CN105754922A - Construction method of corynebacterium glutamicum mutant strain of high-yield L-lysine - Google Patents

Construction method of corynebacterium glutamicum mutant strain of high-yield L-lysine Download PDF

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CN105754922A
CN105754922A CN201610272266.0A CN201610272266A CN105754922A CN 105754922 A CN105754922 A CN 105754922A CN 201610272266 A CN201610272266 A CN 201610272266A CN 105754922 A CN105754922 A CN 105754922A
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corynebacterium glutamicum
construction method
lysine
pcr
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王瑞明
汪俊卿
王蕾
王腾飞
李桂华
王建彬
隋松森
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Shandong feed quality inspection institute
ZHUCHENG DONGXIAO BIOTECHNOLOGY CO Ltd
Qilu University of Technology
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Shandong feed quality inspection institute
ZHUCHENG DONGXIAO BIOTECHNOLOGY CO Ltd
Qilu University of Technology
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    • 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)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine

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Abstract

The invention relates to a construction method of a corynebacterium glutamicum mutant strain of high-yield L-lysine.According to the method, the corynebacterium glutamicum mutant strain is obtained through construction after inactivation of an NCgl0929 gene of corynebacterium glutamicum.Due to the fact that inactivation of the endogenous gene NCgl0929 causes obvious reduction of the intracellular transfer rate of lysine and compared with wild strains, the recombinant strain constructed by adopting the construction method of the corynebacterium glutamicum mutant strain of the high-yield L-lysine is more advantageous to production of high-concentration L-lysine and can further reduce the production costs by serving as an L-lysine production strain.

Description

A kind of construction method of the corynebacterium glutamicum mutant bacteria of high-yield L-lysine
Technical field
The present invention relates to the construction method of the corynebacterium glutamicum mutant bacteria of a kind of high-yield L-lysine, belong to biotechnology skill Art field.
Background technology
1B is one of essential amino acid, can promote human development, strengthen immunologic function, and be improved nervus centralis The effect of function of organization.1B is white or the free-pouring crystalline powder of near-white；Almost odorless.Soluble in water and Formic acid, is insoluble in ethanol and ether.1B molecular structure exists two amine-based basic relatively strong, the carboxylic acid on C4 position Property is more weak, and therefore 1B normally behaves as basic amino acid.1B is mainly used as feed additive, Food fortification Agent and pharmacy.
At present, 1B can be produced by different methods, including microbe fermentation method (one-step fermentation, two steps Fermentation method), enzyme process, albumen hydrolysis etc..Most common method is microbe fermentation method, is made by microorganism and has biological conjunction Become the ability of self institute's essential amino acids, utilize the metabolism of microorganism, by bacterial strain is transformed, select various ammonia Base acid auxotroph and Amino acid analogue resistant mutant strain, thus release the feedback repression in biosynthesis pathway and press down Make use, reach the purpose of 1B in excess accumulation metabolic pathway.
Many kinds are included, if corynebacterium is (such as corynebacterium glutamicum for producing the microorganism of 1B Corynebacterium glutamicum and Corynebacterium crenatum Corynebacterium crenatum), brevibacterium is (such as Huang Color brevibacterium Breibacterium flavum and brevibacterium lactofermentus Brevibacterium lactofermentus), Rocca Salmonella, read coccus, pseudomonas, Escherichia (such as escherichia coli Escherichia coli) and bacillus cereus (as Bacillus macerans Bacillus macerans), Archimycetes (such as thermal deformation bacterium Thermoprotens neutrophilus) And the yeast (such as Cryptococcus Cryptococcus neoformans) etc. in fungus.At present, both at home and abroad for industry Metaplasia produce 1B bacterial strain mostly be corynebacterium glutamicum (C.glutamicum), brevibacterium flavum (B.flavum), Brevibacterium lactofermentus (B.lactofermentus) and the reworked bacterial strain of escherichia coli (E.coli).Both at home and abroad to L- Lysine produces the selection-breeding of strain mainly by mutation breeding technologies and metabolic engineering breeding technique.Metabolic engineering breeding is from molecule Selection-breeding bacterial strain is optimized by minimal, the clearest and the most definite genetic modification in level.But, compared with wild-type strain, There is the biggest defect in these bacterial strains, as the speed of growth is slow, sugar consumption speed is low, to external world poor environment tolerance difference etc..With Development and the parsing of C. glutamicum gene group of biotechnology, classic mutagenesis breeding obtains 1B Producing Strain side Method is gradually metabolized Engineering Breeding method and is replaced.Numerous studies show simultaneously, the genetic engineering obtained by technique for gene engineering Bacterium, in addition to some advantages remaining wild-type strain, moreover it is possible to largely improve 1B yield.Such as Chinese patent Document CN1539015 (application number 02815446.0) discloses including accBC, accDA, catA, cysD, cysE Multiple can be used for lysine production improve genes improve sites.Chinese patent literature CN101600796 (application number 200780048626.8), CN1974760 (application number 200610163142.5), CN103243042 (application number 201310092638.8) respectively gene NCg22534, NCgl1835, NCg21090 are carried out the inactivation a series of L-of acquisition and relies ammonia The corynebacterium glutamicum engineering bacteria that acid productivity improves.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, it is provided that the structure of the corynebacterium glutamicum mutant bacteria of a kind of high-yield L-lysine Method.The present invention is obtained by transformation bacterial strain corynebacterium glutamicum, and specific strategy is inactivation corynebacterium glutamicum endogenous gene NCgl0929 (coding lysine intracellular transport albumen) is to reduce the redundancy transported outside lysine intracellular born of the same parents in lysine fermentation process Circulation, makes more energy flow to 1B compound direction, improves 1B productivity.
Term explanation
In the present invention, " inactivation " can be induced by any method for deactivating known in the art." inactivate " effect produced Refer to that the expression of endogenous gene NCgl0929 is lowered to the lowest level, although or producing not expressing gene and being expressed But express the product of the most active or active reduction.
In the present invention, " inactivation " can insert one or more alkali by genetic engineering means in endogenous gene NCgl0929 Base pair, or lack the one or more base pairs in this endogenous gene NCgl0929, or changed by gene internal sequence or Transversion obtains.
Technical solution of the present invention is as follows:
The construction method of the corynebacterium glutamicum mutant bacteria of a kind of high-yield L-lysine, is by corynebacterium glutamicum Build after the NCgl0929 gene inactivation of (Corynebacterium glutamicum) and obtain, NCgl0929 gene code frame Nucleotide sequence is as shown in SEQ ID NO.1.
According to currently preferred, described corynebacterium glutamicum (Corynebacterium glutamicum) derives from China's work Industry Microbiological Culture Collection administrative center, bacterium numbering CICC 23604.
According to currently preferred, above-mentioned construction method, step is as follows:
(1) by the segment length genetic fragment more than 400bp in PCR amplification NCgl0929 gene code frame as homology Arm sequence, the nucleotide sequence of NCgl0929 gene code frame is as shown in SEQ ID NO.1；
(2) resistance label genetic fragment is expanded by PCR；
(3) the resistance label genetic fragment that homology arm sequence step (1) prepared and step (2) prepare carries out over-lap PCR Being attached, two ends preparing fusion fragment all contain identical restriction enzyme digestion sites, and this restriction enzyme site is not Plan can be occurred in knock out in gene and resistance label gene；
(4) prepare corynebacterium glutamicum competent cell, the fusion gene that step (3) prepares is converted paddy ammonia after enzyme action Acid corynebacterium competent cell, to obtain final product.
According to the present invention it is further preferred that in described step (1), PCR expands with corynebacterium glutamicum The genomic DNA of (Corynebacterium glutamicum) is template, and the nucleotide sequence of pcr amplification primer thing is as follows:
F1:CGGGATCCTCGTACTGTATCTATTAGAACCCT；
R1:AAGGCCAGCAAAA GCTAAAGCCCAAGAATGCAAC；
The reaction system of PCR amplification is as follows, and total system is 50 μ l:
PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 54 DEG C of annealing 30sec, 72 DEG C extend 30sec, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations.
According to currently preferred, in described step (2), PCR amplification template is that shuttle plasmid pHT01 is (precious purchased from Hangzhou Match bio tech ltd) DNA；The nucleotide sequence of pcr amplification primer thing is as follows:
F2:GGGCTTTAGC TTTTGCTGGCCTTTTGCTCA；
R2:GCTGGATCCTAGTGACTGGCGATGCT；
The reaction system of PCR amplification is as follows, and total system is 50 μ l:
Described PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 3min, 30 circulations；72℃ Extend 10min, 4 DEG C of preservations.
According to currently preferred, in described step (3), the first stage amplification system of over-lap PCR is as follows, and total system is 25 μ l:
The first stage amplification program of over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 54 DEG C of annealing 30sec, 72 DEG C extend 2min, 5 circulations；72℃ Extend 10min；
The second stage amplification system of over-lap PCR be the first stage expand after product on the basis of add following composition, totally System is 50 μ l:
The second stage amplification program of over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 4min, 30 circulations；72℃ Extend 10min, 4 DEG C of preservations.
According to currently preferred, specifically comprising the following steps that of described step (4)
I the single bacterium colony of () picking corynebacterium glutamicum (Corynebacterium glutamicum), trains in seed culture medium Support to cell concentration OD600It is 0.7～0.9, is placed in cooled on ice, centrifugal after cooling, turn buffer solution bacterium with the electricity of pre-cooling Body 3～5 times, electricity turns the resuspended thalline of buffer, prepares competent cell；
(ii) fusion gene preparing step (3) carries out single endonuclease digestion, and 28～32 DEG C of enzyme action 2～5h, electroporation is extremely In the competent cell that step (i) is prepared, move into liquid resuscitation culture medium, after cultivating 0.5～2h at 28～32 DEG C, screening, Obtain.
According to the present invention it is further preferred that in described step (ii) single endonuclease digestion system as follows, total system is 40 μ l:
According to the present invention it is further preferred that in described step (i), seed culture medium, every liter of component is as follows:
Peptone 8～12g, yeast powder 4～6g, sodium chloride 8～12g, sorbitol 85～96g.
According to the present invention it is further preferred that in described step (i), electricity turns buffer, and every liter of component is as follows:
Sorbitol 85～96g, mannitol 85～96g, glycerol 95～105mL.
According to the present invention it is further preferred that in described step (i), the condition of electroporation is: 2100V shocks by electricity 5ms.
According to the present invention it is further preferred that liquid resuscitation culture medium in described step (ii), every liter of component is as follows:
Peptone 8～12g, yeast powder 4～6g, sodium chloride 8～12g, sorbitol 85～96g, mannitol 65～73g.
The application in producing 1B of the corynebacterium glutamicum mutant bacteria of the high-yield L-lysine that above-mentioned construction method prepares.
Build principle
LysP is the lysine transport protein of corynebacterium glutamicum, is responsible for the absorption of lysine, but high-yield L-lysine bacterium In kind, by research, inventor finds that the expression of this gene then can cause the circulation outside lysine intracellular born of the same parents, due to the suction of lysine Receiving and secretion is required to consumed energy, therefore this process can increase cell energy consumption, and then impact includes that the synthesis of lysine exists Interior other metabolic process of cell.Therefore, by building NCgl0929 gene inactivation (expressing LysP), thus reach to obtain The recombinant bacterium of 1B high yield.
Beneficial effect
The present invention provides the construction method of the corynebacterium glutamicum mutant strain of a kind of high-yield L-lysine, the recombinant bacterium of structure Strain causes lysine intracellular transport speed substantially to reduce due to endogenous gene NCgl0929 inactivation, compared with wild type, and this bacterial strain It is more beneficial for producing the 1B of high concentration, produces bacterial strain as 1B and can reduce production cost further.
Detailed description of the invention
Below in conjunction with embodiment, technical scheme is further elaborated, but institute of the present invention protection domain is not limited to this.
Corynebacterium glutamicum (Corynebacterium glutamicum) 23604 in embodiment derives from the micro-life of Chinese industrial Thing culture presevation administrative center, bacterium numbering CICC 23604；
Shuttle plasmid pHT01 is purchased from Bao Sai bio tech ltd, Hangzhou.
Embodiment 1: gene knockout fragment builds
1) (i) extracts corynebacterium glutamicum (Corynebacterium glutamicum) 23604 genomic DNAs, with this Genomic DNA is template, carries out PCR amplification, obtains homology arm NCg2；
Described PCR primer sequence is as follows:
F1:CGGGATCC TCGTACTGTATCTATTAGAACCCT
R1:AAGGCCAGCAAAA GCTAAAGCCCAAGAATGCAAC
Described PCR amplification system is:
Described PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 54 DEG C of annealing 30sec, 72 DEG C extend 30sec, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations；
Agarose gel electrophoresis inspection PCR primer, a length of 541bp, use SanPrep pillar DNA glue to reclaim test kit (the raw work in Shanghai) carries out glue recovery, reclaims product-20 DEG C preservation, standby；
(ii) DNA of shuttle plasmid pHT01 (purchased from Bao Sai bio tech ltd, Hangzhou) is extracted, with DNA as mould Plate, carries out PCR amplification, obtains CmrFragment；
Described PCR primer sequence is as follows:
F2:GGGCTTTAGC TTTTGCTGGCCTTTTGCTCA
R2:GCTGGATCCTAGTGACTGGCGATGCT
Described PCR amplification system is:
Described PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 3min, 30 circulations；72℃ Extend 10min, 4 DEG C of preservations；
Agarose gel electrophoresis inspection PCR primer, a length of 1264bp, use SanPrep pillar DNA glue to reclaim reagent Box (the raw work in Shanghai) carries out glue recovery, reclaims product-20 DEG C preservation, standby；
(iii) by Cm prepared with step (ii) for homology arm NCg2 prepared for step (i)rFragment carries out over-lap PCR, system Obtain NCg2-CmrFragment；
The amplification system of described over-lap PCR is:
The first stage amplification program of described over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 54 DEG C of annealing 30sec, 72 DEG C extend 2min, 5 circulations；72℃ Extend 10min；
The second stage amplification program of described over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 4min, 30 circulations；72℃ Extend 10min, 4 DEG C of preservations；
Agarose gel electrophoresis inspection PCR primer, a length of 1787bp, use SanPrep pillar DNA glue to reclaim reagent Box (the raw work in Shanghai) carries out glue recovery, reclaims product-20 DEG C preservation, standby；
Embodiment 2: preparation Bacillus licheniformis competence
I the single bacterium colony of () picking corynebacterium glutamicum (Corynebacterium glutamicum) 23604, is inoculated in 10mL kind In sub-culture medium, 37 DEG C, 220r/min, incubated overnight；
Described seed culture medium, every liter of component is as follows:
Peptone 10g, yeast powder 5g, sodium chloride 10g, sorbitol 91g.
(ii) take the above-mentioned bacterium solution of 1mL to be transferred in 100mL seed culture medium, 37 DEG C, 220r/min cultivation to OD600=0.9；
(iii) bacterium solution is transferred to 100mL centrifuge tube, ice bath 15～20min, make thalline stop growing；
(iv) after ice bath 4 DEG C, 5000g, 5min centrifugal, collect thalline；
The electricity of the thalline pre-cooling v () is centrifugal after turns buffer solution 3 times；
Electricity turns buffer, and every liter of component is as follows:
Sorbitol 91g, mannitol 91g, glycerol 100mL.
(vi), after washing terminates, 1000 μ L electricity are used to turn the resuspended thalline of buffer；
(vii) the competent cell subpackage 100 μ L prepared often is managed ,-80 DEG C of preservations, standby.
Embodiment 3:NCg2-CmrFragment electricity converts corynebacterium glutamicum (Corynebacterium glutamicum) 23604
I () is by NCg2-CmrFragment restricted enzyme BamH I, 30 DEG C of enzyme action 3h；
Enzyme action system (40 μ l) is as follows:
(ii) digestion products is concentrated and purified
(1) add 1/10 volume 3M sodium acetate and 2.5 times of volume dehydrated alcohol, be placed in-20 DEG C of refrigerator 20min；
(2) 12000r/min, centrifugal 5min must precipitate；
(3) the 300 resuspended precipitations of μ L75% (percent by volume) ethanol；
(4) 12000r/min, centrifugal 5min, removing ethanol, 37 DEG C of air-dried 30min,
(5) 15～18 μ L ddH are added2The resuspended DNA of O, is placed in-20 DEG C of preservations.
(iii) electricity converts
NCg2-Cm is measured first with nucleic acid ultramicrospectrophotometerrFragment concentrations, reaches 2100V after 300 μ g/mL Electric shock 5ms, carries out electricity conversion, after the cell obtained uses recovery medium 30 DEG C recovery to cultivate 1h, takes 100 μ L and is coated on On LB solid medium containing 200 μ g/mL chloromycetin, cultivating 2 days at 37 DEG C, screening has the conversion of chlorampenicol resistant Son.
Liquid resuscitation culture medium, every liter of component is as follows:
Peptone 10g, yeast powder 5g, sodium chloride 10g, sorbitol 91g, mannitol 69.4g.
Embodiment 4: the cultivation of positive recombinant bacterium and qualification
The above-mentioned positive restructuring bacterium colony of picking, is inoculated into 37 DEG C of trainings in the LB liquid medium containing 200 μ g/mL chlorampenicol resistants Supporting overnight, after having cultivated, the test kit using Shanghai biological engineering company limited to provide extracts recombinant bacterium DNA, and to obtain The genome obtained is template, F1And R2Carrying out PCR amplification for primer, amplified production utilizes agarose gel electrophoresis to verify；
Described PCR primer sequence is as follows:
F1:CGGGATCC TCGTACTGTATCTATTAGAACCCT
R2:GCTGGATCCTAGTGACTGGCGATGCT
Wherein, underscore mark for restriction enzyme site
Described PCR amplification system is 20 μ l:
Described PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 4min, 30 circulations；72℃ Extend 10min, 4 DEG C of preservations；
Agarose gel electrophoresis inspection PCR primer, result shows, uses primers F1And R2A specificity base can be amplified Because of band, size is about 1800b, close with theoretical value 1787bp, shows that genes of interest is the most successfully incorporated into glutamic acid bar-shaped In bacillus gene group, prepare the corynebacterium glutamicum mutant bacteria of high-yield L-lysine.
Embodiment 5:L-fermenting lysine is tested
The corynebacterium glutamicum mutant bacteria of the high-yield L-lysine of preparation is seeded to 100mL LBG culture medium (glucose 5g/L, peptone 10g/L, yeast extract 5g/L, NaCl 10g/L) in 220rpm, carry out seed culture 20h, so at 30 DEG C After by volume percentage ratio 2% inoculum concentration be seeded to 100mL fermentation medium (glucose 100g/L, peptone 20g/L, beautiful Rice & peanut milk 30mL, carbamide 5g/L, (NH4)2SO425g/L, L-Leu 0.34g/L, KH2PO42g/L, MgSO4·7H2O 1.5g/L, biotin 0.001g/L) fermentation culture 36h, and measured by Hitachi L-8900 type high speed automatic amino acid analyzer Amino acid whose content in fermentation liquid.
Result shows compared with original bacteria, the product of lysine in the corynebacterium glutamicum mutant bacteria fermentation liquid of high-yield L-lysine Amount is promoted to 45g/L by 40g/L.

Claims (10)

1. the construction method of the corynebacterium glutamicum mutant bacteria of a high-yield L-lysine, it is characterised in that by glutamic acid rod Building after the NCgl0929 gene inactivation of shape bacillus (Corynebacterium glutamicum) and obtain, NCgl0929 gene is compiled The nucleotide sequence of code frame is as shown in SEQ ID NO.1.
2. construction method as claimed in claim 1, it is characterised in that described corynebacterium glutamicum (Corynebacterium Glutamicum) Chinese industrial Microbiological Culture Collection administrative center, bacterium numbering CICC 23604 are derived from.
3. construction method as claimed in claim 1 or 2, it is characterised in that step is as follows:
(1) by the segment length genetic fragment more than 400bp in PCR amplification NCgl0929 gene code frame as homology Arm sequence, the nucleotide sequence of NCgl0929 gene code frame is as shown in SEQ ID NO.1；
(2) resistance label genetic fragment is expanded by PCR；
(3) the resistance label genetic fragment that homology arm sequence step (1) prepared and step (2) prepare carries out over-lap PCR Being attached, two ends preparing fusion fragment all contain identical restriction enzyme digestion sites, and this restriction enzyme site is not Plan can be occurred in knock out in gene and resistance label gene；
(4) prepare corynebacterium glutamicum competent cell, the fusion gene that step (3) prepares is converted paddy ammonia after enzyme action Acid corynebacterium competent cell, to obtain final product.
4. construction method as claimed in claim 3, it is characterised in that in described step (1), PCR expands with glutamic acid The genomic DNA of corynebacterium (Corynebacterium glutamicum) is template, the nucleotides sequence of pcr amplification primer thing Arrange as follows:
F1:CGGGATCCTCGTACTGTATCTATTAGAACCCT；
R1:AAGGCCAGCAAAA GCTAAAGCCCAAGAATGCAAC；
The reaction system of PCR amplification is as follows, and total system is 50 μ l:
PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 54 DEG C of annealing 30sec, 72 DEG C extend 30sec, 30 circulations；72 DEG C extend 10min, 4 DEG C of preservations.
5. construction method as claimed in claim 3, it is characterised in that in described step (2), PCR amplification template is for wearing The DNA of shuttle plasmid pHT01；The nucleotide sequence of pcr amplification primer thing is as follows:
F2:GGGCTTTAGC TTTTGCTGGCCTTTTGCTCA；
R2:GCTGGATCCTAGTGACTGGCGATGCT；
The reaction system of PCR amplification is as follows, and total system is 50 μ l:
Described PCR amplification program is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 55 DEG C of annealing 30sec, 72 DEG C extend 3min, 30 circulations；72℃ Extend 10min, 4 DEG C of preservations.
6. construction method as claimed in claim 3, it is characterised in that in described step (3), the first of over-lap PCR Stage amplification system is as follows, and total system is 25 μ l:
The first stage amplification program of over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 54 DEG C of annealing 30sec, 72 DEG C extend 2min, 5 circulations；72℃ Extend 10min；
The second stage amplification system of over-lap PCR be the first stage expand after product on the basis of add following composition, totally System is 50 μ l:
The second stage amplification program of over-lap PCR is as follows:
95 DEG C of denaturations 5min；94 DEG C of degeneration 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend 4min, 30 circulations；72℃ Extend 10min, 4 DEG C of preservations.
7. construction method as claimed in claim 3, it is characterised in that specifically comprising the following steps that of described step (4)
I the single bacterium colony of () picking corynebacterium glutamicum (Corynebacterium glutamicum), trains in seed culture medium Support to cell concentration OD600It is 0.7～0.9, is placed in cooled on ice, centrifugal after cooling, turn buffer solution bacterium with the electricity of pre-cooling Body 3～5 times, electricity turns the resuspended thalline of buffer, prepares competent cell；
(ii) fusion gene preparing step (3) carries out single endonuclease digestion, and 28～32 DEG C of enzyme action 2～5h, electroporation is extremely In the competent cell that step (i) is prepared, move into liquid resuscitation culture medium, after cultivating 0.5～2h at 28～32 DEG C, screening, Obtain.
8. construction method as claimed in claim 7, it is characterised in that in described step (ii), single endonuclease digestion system is as follows, always System is 40 μ l:
9. construction method as claimed in claim 7, it is characterised in that in described step (i), seed culture medium, every liter Component is as follows:
Peptone 8～12g, yeast powder 4～6g, sodium chloride 8～12g, sorbitol 85～96g；
Preferably, in described step (i), electricity turns buffer, and every liter of component is as follows:
Sorbitol 85～96g, mannitol 85～96g, glycerol 95～105mL；
Preferably, in described step (i), the condition of electroporation is, and: 2100V shocks by electricity 5ms；
Preferably, liquid resuscitation culture medium in described step (ii), every liter of component is as follows:
Peptone 8～12g, yeast powder 4～6g, sodium chloride 8～12g, sorbitol 85～96g, mannitol 65～73g.
10. the corynebacterium glutamicum mutant bacteria of the high-yield L-lysine that construction method described in claim 1 prepares is producing L- Application in lysine.
CN201610272266.0A 2016-04-27 2016-04-27 Construction method of corynebacterium glutamicum mutant strain of high-yield L-lysine Pending CN105754922A (en)

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CN109517751A (en) * 2017-09-18 2019-03-26 赢创德固赛有限公司 The method of fermentation producing L-amino-acid
CN109694841A (en) * 2019-02-02 2019-04-30 江南大学 A kind of corynebacterium glutamicum recombinant bacterium, preparation method and application
CN110218749A (en) * 2019-05-16 2019-09-10 内蒙古伊品生物科技有限公司 With the method for the bacterial fermentation production lysine for changing NCgl1859
CN110218749B (en) * 2019-05-16 2023-05-12 内蒙古伊品生物科技有限公司 Method for producing lysine by fermentation using NCgl 1859-modified bacteria
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
CN114134062A (en) * 2020-09-03 2022-03-04 大象株式会社 Corynebacterium glutamicum mutant strain having improved L-lysine productivity and method for producing L-lysine using the same
CN114540261A (en) * 2020-11-24 2022-05-27 北京化工大学 Genetically engineered bacterium for producing aminoadipic acid
CN114540261B (en) * 2020-11-24 2024-02-02 北京化工大学 Gene engineering bacteria for producing amino adipic acid
CN114867851A (en) * 2021-04-12 2022-08-05 Cj第一制糖株式会社 Novel cell division membrane protein variants and method for producing L-lysine using same
CN116496950A (en) * 2022-09-27 2023-07-28 欧铭庄生物科技(天津)有限公司滨海新区分公司 Lysine production strain and application thereof, and lysine production method
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