claim 6, it is characterised in that seed liquor incubation is as follows：
(S1) it is transferred in LB culture mediums from cryopreservation tube for 1~2% by volume fraction, 10~12h of aerobic culture；
(S2) it is 1~2% to be transferred in the LB culture mediums of seed fermentation tank by volume fraction；
(S3) thalline OD is treated600It is by volume 5~10% inoculation fermentation culture mediums during to 2.5~4, the fermentation medium
It is formulated and is：JSP culture mediums, nicotinic acid 0.1mM；Citric acid 3.0g/L；Na2HPO4·7H2O 3.00g/L；KH2PO48.00g/L；
(NH4)2HPO420.00g/L；NH4Cl 10g/L；(NH4)2SO45g/L；MgSO4·7H2O 1.00g/L；CaCl2·2H2O
10.0mg/L；ZnSO4·7H2O 0.5mg/L；CuCl2·2H2O 0.25mg/L；MnSO4·H2O 2.5mg/L；CoCl2·6H2O
1.75mg/L；H3BO30.12mg/L；Al2(SO4)3·xH2O 1.77mg/L；Na2MoO4·2H2O 0.5mg/L；Fe(III)
Citrate 16.1mg/L, solvent is water, and it is 8.0 to adjust pH with ammoniacal liquor after sterilizing, and wherein glucose is divided into 3 times after individually sterilizing
Add.
8. application according to claim 7, it is characterised in that in seed liquor incubation, in step (S1) and (S2), training
It is 35~37 DEG C to support temperature.
9. application according to claim 7, it is characterised in that two benches fermentation pattern is used in step (S3), works as thalline
OD600For less than 20 when, logical oxygen carries out aerobic fermentation, and dissolved oxygen is 5%~40%；As thalline OD600Change logical during to more than 20
Carbon dioxide carries out anaerobic fermentation.
10. application according to claim 9, it is characterised in that temperature is 30~32 DEG C in two benches fermentation process, culture
Process pH ammoniacal liquor is adjusted to 7.8~8.1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111411130A (en) * | 2020-03-04 | 2020-07-14 | 南京凯诺生物科技有限公司 | Method for producing β -alanine by mixed fermentation |
| CN112458032A (en) * | 2019-09-06 | 2021-03-09 | 南京盛德生物科技研究院有限公司 | Construction and application of escherichia coli recombinant bacteria for synthesizing glycine by using glucose |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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