CN106635944A - Glutamic acid corynebacterium and construction method and application thereof - Google Patents
Glutamic acid corynebacterium and construction method and application thereof Download PDFInfo
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- CN106635944A CN106635944A CN201611249268.4A CN201611249268A CN106635944A CN 106635944 A CN106635944 A CN 106635944A CN 201611249268 A CN201611249268 A CN 201611249268A CN 106635944 A CN106635944 A CN 106635944A
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- lysine
- corynebacterium glutamicum
- pyc
- lyse
- glutamic acid
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- 238000010276 construction Methods 0.000 title claims abstract description 13
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 title claims abstract 7
- 235000013922 glutamic acid Nutrition 0.000 title claims abstract 7
- 239000004220 glutamic acid Substances 0.000 title claims abstract 7
- 241000186216 Corynebacterium Species 0.000 title abstract 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims abstract description 36
- 239000004472 Lysine Substances 0.000 claims abstract description 26
- 238000004519 manufacturing process Methods 0.000 claims abstract description 21
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 108010053763 Pyruvate Carboxylase Proteins 0.000 claims abstract description 15
- 102100039895 Pyruvate carboxylase, mitochondrial Human genes 0.000 claims abstract description 15
- 230000000694 effects Effects 0.000 claims abstract description 12
- 235000019766 L-Lysine Nutrition 0.000 claims abstract description 10
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 38
- 230000001580 bacterial effect Effects 0.000 claims description 20
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 16
- 235000018977 lysine Nutrition 0.000 claims description 16
- 101150096049 pyc gene Proteins 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 6
- 230000003834 intracellular effect Effects 0.000 claims description 6
- 230000014509 gene expression Effects 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 3
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 235000019764 Soybean Meal Nutrition 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 229910052564 epsomite Inorganic materials 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000000413 hydrolysate Substances 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 239000004455 soybean meal Substances 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 238000009825 accumulation Methods 0.000 abstract description 8
- 244000005700 microbiome Species 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000002474 experimental method Methods 0.000 abstract description 3
- 239000002243 precursor Substances 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 239000001963 growth medium Substances 0.000 abstract description 2
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 abstract 3
- 102000011781 Karyopherins Human genes 0.000 abstract 2
- 108010062228 Karyopherins Proteins 0.000 abstract 2
- 239000012634 fragment Substances 0.000 description 20
- 238000000034 method Methods 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 12
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- 230000035772 mutation Effects 0.000 description 8
- 230000029087 digestion Effects 0.000 description 6
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- 102000004190 Enzymes Human genes 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 description 3
- 102000012410 DNA Ligases Human genes 0.000 description 3
- 108010061982 DNA Ligases Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
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- 235000013305 food Nutrition 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000013207 serial dilution Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 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 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 1
- 239000005996 Blood meal Substances 0.000 description 1
- 241000186146 Brevibacterium Species 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 101100309436 Streptococcus mutans serotype c (strain ATCC 700610 / UA159) ftf gene Proteins 0.000 description 1
- 241000319304 [Brevibacterium] flavum Species 0.000 description 1
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 1
- 235000019730 animal feed additive Nutrition 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 101150025220 sacB gene Proteins 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 238000012549 training Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
-
- 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
- C12N9/93—Ligases (6)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
- C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
- C12Y604/01001—Pyruvate carboxylase (6.4.1.1)
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Engineering & Computer Science (AREA)
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- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
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- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the field of microorganisms, and particularly relates to a glutamic acid corynebacterium and a construction method and application of the glutamic acid corynebacterium. The glutamic acid corynebacterium has the capability of producing L-lysine, and the activities of pyruvic carboxylase exportin and pyruvic carboxylase in a cell are both strengthened. The glutamic acid corynebacterium strengthens the activity of the pyruvic carboxylase exportin, so that the accumulation of the L-lysine is promoted, the yield of the L-lysine is increased, the activity of pyruvic carboxylase is strengthened at the same time, more precursors are provided for the synthesis of the L-lysine, the accumulation capability of the L-lysine is further improved, the amount of the L-lysine in a culture medium is increased, and the capability of production of the L-lysine through the fermentation of the strain is further improved. The experiment shows that the glutamic acid corynebacterium is a strain with high yield of L-lysine, the L-lysine is effectively accumulated, the yield of the L-lysine is improved, and a foundation is laid for the industrialized production of the L-lysine.
Description
Technical field
The invention belongs to microorganism field, and in particular to a kind of Corynebacterium glutamicum and its construction method and application, especially
It is Corynebacterium glutamicum and its construction method and the application for being related to a kind of product 1B
Background technology
1B (L-Lysine), scientific name is L-2, and 6- diaminocaproic acids, molecular formula is C6H14N2O2.HCL, molecular weight
For 182.65.1B is one of important component of protein, is that human body itself can not synthesize but highly desirable eight kinds
One of amino acid, can improve the utilization rate of protein, so as to the nutrition of condensed food significantly, play enhancing development, increase
Plus appetite, reduction disease, constitutional effect.
1B is second largest amino acids production kind in the world, is widely used in animal feed, medicine and food work
Industry.Wherein about 90% is used for feed industry, and 10% is used for food and medicine industry.For animal feed additive, 1B
Body can be helped to absorb other amino acid, so as to improve feeding quality.
1B is initially isolated from protein hydrolysate, and protein Hydrolyze method is typically with animal blood meal
Raw material, is that technological process is simple the characteristics of this method is most, but raw material sources are very limited, are suitable only for small-scale production.Hereafter again
Occur in that chemical synthesis, Hydrolyze method, enzyme process.Gilvarg is equal to and first reported one plant of Enterobacter the fifties in last century
Representative strain colon bacillus biosynthesis lysine by way of.Nineteen fifty-seven, Japan started to produce paddy using wild strain fermentation method
Propylhomoserin, nineteen sixty Japan this under wish youth etc. with ultraviolet irradiation Corynebacterium glutamicum obtain one plant of auxotropic variant,
Fermentation method industrial production commodity lysine from that point on.
Now, it is industrial mainly lysine to be produced using fermentation method.For the bacterial strain master of industrial fermenting and producing lysine
If the variant of corynebacteria and brevibacterium etc., corynebacteria has high economic worth, wherein corynebacterium glutamicum
It is most widely used.Additionally, Escherichia coli, brevibacterium flavum, saccharomyces cerevisiae, lactic fermentation quarter butt are also applied in lysine production
The report of bacterium, Candida etc..
Corynebacterium glutamicum is gram-positive microorganism, with fast growth, it is non-cause a disease, to the weak drop of own metabolism thing
The characteristic of solution ability, as traditional industry microorganism, Corynebacterium glutamicum be widely used in production l-amino acid, nucleotides and other
Organic acid.But the fermenting property of the bacterial classification of current 1B is still poor, acid production rate is still relatively low, can not still meet large-scale industry
The demand that metaplasia is produced.
The content of the invention
In view of this, present invention aims to the defect of prior art presence, there is provided a kind of 1B yield
High Corynebacterium glutamicum and its construction method and application.
To realize the purpose of the present invention, the present invention is adopted the following technical scheme that：
The invention provides a kind of Corynebacterium glutamicum, with 1B production capacity and its intracellular lysine is defeated
Go out albumen and pyruvate carboxylase activity strengthens simultaneously.
Lysine exports the increased activity of albumen, and the accumulation to 1B has facilitation, can improve L- and rely ammonia
The yield of acid.And pyruvate carboxylase is anaplerotic pathway key enzyme, pyruvate carboxylase activity strengthens can be synthesized for 1B
More precursors are provided, the accumulation ability of 1B is further improved.
Preferably, the Corynebacterium glutamicum has 1B production capacity and its cell interior coding lysine output
The lysE genes of albumen and the pyc gene expressions of encoding pyruvate carboxylase strengthen simultaneously.
It will be appreciated by those skilled in the art that the enhancing of the lysine output albumen and pyruvate carboxylase enzymatic activity is not
It is limited to the enhancing of gene expression.
Further, the enhanced gene editing technology of gene expression include but is not limited to encoding gene copy number increase,
Point mutation.
Preferably, the Corynebacterium glutamicum has 1B production capacity and its cell interior coding lysine output
The copy number of the lysE genes of albumen increases the pyc with encoding pyruvate carboxylaseP458SThe expression of gene strengthens.
In one embodiment, the invention provides a kind of Corynebacterium glutamicum MHZ-0913-3, its intracellular increase
The lysE genes and encoding pyruvate carboxylase with a site mutation of one copy coding lysine output albumen
pycP458SGene, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC
No.13407。
The invention provides a kind of construction method of the Corynebacterium glutamicum, including：
Step A, the recombinant vector for building lysE genes, in conversion glutaric acid bar bacterium MHZ-0912-1 recombinant bacterial strain is obtained；
Step B, structure pycP458SRecombinant vector, step of converting 1 obtain recombinant bacterial strain obtain final product.
Preferably, the introducing of pyc point mutations P458S and insertion mutation is completed by sacB recombination systems.
Preferably, the carrier is pK18mobsacB.The recombinant vector of i.e. described lysE genes is pK18mobsacB-
lysE.The pycP458SRecombinant vector be pK18mobsacB-pycP458S。
The Corynebacterium glutamicum obtained using the present invention carries out fermenting and producing, can obtain effective accumulation of 1B, is
The industrialized production of 1B lays the foundation.Therefore present invention also offers the Corynebacterium glutamicum relies ammonia in fermenting and producing
Application in acid.Preferably, the Corynebacterium glutamicum is the Corynebacterium glutamicum that deposit number is CGMCC No.13407
MHZ-0913-3
Further, present invention also offers a kind of production method of 1B, is CGMCC by deposit number
The Corynebacterium glutamicum of No.13407 is inoculated in fermentation medium ferments.
Wherein, it is preferred that the fermentation medium is by glucose 60g/L, (NH4)2SO425g/L, KH2PO42.0g/L,
MgSO4·7H2O 1.0g/L, soybean meal hydrolysate 10g/L, CaCO330g/L is constituted, pH 7.0.
Preferably, the fermentation condition is 33 DEG C, and ferment 14-15h.
As shown from the above technical solution, the invention provides a kind of Corynebacterium glutamicum and its construction method and application.This
The Corynebacterium glutamicum is invented, with 1B production capacity and its intracellular lysine output albumen and pyruvic acid carboxylic
Change enzymatic activity strengthens simultaneously.Corynebacterium glutamicum of the present invention enhances the activity of lysine output albumen so as to promote L- to rely
The accumulation of propylhomoserin, increases the yield of 1B, while strengthening pyruvate carboxylase activity, provides more for 1B synthesis
Precursor, further improves the accumulation ability of 1B, and the amount for making 1B in culture medium increases, and then improves strain fermentation
Produce the ability of 1B.Experiment shows that Corynebacterium glutamicum of the present invention is 1B superior strain, can effectively accumulate
1B, improves the yield of 1B, is that the industrialized production of 1B is laid a good foundation.
Biological deposits explanation
MHZ-0913-3, Classification And Nomenclature：Corynebacterium glutamicum, Corynebacterium glutamicum, in 2016
November 30 was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and address is Chaoyang District, Beijing City north
No. 3 Institute of Microorganism, Academia Sinica of institute of occasion West Road 1, deposit number is CGMCC No.13407.
Description of the drawings
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing to be used needed for having technology description is briefly described.
Fig. 1 is the schematic diagram of recombinant plasmid pK18mobsacB-lysE.
Fig. 2 is recombinant plasmid pK18mobsacB-pycP458SSchematic diagram.
Fig. 3 shows 1B yield system after MHZ-0912-1 and MHZ-0913-1, MHZ-0913-2, MHZ-0913-3 fermentation
Meter figure.
Specific embodiment
The invention discloses a kind of Corynebacterium glutamicum and its construction method and application.Those skilled in the art can use for reference
Present disclosure, is suitably modified technological parameter realization.Specifically, all similar replacements and change are to this area skill
It is it will be apparent that they are considered as being included in the present invention for art personnel.The present invention is carried out by preferred embodiment
Description, related personnel substantially can be modified in without departing from present invention, spirit and scope to method described herein
Or suitably change with combining to realize and apply the technology of the present invention.
For a further understanding of the present invention, with reference to specific embodiment, the present invention will be described in detail, such as without special
Illustrate, involved reagent is commercially available prod in the embodiment of the present invention, can be obtained by commercial channel purchase.Wherein,
Bacterial strain MHZ-0912-1 is disclosed in Chinese CN 105734004A.Primer sequence information used in following examples such as table 1
It is shown.
The primer sequence information of table 1
| Primer | Primer sequence (5 ' -3 ') | SEQ ID NO. |
| lysE-1f | cgcggatcctctagtttcccatcaaccatgta | 1 |
| lysE-1r | cgagcatcacgtgggtcgagtgtatttgggcgaaggccaccgattc | 2 |
| lysE-2f | cccaaatacactcgacccacgtgatgctcgagagctttaacgcgc | 3 |
| lysE-2r | gatttcccacgacgccggcatcaacctaacccatcaacatcagtt | 4 |
| lysE-3f | gatgggttaggttgatgccggcgtcgtgggaaatctcatcgatcg | 5 |
| lysE-3r | tggctgcag ttggaagaaaccatgtacgc | 6 |
| pyc-1f | gagcatgcgggcaaccacgtcttcatcgaaa | 7 |
| pyc-1r | ctgaaggaggtgcgagtgatcggcaatga | 8 |
| pyc-2f | tcattgccgatcactcgcacctccttcag | 9 |
| pyc-2r | ctagctagcggggtgtatcccacggtgttgcg | 10 |
Embodiment 1：The structure of recombinant plasmid pK18mobsacB-lysE and increase by one in starting strain MHZ-0912-1
Copy lysE
With the genomes of ATCC 13032 as template, performing PCR amplification is entered with lysE-1f/lysE-1r primer pairs, obtain upstream
Fragment lysE-up+lysE；With the genomes of ATCC 13032 as template, performing PCR amplification is entered with lysE-2f/lysE-2r primer pairs,
Fragment lysE is obtained, with the genomes of ATCC 13032 as template, performing PCR amplification is entered with lysE-3f/lysE-3r primer pairs, obtained
Fragment lysE-dn.With lysE-up+lysE, the fragment mixture of lysE, lysE-dn tri- is template, is drawn with lysE-1f/lysE-3r
Thing obtains the lysE fragments with two copies to entering performing PCR amplification.Fragment carries out double digestion, carrier with BamHI, PstI
PK18mobsacB is cut with same enzyme is double.Two digestion products are attached with T4DNA Ligase, convert Trans1T1 competence
Cell, obtains recombinant plasmid pK18mobsacB-lysE.
MHZ-0912-1 is prepared according to Corynebacterium glutamicum handbook (C.glutamicum Handbook, Charpter 23)
Competent cell.Recombinant plasmid pK18mobsacB-lysE converts the competent cell with electroporation method, and is containing 15mg/
Screen transformant on the BHI Selective agar mediums of L kanamycins, wherein the lysE genetic fragments of two copies are inserted into due to homology
To in chromosome.By the transformant incubated overnight sieved in common BHI fluid nutrient mediums, cultivation temperature is 33 DEG C, and revolution is shaken
Bed 220rpm shaken cultivations.In this incubation, transformant occurs second and recombinates, by gene swapping by carrier sequence from base
Because removing in group.Culture is done into continuous gradient dilutions (10-2Serial dilution is to 10-4), dilution is coated on containing 10% sucrose
Common BHI solid mediums on, 33 DEG C of quiescent culture 48h.The bacterial strain for growing does not carry the carrier of insertion in its genome
Sequence.Aim sequence, and nucleotide sequencing analysis are expanded by PCR, obtaining increases the bacterial strain of a copy lysE, and is named as
MHZ-0913-1。
Embodiment 2：Recombinant plasmid pK18mobsacB-pycP458SStructure and point mutation is introduced in MHZ-0912-1, increase
The activity of strong pyruvate carboxylase
With the genomes of ATCC 13032 as template, performing PCR amplification is entered with pyc-1f/pyc-1r primer pairs, obtain upstream piece
Section pyc-up；With the genomes of ATCC 13032 as template, performing PCR amplification is entered with pyc-2f/pyc-2r primer pairs, obtain downstream piece
Section pyc-dn.With the fragment mixture of pyc-up, pyc-dn two as template, performing PCR amplification is entered with pyc-1f/pyc-2r primer pairs,
Obtain the pyc being mutatedP458SFragment.pycP458SFragment SphI, NheI carry out double digestion, and pK18mobsacB is double with same enzyme
Cut.Two digestion products are attached with T4DNA Ligase, convert Trans1T1 competent cells, obtain recombinant plasmid
pK18mobsacB-pycP458S。
MHZ-0912-1 competent cells, recombinant plasmid pK18mobsacB- are prepared according to Corynebacterium glutamicum handbook
pycP458SThe competent cell is converted with electroporation method, and is sieved on the BHI Selective agar mediums containing 15mg/L kanamycins
Transformant is selected, wherein the pyc being mutatedP458SFragment is inserted into chromosome due to homology.The transformant for sieving overnight is trained
Support in common BHI fluid nutrient mediums, cultivation temperature is 33 DEG C, turns round shaking table 220rpm shaken cultivations.In this incubation, turn
Beggar occurs second and recombinates, and is removed carrier sequence from genome by gene swapping.Culture is done into continuous gradient dilute
Release (10-2Serial dilution is to 10-4), dilution is coated on the common BHI solid mediums containing 10% sucrose, 33 DEG C of standings
Culture 48h.The bacterial strain for growing does not carry the carrier sequence of insertion in its genome.Aim sequence, and nucleosides are expanded by PCR
Sour sequencing analysis, obtain the pyc of mutationP458SThe bacterial strain of fragment, and it is named as MHZ-0913-2.
Embodiment 3：Recombinant plasmid pK18mobsacB-pycP458SStructure and point mutation is introduced in MHZ-0913-1, increase
The activity of strong pyruvate carboxylase
According to the method construction recombination plasmid pK18mobsacB-pyc described in embodiment 2P458S：With ATCC13032 genomes
For template, performing PCR amplification is entered with pyc-1f/pyc-1r primer pairs, obtain fragment upstream pyc-up；With the genomes of ATCC 13032
For template, performing PCR amplification is entered with pyc-2f/pyc-2r primer pairs, obtain segments downstream pyc-dn.With pyc-up, pyc-dn two
Fragment mixture is template, and with pyc-1f/pyc-2r primer pairs performing PCR amplification is entered, and obtains the pyc being mutatedP458SFragment.
pycP458SFragment SphI, NheI carry out double digestion, and pK18mobsacB is cut with same enzyme is double.Two digestion products are with T4 DNA
Ligase is attached, and converts Trans1T1 competent cells, obtains recombinant plasmid pK18 mobsacB-pycP458S。
MHZ-0913-1 competent cells are prepared according to Corynebacterium glutamicum handbook.Recombinant plasmid pK18mobsacB-
pycP458SMHZ-0913-1 competent cells are converted with electroporation method, and training is selected in the BHI containing 15mg/L kanamycins
Transformant is screened on foster base, wherein the pyc being mutatedP458SFragment is inserted into chromosome due to homology.By the conversion sieved
In common BHI fluid nutrient mediums, cultivation temperature is 33 DEG C to sub- incubated overnight, turns round shaking table 220rpm shaken cultivations.This culture
During, transformant occurs second and recombinates, and is removed carrier sequence from genome by gene swapping.Culture is done and is connected
Continuous gradient dilution (10-2Serial dilution is to 10-4), dilution is coated on the common BHI solid mediums containing 10% sucrose,
33 DEG C of quiescent culture 48h.The bacterial strain grown on SM does not carry the carrier sequence of insertion in its genome.Pass through
PCR expands aim sequence, and nucleotide sequencing analysis, obtains the pyc with mutationP458SThe bacterial strain of fragment, and it is named as MHZ-
0913-3。
Embodiment 4：1B engineering bacteria fermentation produces 1B
MHZ-0912-1 bacterial strains and bacterial strain described in embodiment 1 to embodiment 3 are carried out in 500ml triangle shaking flasks respectively
Fermented and cultured, 33 DEG C of cultivation temperature, incubation time 14-15 hours, every group of Setup Experiments three are parallel.The fermentation medium group
Divide as follows：Glucose 60g/L, (NH4)2SO425g/L, KH2PO42.0g/L, MgSO4·7H2O 1.0g/L, soybean meal hydrolysate
10g/L, CaCO330g/L, NaOH adjust pH=7.0.Each strain fermentation produces 1B and the results are shown in Table 2.
The each strain fermentation of table 2 produces 1B result
As shown in Table 2, increase by one copy lysE bacterial strain MHZ-0913-1 1B yield and saccharic acid conversion ratio with
Control group bacterial strain MHZ-0912-1 is compared and increased, with significant difference.Same mutation pycP458SThe bacterial strain MHZ- of fragment
The 1B yield and saccharic acid conversion ratio of 0913-2 also increases compared with control group bacterial strain MHZ-0912-1, with notable
Difference.
Increase by one simultaneously and copy lysE genes and mutation pycP458SThe bacterial strain MHZ-0913-3 of fragment, compares control group bacterium
Strain, yield and conversion ratio be improved largely, and 1B output increased 7g/L, conversion ratio improves 11.6%.
In sum, (deposit number is CGMCC to the 1B genetic engineering bacterium MHZ-0913-3 constructed by the present invention
No.13407 effective accumulation of 1B in sweat) can be realized, and with higher saccharic acid conversion ratio, the bacterial strain tool
There is extensive prospects for commercial application.
SEQUENCE LISTING
<110>Langfang plum blossom biotechnology development corporation, Ltd.
<120>A kind of Corynebacterium glutamicum and its construction method and application
<130> MP1623785
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 32
<212> DNA
<213>Artificial sequence
<400> 1
cgcggatcct ctagtttccc atcaaccatg ta 32
<210> 2
<211> 46
<212> DNA
<213>Artificial sequence
<400> 2
cgagcatcac gtgggtcgag tgtatttggg cgaaggccac cgattc 46
<210> 3
<211> 45
<212> DNA
<213>Artificial sequence
<400> 3
cccaaataca ctcgacccac gtgatgctcg agagctttaa cgcgc 45
<210> 4
<211> 45
<212> DNA
<213>Artificial sequence
<400> 4
gatttcccac gacgccggca tcaacctaac ccatcaacat cagtt 45
<210> 5
<211> 45
<212> DNA
<213>Artificial sequence
<400> 5
gatgggttag gttgatgccg gcgtcgtggg aaatctcatc gatcg 45
<210> 6
<211> 29
<212> DNA
<213>Artificial sequence
<400> 6
tggctgcagt tggaagaaac catgtacgc 29
<210> 7
<211> 31
<212> DNA
<213>Artificial sequence
<400> 7
gagcatgcgg gcaaccacgt cttcatcgaa a 31
<210> 8
<211> 29
<212> DNA
<213>Artificial sequence
<400> 8
ctgaaggagg tgcgagtgat cggcaatga 29
<210> 9
<211> 29
<212> DNA
<213>Artificial sequence
<400> 9
tcattgccga tcactcgcac ctccttcag 29
<210> 10
<211> 32
<212> DNA
<213>Artificial sequence
<400> 10
ctagctagcg gggtgtatcc cacggtgttg cg 32
Claims (10)
1. a kind of Corynebacterium glutamicum, it is characterised in that with 1B production capacity and its intracellular lysine output
Albumen and pyruvate carboxylase activity strengthen simultaneously.
2. Corynebacterium glutamicum according to claim 1, it is characterised in that with 1B production capacity and its is thin
The pyc gene expressions of the lysE genes and encoding pyruvate carboxylase of intracellular coding lysine output albumen strengthen simultaneously.
3. Corynebacterium glutamicum according to claim 1, it is characterised in that with 1B production capacity and its is thin
The copy number of the lysE genes of intracellular coding lysine output albumen increases the pyc with encoding pyruvate carboxylaseP458SGene
Strengthen.
4. Corynebacterium glutamicum according to claim 1, it is characterised in that the Corynebacterium glutamicum is deposited in Chinese micro- life
Thing culture presevation administration committee common micro-organisms center, deposit number is CGMCC No.13407.
5. the construction method of Corynebacterium glutamicum described in claim 1, it is characterised in that include：
Step A, the recombinant vector for building coding lysE genes, in conversion Corynebacterium glutamicum MHZ-0912-1 recombinant bacterium is obtained
Strain；
Step B, structure pycP458SRecombinant vector, step of converting 1 obtain recombinant bacterial strain obtain final product.
6. construction method according to claim 5, it is characterised in that the carrier is pK18mobsacB.
7. application of the Corynebacterium glutamicum in fermentation production of L-lysine described in claim 1-4 any one.
8. a kind of production method of 1B, it is characterised in that by deposit number for CGMCC No.13407 glutamic acid rod
Bacillus is inoculated in fermentation medium ferments.
9. production method according to claim 8, it is characterised in that the fermentation medium is by glucose 60g/L, (NH4)2SO425g/L, KH2PO42.0g/L, MgSO4·7H2O 1.0g/L, soybean meal hydrolysate 10g/L, CaCO330g/L is constituted, pH
7.0。
10. production method according to claim 8, it is characterised in that the fermentation condition is 33 DEG C, and ferment 14-15h.
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Cited By (4)
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| CN110951662A (en) * | 2019-12-26 | 2020-04-03 | 新疆梅花氨基酸有限责任公司 | Coryneform bacterium capable of highly producing lysine, and construction method and application thereof |
| CN112063571A (en) * | 2020-08-14 | 2020-12-11 | 廊坊梅花生物技术开发有限公司 | Engineering bacterium for high yield of L-amino acid and construction method and application thereof |
| CN113544141A (en) * | 2020-02-13 | 2021-10-22 | Cj第一制糖株式会社 | Microorganism comprising mutant LysE and method for producing L-amino acid using the same |
| CN115490761A (en) * | 2021-11-01 | 2022-12-20 | 中国科学院天津工业生物技术研究所 | Recombinant microorganism constructed based on lysine efflux protein and method for producing lysine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110951662A (en) * | 2019-12-26 | 2020-04-03 | 新疆梅花氨基酸有限责任公司 | Coryneform bacterium capable of highly producing lysine, and construction method and application thereof |
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| CN113544141A (en) * | 2020-02-13 | 2021-10-22 | Cj第一制糖株式会社 | Microorganism comprising mutant LysE and method for producing L-amino acid using the same |
| CN113544141B (en) * | 2020-02-13 | 2023-10-31 | Cj第一制糖株式会社 | Microorganism comprising mutant LysE and method for producing L-amino acid using the same |
| CN112063571A (en) * | 2020-08-14 | 2020-12-11 | 廊坊梅花生物技术开发有限公司 | Engineering bacterium for high yield of L-amino acid and construction method and application thereof |
| CN115490761A (en) * | 2021-11-01 | 2022-12-20 | 中国科学院天津工业生物技术研究所 | Recombinant microorganism constructed based on lysine efflux protein and method for producing lysine |
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