CN114395518A - Recombinant escherichia coli and construction method and application thereof - Google Patents
Recombinant escherichia coli and construction method and application thereof Download PDFInfo
- Publication number
- CN114395518A CN114395518A CN202111485206.4A CN202111485206A CN114395518A CN 114395518 A CN114395518 A CN 114395518A CN 202111485206 A CN202111485206 A CN 202111485206A CN 114395518 A CN114395518 A CN 114395518A
- Authority
- CN
- China
- Prior art keywords
- escherichia coli
- gene
- whib
- cti
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 48
- 238000010276 construction Methods 0.000 title claims description 7
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 claims abstract description 46
- 238000000855 fermentation Methods 0.000 claims abstract description 34
- 230000004151 fermentation Effects 0.000 claims abstract description 33
- 239000001384 succinic acid Substances 0.000 claims abstract description 22
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 230000005764 inhibitory process Effects 0.000 claims abstract description 12
- 230000035882 stress Effects 0.000 claims abstract description 11
- 239000000758 substrate Substances 0.000 claims abstract description 10
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 claims abstract description 9
- 235000021122 unsaturated fatty acids Nutrition 0.000 claims abstract description 9
- 150000004670 unsaturated fatty acids Chemical class 0.000 claims abstract description 9
- 108090000175 Cis-trans-isomerases Proteins 0.000 claims abstract description 8
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 6
- 230000036542 oxidative stress Effects 0.000 claims abstract description 5
- 230000001105 regulatory effect Effects 0.000 claims abstract description 5
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 5
- 241000589516 Pseudomonas Species 0.000 claims abstract 4
- 241000186216 Corynebacterium Species 0.000 claims abstract 2
- 239000013612 plasmid Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 12
- 238000004321 preservation Methods 0.000 claims description 10
- 238000010367 cloning Methods 0.000 claims description 9
- 239000012634 fragment Substances 0.000 claims description 9
- 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 8
- 239000008103 glucose Substances 0.000 claims description 8
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 6
- 238000012163 sequencing technique Methods 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 230000001590 oxidative effect Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 229940074404 sodium succinate Drugs 0.000 claims description 3
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims 1
- 238000012262 fermentative production Methods 0.000 claims 1
- 230000007017 scission Effects 0.000 claims 1
- 210000000170 cell membrane Anatomy 0.000 abstract description 5
- 238000009825 accumulation Methods 0.000 abstract description 4
- 238000012986 modification Methods 0.000 abstract description 4
- 230000004048 modification Effects 0.000 abstract description 4
- 238000011218 seed culture Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 11
- 101100102960 Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145) whiB gene Proteins 0.000 description 10
- 238000012360 testing method Methods 0.000 description 8
- 238000011081 inoculation Methods 0.000 description 7
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 6
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 102000003813 Cis-trans-isomerases Human genes 0.000 description 4
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000010170 biological method Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 2
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 2
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 2
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 2
- 229910004619 Na2MoO4 Inorganic materials 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 229960003237 betaine Drugs 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 description 2
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000011780 sodium chloride Substances 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
- 159000000000 sodium salts Chemical class 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 239000011592 zinc chloride Substances 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- ZMKVBUOZONDYBW-UHFFFAOYSA-N 1,6-dioxecane-2,5-dione Chemical compound O=C1CCC(=O)OCCCCO1 ZMKVBUOZONDYBW-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229910017677 NH4H2 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
- 230000001580 bacterial effect Effects 0.000 description 1
- 229920000229 biodegradable polyester Polymers 0.000 description 1
- 239000004622 biodegradable polyester Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 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
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 101150070536 whiB gene Proteins 0.000 description 1
Images
Classifications
-
- 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
- C12N9/90—Isomerases (5.)
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a recombinant escherichia coli, which improves the stress resistance and product synthesis performance of escherichia coli based on cell membrane modification, and particularly introduces and expresses a gene of an oxidative stress regulating enzyme from corynebacterium glutamicumwhiB(NCBI ID: AKL 15-01645) and unsaturated fatty acid cis-trans isomerase gene derived from Pseudomonas aeruginosacti(NCBI ID:CWI20_17990)To be implemented. The recombinant strain can tolerate the inhibition of high-concentration substrates and products under the anaerobic condition, and can improve the growth amount of the strain and the accumulation of succinic acid under the anaerobic condition of a 5L fermentation tank.
Description
Technical Field
The invention belongs to the technical field of bioengineering and fermentation thereof, and relates to recombinant escherichia coli, a construction method and application thereof.
Background
Succinic acid, also known as succinic acid, is a common natural organic acid that is widely found in humans, animals, plants and microorganisms. Succinic acid is not only an intermediate product of the TCA cycle, but also one of the terminal reduction products of anaerobic metabolism, and has a very important role in the biological metabolic process. The succinic acid and derivatives thereof are widely applied, can be used as a C4 platform compound to synthesize 1, 4-butanediol, tetrahydrofuran and poly (butylene succinate) (PBS) biodegradable polyesters, and are widely applied to various industries such as food, medicine, printing and the like. In recent years, with the increasing conflict between fossil resource utilization and environmental protection, the adoption of a biological method for preparing succinic acid meets the environment-friendly production regulations of green, renewable and the like, and has a wide prospect.
Compared with the defects of complex process, harsh conditions, dependence on petroleum and the like of the traditional chemical synthesis method, the biological method has the advantages of low raw material price, small pollution and the like. However, the fermentation strain is one of the key points of succinic acid biosynthesis, and only a part of strains can produce high-concentration succinic acid. And the biosynthesis method needs to continuously optimize the synthesis path and modify engineering strains so as to achieve the purposes of industrial high yield and high efficiency. The Escherichia coli has clear genetic background, simple gene modification, high growth speed and simple and cheap culture medium, and becomes a research hotspot for preparing succinic acid by a biological method in recent years.
Due to the continuous accumulation of products during the fermentation process of the strains, alkali is added to adjust the pH value to maintain the optimal growth of the strains. However, in the later stage of fermentation, the growth of the strain is still greatly inhibited, and the inhibition brought by high-concentration substrates and high-concentration products cannot be completely relieved by the regulation of alkali liquor. Therefore, the improvement of the stress resistance of the strain becomes a new breakthrough.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems in the prior art, the invention provides a recombinant escherichia coli and application thereof.
In order to achieve the above object, the present invention provides a recombinant Escherichia coli into which genes whiB (NCBI ID: AKL 15-01645) expressing an oxidative stress-regulating enzyme derived from Corynebacterium glutamicum and cti (NCBI ID: CWI 20-17990) expressing an unsaturated fatty acid cis-trans isomerase derived from Pseudomonas aeruginosa are introduced, wherein the sequence number of the gene WhiB is NCBI ID: AKL 15-01645; the sequence number of the gene cti is NCBI ID: CWI20_ 17990; the host bacteria have a preservation number of CCTCC NO: escherichia coli (Escherichia coli) BER208 of M2012351. The preservation date of the Escherichia coli BER208 is 9/14 days 2012, the preservation unit is totally called China center for type culture Collection and the address is Wuhan, Wuhan university, and the details thereof are disclosed in patent CN 102864113A.
The invention further provides a construction method of the recombinant escherichia coli, which constructs a gene whiB for expressing the oxidative stress regulating enzyme from corynebacterium glutamicum and a gene cti for expressing the unsaturated fatty acid cis-trans isomerase from pseudomonas aeruginosa into a recombinant escherichia coli with a preservation number of CCTCCNO: m2012351, in Escherichia coli (Escherichia coli) BER 208.
Specifically, the construction method comprises the following steps:
(1) obtaining target gene fragments whiB and cti;
(2) cloning a target gene fragment to a starting plasmid by using a one-step cloning method to obtain a recombinant plasmid;
(3) and (3) converting the positive cloning plasmid with correct sequencing into a host bacterium Escherichia coli (Escherichia coli) BER208, and screening by Amp resistance to obtain the succinic acid-producing recombinant Escherichia coli with improved anti-reverse performance.
Preferably, the starting plasmid is the pTrc99A plasmid.
Specifically, in the step (2), primers carrying enzyme cutting sites Hind III and homologous arms of a vector pTrc99A are designed and inserted into linearized gene fragments whiB and ct to obtain a cloning plasmid pTrc 99A-whiB-cti.
The invention further provides application of the recombinant escherichia coli in preparation of succinic acid.
Furthermore, the invention provides the inhibition effect of the recombinant escherichia coli on the high-concentration substrate and high-concentration product in the succinic acid produced by the fermentation method.
The recombinant escherichia coli can be used for producing succinic acid by fermentation in a fermentation system in which 90g/L of substrate is put into glucose or 30g/L of sodium succinate in a product and 60g/L of glucose are used as carbon sources. The continuous accumulation of succinic acid in the later fermentation period causes the adverse environment of hypertonic and high-yield product inhibition, influences the growth and production of the strain, relieves the inhibition by improving the metabolic flux, improves the strength of cell membranes by improving the proportion of unsaturated fatty acid of the cell membranes, improves the tolerance performance of the strain and ensures the growth.
The invention further provides a method for improving the stress resistance of escherichia coli and the synthesis performance of products, which is characterized in that a gene whiB for expressing oxidative stress regulating enzyme from corynebacterium glutamicum and a gene cti for expressing unsaturated fatty acid cis-trans isomerase from pseudomonas aeruginosa are introduced into the escherichia coli, wherein the sequence number of the gene whiB is NCBI ID: AKL15_ 01645; the sequence number of gene cti is NCBI ID: CWI 20-17990.
Preferably, the preservation number of the escherichia coli is CCTCC NO: escherichia coli (Escherichia coli) BER208 of M2012351.
Has the advantages that: compared with the prior art, the pTrc99A plasmid is introduced into host bacteria, and the oxidative stress regulating enzyme whiB from corynebacterium glutamicum and the unsaturated fatty acid cis-trans isomerase cti from pseudomonas aeruginosa are expressed to carry out cell membrane modification, so that the recombinant microorganism can resist the inhibition of high-concentration substrates and products under anaerobic conditions. The strain can improve the growth amount of the strain and the accumulation of succinic acid under the anaerobic condition of a 5L fermentation tank. In the process of adjusting pH by using sodium salt and carrying out anaerobic fermentation for 180 hours, the final succinic acid yield of the modified strain is 78.05g/L, and compared with the original strain, the succinic acid yield is improved by 41.91%, so that the modified strain has great economic value.
Drawings
FIG. 1 shows the results of colony PCR verification;
FIG. 2 shows the stress resistance results of the strains under the inhibition of a high concentration of substrate;
FIG. 3 shows the strain stress resistance results under the inhibition of high concentration products;
FIG. 4 shows the results of the increase in the productivity of the fermenter strains.
Detailed Description
The present invention will be described in further detail with reference to specific examples, which will help understanding the present invention, but the scope of the present invention is not limited to the following examples.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1: and (3) constructing recombinant Escherichia coli.
The plasmid pTrc99A-whiB-cti was constructed by a synthetic biological method and introduced into the target strain.
(1) Designing upstream and downstream primers, connecting the whiB and cti gene fragments with the digested linear pTrc99A plasmid by a multi-fragment one-step cloning method, and transforming the fragments into E.coli DH5 alpha. After plasmid cutting and colony PCR verification, the correctly verified plasmid is sent to a sequencing company for sequencing. The sequences of the upstream and downstream primers are shown in Table 1. The restriction site is Hind III. The PCR conditions are shown in Table 2. The results of colony PCR validation are shown in FIG. 1. For preparation of competent cells, gene extraction, and extraction of plasmids such as pTrc99A, please refer to the third edition of molecular cloning Experimental guidelines.
TABLE 1
TABLE 2
Temperature of | Time |
95℃ | 5min |
95℃ | 15s |
56℃ | 15s |
72℃ | 2min30s |
72℃ | 10min |
16℃ | 1h (for temporary preservation only) |
(2) Mixing the recombinant plasmid with correct sequencing with the competent cells of a target strain (Escherichia coli BER208 with the preservation number of CCTCC NO: M2012351), and carrying out ice bath for 30 min; then putting the mixture into a 42 ℃ water bath kettle, performing heat shock for 90s, and performing ice bath for 2 min; adding 900 mu L of sterile LB culture medium, mixing uniformly, and putting in a shaking table for resuscitation for 1 h; finally, 150. mu.L of the bacterial solution was pipetted and spread on LB solid medium containing ampicillin in a sterile operating table. The plate was inverted and placed in a 37 ℃ incubator. To screen out positive single colonies from hundreds of single clones on the plate, sequencing was performed.
Example 2: and constructing stress resistance fermentation experiments of the strains in adverse environment.
(1) And (3) test tube seed culture: inoculating the genetically engineered bacteria into a seed culture medium of a test tube for culture. The culture temperature is 37 ℃, the rotation speed is 200rpm, and the culture time is 12 h.
Wherein the test tube seed culture medium comprises the following components: 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride. Amp 100. mu.g/ml was added at the time of inoculation.
(2) And (3) seed culture in a shaking flask: inoculating the test tube seed culture solution into a seed culture medium of a shake flask for culture, wherein the liquid loading of the shake flask is 50 mL. When inoculating, 100 mug/ml Amp and 30g/L glucose are added, and the inoculation amount is 2%. The culture temperature is 37 ℃, the rotation speed is 200r/min, and the culture time is 18-24 h.
Wherein the formula of the shake flask culture medium is as follows: betaine 0.12g/L, KH2PO4 3.5g/L,K2HPO4·3H2O 6.54g/L,(NH4)2HPO4 3.5g/L,MgSO4·7H2O 0.25g/L,CaCl2·2H2O15 mg/L, and adding trace element FeCl3·6H2O 1.6mg/L,CoCl2·2H2O 0.2mg/L,CuCl2·2H2O 0.1mg/L,ZnCl2·4H2O 0.2mg/L,Na2MoO4·2H2O 0.2mg/L,H3BO3 0.05mg/L。
(3) Reverse-environment fermentation: inoculating the shake flask seed culture solution into an anaerobic flask in a fermentation culture medium for fermentation culture under the conditions of high-concentration substrate and high-concentration product. When in inoculation, 100 mu g/mL Amp, 0.5mmol/L IPTG and 10% of the inoculation amount of an anaerobic bottle are added, the liquid loading amount of a 100mL anaerobic shaking bottle is 30mL, basic magnesium carbonate is added as a pH regulator, and carbon dioxide is added for 3 min; the culture temperature is 37 ℃, 200r/min, and the fermentation time is 48 h.
Wherein the anaerobic bottle culture medium comprises the following components in parts by weight: betaine 0.12g/L, (NH)4)2HPO4 2.6g/L,NH4H2PO40.87g/L,KCl 0.15g/L,MgSO4·7H2O0.37 g/L, and trace element FeCl3·6H2O 2.4mg/L,CoCl2·2H2O 0.3mg/L,CuCl2·2H2O 0.15mg/L,ZnCl2·4H2O 0.5mg/L,Na2MoO4·2H2O 0.5mg/L,MnCl2·4H2O0.5 mg/L and H3BO3 0.075g/L。
The fermentation result under the high substrate concentration inhibition is shown in figure 3, compared with a control strain G1, the OD growth amount of the strain G2 is obviously improved, the succinic acid yield is improved to some extent, the expression of whiB successfully improves the glucose utilization rate of the strain, the high sugar inhibition is relieved, and the expression of cti resists the hypertonic environment caused by high sugar, the growth of the strain is ensured, and the stress resistance of the strain is improved.
The fermentation results under the inhibition of high product concentration are shown in fig. 4, and compared with a control strain G1, the OD (optical density) growth amount of the strain G2 is obviously improved under the stress of adding 30G/L sodium succinate, and the succinic acid yield is obviously improved, so that the stress resistance of the strain is obviously improved by modifying cell membranes.
Example 3: fermentation experiments and yield improvement of the constructed strain under a 5L fermentation tank.
(1) And (3) test tube seed culture: inoculating the genetically engineered bacteria into a seed culture medium of a test tube for culture. The culture temperature is 37 ℃, the rotation speed is 200rpm, and the culture time is 12 h.
Wherein the test tube seed culture medium comprises the following components: 10g/L of peptone, 5g/L of yeast powder and 5g/L of sodium chloride. Amp 100. mu.g/ml was added at the time of inoculation.
(2) And (3) seed culture in a shaking flask: inoculating the test tube seed culture solution into a seed culture medium of a shake flask for culture, wherein the liquid loading of the shake flask is 50 mL. When inoculating, 100 mug/ml Amp and 30g/L glucose are added, and the inoculation amount is 2%. The culture temperature is 37 ℃, the rotation speed is 200r/min, and the culture time is 18-24 h.
(3) Fermentation in a fermentation tank: inoculating the shake flask seed culture solution into a 5L fermentation tank filled with 2L fermentation medium for fermentation culture. The inoculum size was 10%, Amp 100. mu.g/ml and IPTG 0.5mmol/L were added at the time of inoculation, and glucose was added so that the initial sugar concentration was 50 g/L. And continuously introducing sterile carbon dioxide gas to maintain the anaerobic environment of fermentation. The fermentation temperature is maintained at 37 ℃, and the stirring speed is 200 r/min. The pH value of the fermentation is kept to be 6.75-6.80 by using mixed sodium salt, sampling is carried out once every 12h, and the fermentation time is 180 h.
The fermentation result of the fermentation tank is shown in figure 4, compared with a control strain G1, the total sugar consumption of the strain G2 is obviously improved, the succinic acid yield is improved to 78.05G/L, and the fermentation tank has an industrial prospect.
The present invention provides a new idea and method for recombinant escherichia coli, and a number of methods and ways for implementing the technical solution, and the above description is only a preferred embodiment of the present invention, it should be noted that, for those skilled in the art, many modifications and embellishments can be made without departing from the principle of the present invention, and these modifications and embellishments should also be regarded as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.
Sequence listing
<110> Nanjing university of industry
<120> recombinant escherichia coli and construction method and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 58
<212> DNA
<213> upstream primer of gene whiB (Artificial Sequence)
<400> 1
gtcgacctgc aggcatgcaa gcttggatgg aattcatgac gtctgtgatt ccagagca 58
<210> 2
<211> 42
<212> DNA
<213> downstream primer of whiB Gene (Artificial Sequence)
<400> 2
ccaaaacagc caagctttca gaggttctcg tagcgattca tg 42
<210> 3
<211> 47
<212> DNA
<213> upstream primer of Gene cti (Artificial Sequence)
<400> 3
ccattaacgt ggtcagcttt ttgaatgttg ccaagaccgt tggttgg 47
<210> 4
<211> 42
<212> DNA
<213> downstream primer of Gene cti (Artificial Sequence)
<400> 4
ccaaaacagc caagctttca gaggttctcg tagcgattca tg 42
Claims (10)
1. A recombinant Escherichia coli into which a gene expressing an oxidative stress-regulating enzyme derived from Corynebacterium glutamicum has been introducedwhiBAnd unsaturated fatty acid cis-trans isomerase gene derived from Pseudomonas aeruginosactiWherein, the genewhiBThe sequence number of (1) is NCBI ID: AKL15_ 01645; genectiCWI20_ 17990; the host bacteria have a preservation number of CCTCC NO: escherichia coli (M2012351) (II)Escherichia coli)BER208。
2. The method for constructing recombinant Escherichia coli according to claim 1, wherein the expression of a gene encoding an oxidative stress-regulating enzyme derived from Corynebacterium glutamicum is usedwhiBAnd unsaturated fatty acid cis-trans isomerase gene derived from Pseudomonas aeruginosactiConstructing the culture medium with a preservation number of CCTCC NO: escherichia coli (M2012351) (II)Escherichia coli) BER 208.
3. The construction method according to claim 2, characterized by comprising the steps of:
(1) target gene fragmentwhiBAndctiobtaining;
(2) cloning a target gene fragment to a starting plasmid by using a one-step cloning method to obtain a recombinant plasmid;
(3) transforming the positive cloning plasmid with correct sequencing into the host bacterium Escherichia coli (Escherichia coli) In BER208, the succinic acid-producing recombinant Escherichia coli with improved stress resistance is obtained by Amp resistance screening.
4. The method according to claim 3, wherein the starting plasmid is pTrc99A plasmid.
5. The method of claim 3, wherein in step (2), primers carrying HindIII cleavage sites and homology arms of pTrc99A vector are designed and inserted into the linearized gene fragmentwhiB、ctiTo obtain the cloning plasmid pTrc99A-whiB-cti。
6. Use of the recombinant E.coli of claim 1 or claim 5 for the preparation of succinic acid.
7. The use of claim 6, wherein the recombinant E.coli reduces inhibition of high substrate and high product concentrations in the fermentative production of succinic acid.
8. The use of claim 7, wherein the recombinant Escherichia coli can be used for producing succinic acid by fermentation in a fermentation system with 90g/L glucose as substrate or 30g/L sodium succinate and 60g/L glucose as carbon source.
9. A method for improving the stress resistance of Escherichia coli and the synthesis performance of products, which is characterized in that the method comprises expressing a gene of an oxidative stress regulating enzyme from Corynebacterium glutamicumwhiBAnd unsaturated fatty acid cis-trans isomerase gene derived from Pseudomonas aeruginosactiIntroduced into Escherichia coli, wherein the genewhiBThe sequence number of (1) is NCBI ID: AKL15_ 01645; genectiCWI20_ 17990.
10. The method of claim 9, wherein the E.coli has a preservation number of CCTCC NO: escherichia coli (M2012351) (II)Escherichia coli)BER208。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111485206.4A CN114395518B (en) | 2021-12-07 | 2021-12-07 | Recombinant escherichia coli as well as construction method and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111485206.4A CN114395518B (en) | 2021-12-07 | 2021-12-07 | Recombinant escherichia coli as well as construction method and application thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114395518A true CN114395518A (en) | 2022-04-26 |
CN114395518B CN114395518B (en) | 2024-08-27 |
Family
ID=81225191
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111485206.4A Active CN114395518B (en) | 2021-12-07 | 2021-12-07 | Recombinant escherichia coli as well as construction method and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114395518B (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102864113A (en) * | 2012-10-16 | 2013-01-09 | 南京工业大学 | Bacterial strain for producing succinic acid, method for producing succinic acid by using bacterial strain and application of bacterial strain |
-
2021
- 2021-12-07 CN CN202111485206.4A patent/CN114395518B/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102864113A (en) * | 2012-10-16 | 2013-01-09 | 南京工业大学 | Bacterial strain for producing succinic acid, method for producing succinic acid by using bacterial strain and application of bacterial strain |
Non-Patent Citations (2)
Title |
---|
JUNG HO AHN等: "Membrane engineering via trans-unsaturated fatty acids production improves succinic acid production in Mannheimia succiniciproducens" * |
SOON-CHUN CHUNG等: "Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum" * |
Also Published As
Publication number | Publication date |
---|---|
CN114395518B (en) | 2024-08-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN106434510B (en) | One plant of fermentation produces the genetic engineering bacterium of L-Aspartic acid | |
CN105296411B (en) | Genetically engineered bacterium for producing L-aspartic acid by monosaccharide fermentation and construction method and application thereof | |
CN107815446B (en) | A kind of fermentation process in high density of recombination nitrile hydratase Recombinant organism | |
CN107022515B (en) | Genetically engineered bacterium for producing L-aspartic acid by utilizing anaerobic fermentation of lignocellulose hydrolysate and construction method and application thereof | |
CN112226398B (en) | Recombinant escherichia coli for efficiently producing glutaric acid and construction method thereof | |
CN104232722A (en) | Method for producing 9-alpha-hydroxyandrostenedione by microbial fermentation | |
CN102533626A (en) | Genetic engineering strain for producing succinic acid by using glucose and fermentation acid production method thereof | |
CN105051181B (en) | The preparation method of the increased recombinant microorganism of the generative capacity of 2,3-butanediol and the 2,3-butanediol using it | |
CN102154339A (en) | Construction method of gene engineering strain for producing succinic acid escherichia coli | |
Hu et al. | Efficient production of d-1, 2, 4-butanetriol from d-xylose by engineered Escherichia coli whole-cell biocatalysts | |
CN109370971A (en) | Genetically engineered bacterium for producing L-aspartic acid by fermentation and construction method and application thereof | |
WO2018014488A1 (en) | Escherichia coli engineering bacteria and method for catalyzed synthesis of fumaric acid from maleic acid by using same | |
CN107227286B (en) | Genetically engineered bacterium capable of producing succinic acid at high yield, and construction method and application thereof | |
CN106834128A (en) | Genetically engineered bacterium for producing beta-alanine by glucose fermentation and construction method and application thereof | |
CN105925520B (en) | Recombinant escherichia coli capable of efficiently converting fumaric acid into L-asparagine as well as construction method and application thereof | |
CN116925987A (en) | High-yield stress-resistant strain strengthened by teichoic acid synthesis, construction method thereof and polyol fermentation application | |
CN108004275B (en) | Escherichia coli recombinant bacterium for producing adipic acid and application thereof | |
CN114395518A (en) | Recombinant escherichia coli and construction method and application thereof | |
CN116083332A (en) | Construction and application of recombinant escherichia coli producing adipic acid | |
CN114806991A (en) | Engineering escherichia coli for improving yield of fucosyllactose and production method | |
CN108728389A (en) | One plant of colibacillus engineering and its application for producing 2,3,5,6- Tetramethylpyrazines | |
KR100556099B1 (en) | Variant of Rumen Bacteria and Process for Preparing Succinic Acid Employing the Same | |
CN118165907B (en) | Gamma-aminobutyric acid production strain and construction method and application thereof | |
CN118006652B (en) | Dopamine production strain, construction method and application thereof | |
CN103436477B (en) | Escherichia coli strain for producing succinic acid with glycerol as well as construction method and use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |