CN105925520A - Recombinant escherichia coli efficiently transforming fumaric acid into L-asparagine as well as construction method and application thereof - Google Patents
Recombinant escherichia coli efficiently transforming fumaric acid into L-asparagine as well as construction method and application thereof Download PDFInfo
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- CN105925520A CN105925520A CN201610430229.8A CN201610430229A CN105925520A CN 105925520 A CN105925520 A CN 105925520A CN 201610430229 A CN201610430229 A CN 201610430229A CN 105925520 A CN105925520 A CN 105925520A
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- fumaric acid
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- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 title claims abstract description 65
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 36
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 title claims abstract description 33
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 239000001530 fumaric acid Substances 0.000 title claims abstract description 32
- 229960001230 asparagine Drugs 0.000 title claims abstract description 13
- 238000010276 construction Methods 0.000 title abstract description 5
- 230000001131 transforming effect Effects 0.000 title abstract 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims abstract description 25
- 229960005261 aspartic acid Drugs 0.000 claims abstract description 25
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 claims abstract description 24
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 22
- 230000001580 bacterial effect Effects 0.000 claims abstract description 14
- 230000009466 transformation Effects 0.000 claims abstract description 14
- 101100174518 Escherichia coli (strain K12) fumB gene Proteins 0.000 claims abstract description 13
- 108010036781 Fumarate Hydratase Proteins 0.000 claims abstract description 8
- 102100036160 Fumarate hydratase, mitochondrial Human genes 0.000 claims abstract description 8
- 101100120826 Methylorubrum extorquens (strain ATCC 14718 / DSM 1338 / JCM 2805 / NCIMB 9133 / AM1) fumC gene Proteins 0.000 claims abstract description 3
- 101150004244 fumA gene Proteins 0.000 claims abstract description 3
- 101150028420 fumC gene Proteins 0.000 claims abstract description 3
- 108090000790 Enzymes Proteins 0.000 claims description 33
- 238000006243 chemical reaction Methods 0.000 claims description 21
- 239000012634 fragment Substances 0.000 claims description 20
- 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 17
- 229950006334 apramycin Drugs 0.000 claims description 17
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 210000004027 cell Anatomy 0.000 claims description 14
- 238000005215 recombination Methods 0.000 claims description 13
- 230000006698 induction Effects 0.000 claims description 12
- 230000006798 recombination Effects 0.000 claims description 12
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 9
- 235000009582 asparagine Nutrition 0.000 claims description 9
- 101150085112 fumB gene Proteins 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000013612 plasmid Substances 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 108010046276 FLP recombinase Proteins 0.000 claims description 8
- 108010091086 Recombinases Proteins 0.000 claims description 8
- 102000018120 Recombinases Human genes 0.000 claims description 8
- 230000005611 electricity Effects 0.000 claims description 8
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 7
- 230000015572 biosynthetic process Effects 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 6
- 108091081024 Start codon Proteins 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 230000006801 homologous recombination Effects 0.000 claims description 6
- 238000002744 homologous recombination Methods 0.000 claims description 6
- 230000002779 inactivation Effects 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 210000002706 plastid Anatomy 0.000 claims description 6
- 108020005038 Terminator Codon Proteins 0.000 claims description 5
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims description 5
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 5
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 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 description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 4
- 238000003209 gene knockout Methods 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 210000004243 sweat Anatomy 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- -1 acyl ammonia Chemical compound 0.000 claims description 3
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 claims description 3
- 230000033228 biological regulation Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 241000432824 Asparagus densiflorus Species 0.000 claims description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 125000002252 acyl group Chemical group 0.000 claims description 2
- 150000001412 amines Chemical class 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 2
- QHFQAJHNDKBRBO-UHFFFAOYSA-L calcium chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Ca+2] QHFQAJHNDKBRBO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 210000002429 large intestine Anatomy 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 2
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 235000013619 trace mineral Nutrition 0.000 claims description 2
- 239000011573 trace mineral Substances 0.000 claims description 2
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 7
- 239000000047 product Substances 0.000 abstract description 4
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 abstract description 2
- 101100174521 Bradyrhizobium diazoefficiens (strain JCM 10833 / BCRC 13528 / IAM 13628 / NBRC 14792 / USDA 110) fumC2 gene Proteins 0.000 abstract description 2
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 abstract description 2
- 239000001630 malic acid Substances 0.000 abstract description 2
- 235000011090 malic acid Nutrition 0.000 abstract description 2
- 102100025573 1-alkyl-2-acetylglycerophosphocholine esterase Human genes 0.000 abstract 2
- 108010024976 Asparaginase Proteins 0.000 abstract 2
- 239000006227 byproduct Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 9
- 230000008859 change Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 101100492609 Talaromyces wortmannii astC gene Proteins 0.000 description 4
- 101150116772 aatA gene Proteins 0.000 description 4
- 101150005925 aspC gene Proteins 0.000 description 4
- 101150100742 dapL gene Proteins 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000012797 qualification Methods 0.000 description 3
- 230000035939 shock Effects 0.000 description 3
- 230000009182 swimming Effects 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 241001646716 Escherichia coli K-12 Species 0.000 description 2
- 102220561864 Polyphosphoinositide phosphatase_K236N_mutation Human genes 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 208000007848 Alcoholism Diseases 0.000 description 1
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 description 1
- BHELIUBJHYAEDK-OAIUPTLZSA-N Aspoxicillin Chemical compound C1([C@H](C(=O)N[C@@H]2C(N3[C@H](C(C)(C)S[C@@H]32)C(O)=O)=O)NC(=O)[C@H](N)CC(=O)NC)=CC=C(O)C=C1 BHELIUBJHYAEDK-OAIUPTLZSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 101100380328 Dictyostelium discoideum asns gene Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical class ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000623377 Terminalia elliptica Species 0.000 description 1
- 201000007930 alcohol dependence Diseases 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000005915 ammonolysis reaction Methods 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
- 101150062095 asnA gene Proteins 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 101150100366 end gene Proteins 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
- C12N9/82—Asparaginase (3.5.1.1)
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- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/20—Aspartic acid; Asparagine
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01001—Asparaginase (3.5.1.1)
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- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01002—Fumarate hydratase (4.2.1.2)
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- 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 a recombinant escherichia coli efficiently transforming fumaric acid into L-asparagine as well as a construction method and an application thereof. Fumarase encoding genes fumA, fumB, fumC in an ammonium-tolerant escherichia coli BEW308 are inactivated, and then L-aspartase encoding and L-asparaginase encoding genes are inserted into the positions of the fumarase encoding fumAC genes, thereby obtaining the recombinant escherichia coli having no malic acid by product, less L-aspartic acid accumulated, and a major product L-asparagine. The invention also discloses a construction method and an application of the bacterial strain. The recombinant escherichia coli can realize constitutive high-activity expression of L-aspartase and L-asparaginase, and ultimately achieve transformation of fumaric acid into L-asparagine.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a strain and convert fumaric acid synthesis altheine production
Bacterial strain and construction method thereof and application.
Background technology
Altheine is the hydroxyamide compounds of L-Aspartic acid, in terms of medicine, food, light industry and water environmental protection
Tool has been widely used, and in pharmaceuticals industry mainly as one of 20 kinds in amino acid transfusion, and it is dermopathic to have treatment
Effect.As long as in food service industry as nutritional supplement, anti-alcoholism agent, it is possible to derive further and make flavouring agent.At chemical industry
Industry mainly does the stabilizer of molysite and the film of ceramic surface.It is the important source material of membrane for water treatment at environmental protection industry (epi).
The at present production of altheine mainly with L-Aspartic acid for raw material through esterification ammonolysis, refined form, but its mistake
Journey product yield is on the low side, and relates to substantial amounts of organic solvent, pollutes big, and waste water is difficult.Bioanalysis has reaction gentleness, converts
The advantages such as rate is high, the three wastes are disposable, are the optimal modes of production of altheine, but L-Aspartic acid are converted into altheine
During relate to the participation of coenzyme, it is therefore desirable to solving the supply problem of coenzyme, the present invention constructs based on this to be had efficiently
The bacterial strain of Synthesis altheine, and establish its zymotechnique, production target has significant economy.
Summary of the invention
The technical problem to be solved in the present invention is to provide a strain and utilizes the restructuring large intestine of fumaric acid synthesis altheine
Bacillus.
The present invention also to solve the technical problem that be to provide above-mentioned can Efficient Conversion fumaric acid synthesis altheine
Recombination bacillus coli application in preparing altheine.
For solving above-mentioned technical problem, the present invention adopts the following technical scheme that
One strain Efficient Conversion fumaric acid is-recombination bacillus coli of asparagine, and by resistance to ammonium type Escherichia coli BEW308
Gene fumA, fumB, fumC inactivation of middle coding fumarase, then will coding L-Aspartic acid enzyme gene and coding L-asparagus fern acyl
Ammonia enzyme gene is inserted into the fumAC gene location of coding fumarase, and obtaining Efficient Conversion fumaric acid is the-weight of asparagine
Group Escherichia coli.Described resistance to ammonium type Escherichia coli BEW308, the preserving number of this bacterial strain is CCTCC NO:M2013157, this bacterium
The details of strain are disclosed in the Chinese patent of Application No. 201310279778.6.
Wherein, the gene order of described coding L-Aspartic acid enzyme as shown in SEQ ID NO:1, described coding L-asparagus fern acyl
The gene order of amine enzyme is as shown in SEQ ID NO:2.
Wherein, the upstream from start codon of L-Aspartic acid enzyme gene has a segment signal peptide, the gene order of this signal peptide
As shown in SEQ ID NO:3, the terminator codon of L-Aspartic acid enzyme gene is deleted, at the end of L-Aspartic acid enzyme gene
And having one section of catenation sequence between L-N enzyme gene, this catenation sequence is as shown in SEQ ID NO:4.
Above-mentioned Efficient Conversion fumaric acid is the preparation method of the recombination bacillus coli of asparagine, it is characterised in that bag
Include following steps:
(1) proceed to pKD46 plasmid, in resistance to ammonium type Escherichia coli BEW308, filter out positive transformant Escherichia coli
BEW308-pKD46, utilizes Arabinose to induce it to express λ recombinase, then Escherichia coli BEW308-pKD46 is prepared as sense
By state cell；
(2) with pIJ773 plasmid as template, the sequence shown in SEQ ID NO:5 and SEQ ID NO:6 is primer, and PCR expands
Increase and obtain fumB gene knockout fragment；
(3) fumB gene knockout fragment electricity conversion step (2) obtained is prepared as to Escherichia coli BEW308-pKD46
In competent cell, screen positive recombinant in general resistant panel pacifying；
(4) positive recombinant that step (3) obtains is prepared as competent cell, by pCP20 Plastid transformation wherein, 42 DEG C
Induction FLP recombinase is expressed, and carries out double choosing experiment in apramycin resistant panel and nonresistant flat board, flat in thing resistance
Grow on plate, but the bacterial strain BEW308-that bacterial strain is fumB gene inactivation that can not grow on the flat board having apramycin resistance
ΔfumB；
(5) synthesis homologous recombination fragment: increase signal peptide sequence SEQ ID before SEQ ID NO:1 initiation codon ATG
NO:3, deletes the terminator codon of L-Aspartic acid enzyme gene, and adds catenation sequence SEQ after L-Aspartic acid enzyme gene
ID NO:4, this fragment also includes apramycin resistance gene sequence, and with the addition of the homology arm of fumAC at the two ends of this fragment,
Particular sequence is as shown in SEQ ID NO:7；
(6) the bacterial strain BEW308-Δ fumB of fumB gene inactivation step (4) obtained is prepared as competent cell, will
PKD46 Plastid transformation wherein, utilizes Arabinose to induce it to express λ recombinase, then is prepared into competent cell；
(7) the homologous recombination fragment in step (5) is converted to the competent cell containing λ recombinase of step (6),
Positive recombinant is screened in general resistant panel pacifying；
(8) positive recombinant in step (7) being prepared as competent cell, by pCP20 Plastid transformation wherein, 42 DEG C lure
Lead FLP recombinase to express, apramycin resistant panel and nonresistant flat board are carried out double choose experiment, at nonresistant flat board
Upper growth, but the bacterial strain that can not grow on the flat board having apramycin resistance to be Efficient Conversion fumaric acid to be asparagus fern
The recombination bacillus coli BEW308 Δ fumB-△ fumAC-aspC2-30-asnA of acid amides.
Above-mentioned conversion fumaric acid is-application in producing altheine of the recombination bacillus coli of asparagine.
Wherein, its sweat is divided into induction period and transformation stage.
Wherein, induction period culture medium prescription is: fumaric acid 20g/L, dusty yeast 24g/L, peptone 10g/L, K2HPO4
36mmol/L, MgSO410mmol/L, trace element [CaCl2.6H2O 0.74g/L, ZnSO4.7H2O 0.18g/L,
MnSO4.H2O 20g/L, Na2-EDTA20.1g/L, CuSO40.1g/L, CoCl2 0.104g/L,FeSO4.7H2O2g/L]2mL/
L, solvent is water, and pH is 7.2～7.5.
Transformation stage fed-batch fermentation: induction period adds total glucose 40～60g/L, fumaric acid after terminating by several times
150～200g/L.
Wherein, induction period temperature is 28～30 DEG C, and pH is 7.2～7.8, and dissolved oxygen is 5～40%；Transformation stage temperature is
30～37 DEG C, pH is 7.2～7.8, and dissolved oxygen is 5～40%.
Wherein, glucose fed mode is that glucose is configured to the liquid storage of 600g/L, and according to fermented liquid integration two
Secondary add, amount to 40～60g/L；The additional way of fumaric acid is to be made into the fumaric acid suspension of 400g/L, and uses ammoniacal liquor
Regulation pH, to neutral, divides the fumaric acid adding a total of about 150～200g/L for 5 times.
Beneficial effect:
(1) present invention can realize the composing type high-activity expression of L-Aspartic acid enzyme and L-ASP, and fermentation training
The cell obtained after Yanging has low fumarase activity.
(2) by induction-conversion two benches sweat, its converted product is mainly altheine (rubbing of substrate
That conversion ratio is more than 97.5%), there is a small amount of L-Aspartic acid to accumulate, accumulate without malic acid.The method can be effectively improved substrate and prolong
The conversion yields of fumarate, reduces the production of accessory substance, effectively reduces production cost.
Accompanying drawing explanation
Fig. 1 knocks out the linear fragment PCR figure of fumB gene, and swimming lane M is marker, and swimming lane 1 is linear fragment.
Fig. 2 bacterium colony PCR identifies figure, and wherein BEW308 swimming lane is starting strain, and M is Marker, and 1～8 is the single bacterium identified
Fall.
Detailed description of the invention
According to following embodiment, the present invention be may be better understood.But, as it will be easily appreciated by one skilled in the art that reality
Execute the content described by example and be merely to illustrate the present invention, and should be also without limitation on basis described in detail in claims
Invention.
Embodiment 1:
This example demonstrates that and utilize homologous recombination technique to knock out fumarase in parent resistance to ammonium type Escherichia coli BEW308
FumB gene, the process of the apramycin resistant strain that is eliminated.
(1) utilize LB culture medium, in 37 DEG C, cultivate Escherichia coli BEW308 to OD under aerobic conditions600=0.4～0.6,
It is prepared as electricity and turns competence；
(2) pKD46 plasmid electricity is proceeded to competent Escherichia coli BEW308.Electric shock condition is: 200 Ω, 25 μ F, electric shock
Voltage 2.3kv, shocks by electricity the time 4～5ms.Rapidly thalline is added the SOC culture medium of precooling 1mL, 150r/min, 30 DEG C after electric shock
The LB culture medium flat plate coating band ampicillin (amp) after cultivating 1h filters out positive transformant BEW308 (pKD46)；
(3) adding the Arabinose of 10mM in LB culture medium, at 30 DEG C, inducing plasmid pKD46 gives expression to λ restructuring
Enzyme, makes electricity and turns competence；
(4) with both sides with the apramycin resistance gene in FRT site as template, utilize high-fidelity PCR amplification system, with
Plasmid pIJ773 is template, and designs the two ends amplimer with fumB homologous fragment, amplifies linear DNA homologous fragment,
Primer sequence is as follows:
Upstream belt homology arm primer H1-P1 (SEQ ID NO:5):
5’-CGGCACGCCATTTTCGAATAACAAATACAGAGTTACAGGCTGGAAGCTATTCCGGGGATCCGTCGACC-3’
Downstream belt homology arm primer H2-P2 (SEQ ID NO:6):
5’-TTACTTAGTGCAGTTCGCGCACTGTTTGTTGACGATTTGCTGGAAGAATGTAGGCTGGAGCTGCTTC-3’
Reaction system: each 0.5 μ l of upstream and downstream primer (100pmol/ μ l) of band homology arm；Template DNA (100ng/ μ l) 0.5
μl；10×buffer 5μl；The each 1 μ l of dNTPs (10mM)；DMSO (100%) 2.5 μ l；Pyrobest archaeal dna polymerase (2.5U/ μ
l)1μl；ddH2O 36/35.5μl；Cumulative volume 50 μ l.
Reaction condition: 94 DEG C, 2min；(94 DEG C, 45sec；50 DEG C, 45sec；72 DEG C, 90sec；10 circulations)；(94 DEG C,
45sec；55 DEG C, 45sec；72 DEG C, 90sec；15 circulations)；72 DEG C, 5min.
The qualification of linear DNA fragment such as Fig. 1.
(5) electricity turns linear DNA fragment to BEW308 (pKD46) competence of abduction delivering λ recombinase, and coats band
The LB flat screen of apramycin selects positive recombinant, and has carried out PCR qualification, and electrophoretogram is as shown in Figure 2.
(6) the plasmid pCP20 of energy abduction delivering FLP recombinase is poured after positive recombinant is prepared as competence into, in 42 DEG C
Heat shock can eliminate apramycin resistance after expressing FLP recombinase.Utilize pair of plates, carry out parallel point sample, it is possible in nonreactive
Property flat board on grow, but the bacterium that can not grow in resistant panel is the bacterial strain having knocked out resistance, named BEW308 (Δ
fumB)。
Embodiment 2: the present embodiment has investigated the L-Aspartic acid enzyme gene of sudden change Escherichia coli K12
(aspC) impact on this enzymatic activity after.Specific operation process is: based on original aspC, by 236 and 249 amino acids
Carry out suddenly change (Lys236Asn, Gly249Thr), by the way of Prof. Du Yucang, synthesize this gene, named aspC2.Will sudden change
Gene be connected to pTrc99a expression plasmid and import BEW308 (Δ fumB), build obtain BEW308 (Δ fumB, aspC2),
Think that the recombinant bacterium BEW308 (Δ fumB, aspC) of sudden change compares, result such as table 1:
Zymologic property situation of change before and after table 1 aspartic acid enzyme mutant
| Enzyme | Specific enzyme activity | Optimal reactive temperature | Optimal reaction pH |
| ASPC | 38090 | 37℃ | 8.2 |
| ASPC2 | 45500 | 37℃ | 7.8 |
Embodiment 3:
This example demonstrates that and utilize homologous recombination technique to knock out fumaric acid in Escherichia coli BEW308 (Δ fumB) further
Enzyme fumAC gene, and introduce high activity L-Aspartic acid enzyme and the L-ASP gene of sudden change.
Whole experimental implementation process is consistent with embodiment 1, and only homologous sequence is different.
(1) the present embodiment is with L-Aspartic acid enzyme gene (aspC) of Escherichia coli K12 for sequence of setting out,
236 and 249 amino acids carry out sudden change acquisition aspC2, i.e. Lys236Asn, Gly249Thr simultaneously, simultaneously at atg start codon
ATP upstream adds segment signal peptide sequence a: atgttgaatccgaaggttgcctacatggtctggatgacgtgcctgggtt
Taacgttgcccagccaggca (shown in SEQ ID NO:3), downstream deletes terminator codon；Described coding altheine
Enzyme gene (asnA), have between its initiation codon and L-Aspartic acid enzyme gene end gene the catenation sequence of one section of 30bp with
Guaranteeing that two enzymes have high activity simultaneously, this 30bp sequence is CAATCTGGACCGTGGCATCCTGGAAGCATT (SEQ ID
Shown in NO:4).
Finally increase homology arm and the apramycin resistance gene sequence of fumAC at two ends, whole fragment gene uses artificial conjunction
The mode become synthesizes, shown in concrete nucleotide sequence as SEQ ID NO:5.
(2) electricity turns the linear DNA fragment BEW308 (Δ fumB, pKD46) to abduction delivering λ recombinase of Prof. Du Yucang
Competence, and the LB flat screen coating band apramycin selects positive recombinant, and carried out PCR qualification, positive recombinant
The plasmid pCP20 of energy abduction delivering FLP recombinase is poured into, after FLP recombinase is expressed in 42 DEG C of heat shocks i.e. after being prepared as competence
Apramycin resistance can be eliminated.Utilize pair of plates, carry out parallel point sample, it is possible to grow on non-resistant flat board, but can not be
In resistant panel, the bacterium of growth is the bacterial strain having knocked out resistance, named BEW308 (Δ fumB-Δ fumAC-aspC2-
30-asnA)
Embodiment 4:
Starting strain Escherichia coli BEW308 and recombinant bacterium Escherichia has been investigated in the present embodiment contrast
Fumaric acid after coli BEW308 (Δ fumB-Δ fumAC-aspC2-30-asnA) Fiber differentiation 5h in the fermentation medium
Enzyme, Aspartase, the correction data of altheine enzymatic activity.
And the number of passes excessively of BEW308 (Δ fumB-Δ fumAC-aspC2-30-asnA) two-step method synthesis altheine
According to.
Concrete steps and result are as follows:
(1) use LB culture medium, access triangular flask from cryopreservation tube by 1～2% (v/v) inoculum concentration, aerobic cultivate 10～
12h, is seeded to shaking flask or seed fermentation tank (culture medium is also for LB), seed culture by 1～2% (v/v) inoculum concentration further
Process temperature controls at 30 DEG C, is not required to regulate pH in cultivation, and dissolved oxygen controls 5～40%, treats thalline OD after cultivating 4～6h600Extremely
Between 2.5～4, by 5～10% inducing culture of inoculation fermentation culture medium, sweat temperature controls, at 30 DEG C, to cultivate
Journey pH ammoniacal liquor controls 7.2～7.8, and dissolved oxygen controls 5～40%, cultivates 5h.
Table 2 Escherichia coli BEW308 (Δ fumB-Δ fumAC-aspC2-30-asnA) and starting strain enzyme
Live and compare
(2) add 30g/L glucose after induction and 150g/L fumaric acid (is mended in three times with ammoniacal liquor regulation to neutrality
Add), improve temperature and enter the transformation stage to 37 DEG C, collect the bacterium solution product analysis converting 12h, 16h, 20h and 24h.Experiment knot
Fruit is shown in Table 2.
Table 3 Escherichia coli BEW308 (Δ fumB-Δ fumAC-aspC2-30-asnA) zymotic fluid process
Claims (9)
1. a strain Efficient Conversion fumaric acid is the recombination bacillus coli of asparagine, it is characterised in that by the large intestine of resistance to ammonium type bar
Bacterium BEW308 encodes fumA, fumB, fumC gene inactivation of fumarase, then gene and the volume of L-Aspartic acid enzyme will be encoded
Code L-N enzyme gene be inserted into coding fumarase fumAC gene location, obtain Efficient Conversion fumaric acid for-
The recombination bacillus coli of asparagine.
Efficient Conversion fumaric acid the most according to claim 1 is the recombination bacillus coli of asparagine, it is characterised in that
The gene order of coding L-Aspartic acid enzyme, as shown in SEQ ID NO:1, encodes the gene order such as SEQ of L-ASP
Shown in ID NO:2.
Efficient Conversion fumaric acid the most according to claim 1 is the recombination bacillus coli of asparagine, it is characterised in that
The upstream from start codon of the gene of coding L-Aspartic acid enzyme has a segment signal peptide, the gene order of this signal peptide such as SEQ ID
Shown in NO:3, the terminator codon of L-Aspartic acid enzyme gene is deleted, at end and the L-asparagus fern of L-Aspartic acid enzyme gene
Having one section to insert catenation sequence between acyl ammonia enzyme gene, this catenation sequence is as shown in SEQ ID NO:4.
4. the preparation method of the recombination bacillus coli that Efficient Conversion fumaric acid is asparagine described in claim 1, it is special
Levy and be, comprise the steps:
(1) proceed to pKD46 plasmid, in resistance to ammonium type Escherichia coli BEW308, filter out positive transformant Escherichia coli BEW308-
PKD46, utilizes Arabinose to induce it to express λ recombinase, then it is thin that Escherichia coli BEW308-pKD46 is prepared as competence
Born of the same parents；
(2) with pIJ773 plasmid as template, the sequence shown in SEQ ID NO:5 and SEQ ID NO:6 is primer, and PCR expands
To fumB gene knockout fragment；
(3) fumB gene knockout fragment electricity step (2) obtained converts and is prepared as experiencing to Escherichia coli BEW308-pKD46
In state cell, screen positive recombinant in general resistant panel pacifying；
(4) positive recombinant that step (3) obtains is prepared as competent cell, by pCP20 Plastid transformation wherein, 42 DEG C of inductions
FLP recombinase is expressed, and carries out double choosing experiment, on the flat board of thing resistance in apramycin resistant panel and nonresistant flat board
Growth, but the bacterial strain BEW308-△ that bacterial strain is fumB gene inactivation that can not grow on the flat board having apramycin resistance
fumB；
(5) synthesis homologous recombination fragment: increase signal peptide sequence SEQ ID NO before SEQ ID NO:1 initiation codon ATG:
3, delete the terminator codon of L-Aspartic acid enzyme gene, and after L-Aspartic acid enzyme gene, add catenation sequence SEQ ID
NO:4, this fragment also includes apramycin resistance gene sequence, and with the addition of the homology arm of fumAC at the two ends of this fragment, tool
Body sequence is as shown in SEQ ID NO:7；
(6) the bacterial strain BEW308-△ fumB of fumB gene inactivation step (4) obtained is prepared as competent cell, by pKD46
Plastid transformation wherein, utilizes Arabinose to induce it to express λ recombinase, then is prepared into competent cell；
(7) the homologous recombination fragment in step (5) is converted to the competent cell containing λ recombinase of step (6), in peace
Positive recombinant is screened in general resistant panel；
(8) positive recombinant in step (7) is prepared as competent cell, by pCP20 Plastid transformation wherein, 42 DEG C of inductions
FLP recombinase is expressed, and carries out double choosing experiment, on nonresistant flat board in apramycin resistant panel and nonresistant flat board
Growth, but the bacterial strain that can not grow on the flat board having apramycin resistance to be Efficient Conversion fumaric acid to be asparagus fern acyl
The recombination bacillus coli BEW308 △ fumB-△ fumAC-aspC2-30-asnA of amine.
5. the fumaric acid that converts described in claim 1 is that the recombination bacillus coli of asparagine is in preparing altheine
Application.
Application the most according to claim 5, it is characterised in that its sweat is divided into induction period and transformation stage.
Application the most according to claim 5, it is characterised in that induction period culture medium prescription is: fumaric acid 20g/L,
Dusty yeast 24g/L, peptone 10g/L, K2HPO436mmol/L, MgSO410mmol/L, trace element [CaCl2.6H2O
0.74g/L, ZnSO4.7H2O 0.18g/L, MnSO4.H2O 20g/L, Na2-EDTA 20.1g/L, CuSO40.1g/L, CoCl2
0.104g/L,FeSO4.7H2O 2g/L] 2mL/L, solvent is water, and pH is 7.2～7.5.
Transformation stage fed-batch fermentation: induction period adds by several times total glucose 40～60g/L after terminating, fumaric acid 150～
200g/L。
Application the most according to claim 6, it is characterised in that induction period temperature is 28～30 DEG C, pH is 7.2～7.8,
Dissolved oxygen is 5～40%；Transformation stage temperature is 30～37 DEG C, and pH is 7.2～7.8, and dissolved oxygen is 5～40%.
Application the most according to claim 7, it is characterised in that glucose fed mode is for be configured to 600g/L by glucose
Liquid storage, and add at twice according to fermentating liquid volume, amount to 40～60g/L；The additional way of fumaric acid is for being made into 400g/
The fumaric acid suspension of L, and with ammoniacal liquor regulation pH to neutral, divide the fumaric acid adding a total of about 150～200g/L for 5 times.
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| CN110003039A (en) * | 2019-03-28 | 2019-07-12 | 新泰市佳禾生物科技有限公司 | The isolation and purification method of altheine in a kind of conversion fluid |
| CN110218691A (en) * | 2019-05-21 | 2019-09-10 | 南京工业大学 | One plant of genetic engineering bacterium for synthesizing altheine and its construction method and application |
| CN111088193A (en) * | 2020-01-10 | 2020-05-01 | 南京工业大学 | Method for improving electrotransformation frequency of methylotrophic butyric acid bacillus |
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| CN105154379A (en) * | 2015-07-27 | 2015-12-16 | 中国食品发酵工业研究院 | Engineering bacterium capable of efficiently converting fumaric acid to produce L-asparaginate, and application of engineering bacterium |
| CN105316273A (en) * | 2015-11-24 | 2016-02-10 | 南京工业大学 | L-aspartase recombinant escherichia coli free of malic acid byproducts and construction method and application of L-aspartase recombinant escherichia coli |
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| CN105154379A (en) * | 2015-07-27 | 2015-12-16 | 中国食品发酵工业研究院 | Engineering bacterium capable of efficiently converting fumaric acid to produce L-asparaginate, and application of engineering bacterium |
| CN105316273A (en) * | 2015-11-24 | 2016-02-10 | 南京工业大学 | L-aspartase recombinant escherichia coli free of malic acid byproducts and construction method and application of L-aspartase recombinant escherichia coli |
Cited By (3)
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| CN110003039A (en) * | 2019-03-28 | 2019-07-12 | 新泰市佳禾生物科技有限公司 | The isolation and purification method of altheine in a kind of conversion fluid |
| CN110218691A (en) * | 2019-05-21 | 2019-09-10 | 南京工业大学 | One plant of genetic engineering bacterium for synthesizing altheine and its construction method and application |
| CN111088193A (en) * | 2020-01-10 | 2020-05-01 | 南京工业大学 | Method for improving electrotransformation frequency of methylotrophic butyric acid bacillus |
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