CN110106206A - A kind of corynebacterium glutamicum construction method improving L-lysine yield and stability - Google Patents

A kind of corynebacterium glutamicum construction method improving L-lysine yield and stability Download PDF

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CN110106206A
CN110106206A CN201910398768.1A CN201910398768A CN110106206A CN 110106206 A CN110106206 A CN 110106206A CN 201910398768 A CN201910398768 A CN 201910398768A CN 110106206 A CN110106206 A CN 110106206A
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corynebacterium glutamicum
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pcr
gene
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CN110106206B (en
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王松江
郭传庄
隋松森
王建彬
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Dongxiao Biotechnology Co.,Ltd.
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ZHUCHENG DONGXIAO BIOTECHNOLOGY CO Ltd
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Abstract

The present invention relates to a kind of construction methods of corynebacterium glutamicum for improving L-lysine yield and stability.This method by corynebacterium glutamicum (Corynebacterium glutamicum) Tnp7a gene inactivation after construct obtain, the nucleotide sequence of Tnp7a gene is as shown in SEQ ID NO. 1.The present invention provides a kind of construction method of corynebacterium glutamicum for improving L-lysine yield and stability, the bacterial strain of building is saving and the swivel base effect that insetion sequence is activated because of environment change in fermentation process since endogenous gene Tnp7a inactivation reduces corynebacterium glutamicum, the mutation or frameshit that blocking gene group is generated due to swivel base, or spawn degeneration process caused by the expression of neighboring gene is adjusted by the promoter that it is carried, improve L-lysine yield.

Description

A kind of corynebacterium glutamicum construction method improving L-lysine yield and stability
Technical field
The present invention relates to a kind of construction methods of corynebacterium glutamicum for improving L-lysine yield and stability, belong to Bioengineering field.
Background technique
L-lysine is white or the crystalline powder that near-white flows freely, and is one of important component of protein, Be humans and animals body cannot itself synthesis but highly desirable one of eight kinds of amino acid, because often lacking in food, so again Referred to as " the first essential amino acid ".L-lysine has different physiological roles, such as has amino acid composition, adjusts in vivo Metabolic balance improves body to the absorption of grain protein and utilization rate and promotion body growth development etc., thus is answered extensively For fields such as feed addictive, food additive and pharmacy.Since the market demand is vigorous, L-lysine has become only secondary at present In the second largest amino acid kind of glutamic acid, about 3,500,000 tons of annual output.The method of industrial production L-lysine mainly has three Kind, i.e. albumen hydrolysis, chemical synthesis and microbe fermentation method.Wherein microbe fermentation method is due to having low cost, high yield The advantages such as rate, low pollution become the main stream approach of current L-lysine production.
The microorganism for being presently available for L-lysine production is mainly bacterium, including corynebacteria, brevibacterium, read coccus, Pseudomonad, bacillus, Escherichia etc., wherein being applied at present the most with belonging to the corynebacterium glutamicum of corynebacterium Extensively.Although nature microorganism can direct fermentation generate L-lysine, yield is usually lower, it is therefore desirable to microorganism It is transformed.As Chinese invention patent CN1539015 disclose it is more including accBC, accDA, catA, cysD, cysE A gene that can be used for lysine production raising improves site.Chinese patent literature CN101600796(application number 200780048626.8), CN1974760(application number 200610163142.5), CN103243042(application number 201310092638.8) inactivation is carried out to gene NCg22534, NCgl1835, NCg21090 respectively and obtains a series of L-lysines The corynebacterium glutamicum engineering bacteria that yield improves.Although at present there are many report for improving L-lysine yield, L- relies Propylhomoserin still has the necessity further increased, in addition, existing L-lysine-producing bacteria kind is saving and having bacterium more in fermentation process Kind degradation phenomena, thus still need to improve.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of glutamic acid for improving L-lysine yield and stability is rodlike The construction method of bacillus.The present invention is obtained by transformation bacterial strain corynebacterium glutamicum, and specific strategy is that inactivation glutamic acid is rodlike Bacillus endogenous gene Tnp7a(encodes a kind of ISL3 swivel base zymoprotein, is induced by factors such as poor environments) to reduce glutamic acid rod The swivel base effect that because of environment change activates insetion sequence of the shape bacillus in preservation and fermentation process, blocking gene group is due to turning Seat generate mutation or frameshit and by its carry promoter adjust neighboring gene expression caused by spawn degeneration into Journey, and then improve L-lysine yield.
Term explanation
In the present invention, " inactivation " can be induced by any method for deactivating known in the art." inactivation " generate effect be Refer to that the expression of endogenous gene Tnp7a is lowered to very low level, or generation expressing gene and although is not expressed but table Up to the product for not having activity or activity reduction.
In the present invention, " inactivation " can be inserted into one or more base-pairs by genetic engineering means in gene Tnp7a, Perhaps one or more base-pairs in gene Tnp7a are lacked or converted by gene internal sequence or transversion obtain.
Technical solution of the present invention is as follows:
The construction method for improving the corynebacterium glutamicum of L-lysine yield and stability, is by corynebacterium glutamicum (Corynebacterium glutamicum) Tnp7a gene inactivation after construct obtain, the nucleotide sequence of Tnp7a gene is such as Shown in SEQ ID NO. 1.
It is preferred according to the present invention, the corynebacterium glutamicum (Corynebacterium glutamicum) derive from Chinese industrial Microbiological Culture Collection administrative center, bacterium numbering CICC 23604.
Preferred according to the present invention, above-mentioned construction method, steps are as follows:
(1) genetic fragment by a segment length in PCR amplification Tnp7a gene encoder block greater than 300bp is as homology arm sequence, The nucleotide sequence of Tnp7a gene encoder block is as shown in SEQ ID NO. 2, amino acid such as 3 institute of SEQ ID NO. of coding Show；
(2) pass through PCR amplification resistance label genetic fragment；
(3) by resistance label genetic fragment made from homology arm sequence made from step (1) and step (2) carry out over-lap PCR into Row connection, identical restriction enzyme digestion sites are contained in two ends that fusion segment is made, and the restriction enzyme site is not Quasi- knock out in gene and resistance label gene can be appeared in；
(4) corynebacterium glutamicum competent cell is prepared, fusion made from step (3) is converted into glutamic acid after digestion Corynebacteria competent cell to get.
According to the present invention it is further preferred that PCR amplification is in the step (1) with corynebacterium glutamicum (Corynebacterium glutamicum) genomic DNA be template, obtain homology armTnp1, the core of PCR amplification primer Nucleotide sequence is as follows:
F1: CGGGATCCCGTTTTTCATGCTTGCCGTCT；
R1: AAGGCCAGCAAAA GCTAAAGCCCAAGAATGCAAC；
The reaction system of PCR amplification is as follows, and total system is 50 μ l:
2×HiFi-PCR master 25μl
F1(10 μm of ol/L) 2 μ l
R1(10 μm of ol/L) 2 μ l
2 μ l of template
dd H2O 19μl；
PCR amplification program is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 30sec, 30 recycle；72 DEG C are prolonged Stretch 10min, 4 DEG C of preservations.
Preferred according to the present invention, in the step (2), PCR amplification template is shuttle plasmid pHT01(precious purchased from Hangzhou Match Biotechnology Co., Ltd) DNA, obtain chloramphenicol resistance gene Cmr；The nucleotide sequence of PCR amplification primer is as follows:
F2: GGTGGTCGGCATTTTTGCTGGCCTTTTGCTCA；
R2: CATAATCGGCTGGATCCTAGTGACTGGCGATGCT；
The reaction system of PCR amplification is as follows, and total system is 50 μ l:
2×HiFi-PCR master 25μl
F2(10 μm of ol/L) 2 μ l
R2(10 μm of ol/L) 2 μ l
2 μ l of template
dd H2O 19μl；
The PCR amplification program is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 3min, 30 recycle；72 DEG C of extensions 10min, 4 DEG C of preservations.
Preferred according to the present invention, in the step (3), the first stage amplification system of over-lap PCR is as follows, and total system is 25 μ l:
2×HiFi-PCR master 12.5μl
Tnp1 2μl
Cmr 2μl
dd H2O 8.5μl
The first stage amplification program of over-lap PCR is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 2min, 5 recycle；72 DEG C of extensions 10min；
The second stage amplification system of over-lap PCR is overall for following ingredient is added on the basis of the product after amplification in the first stage System is 50 μ l:
2×HiFi-PCR master 12.5μl
F1(10 μm of ol/L) 2 μ l
R2(10 μm of ol/L) 2 μ l
dd H2O 8.5μl
The second stage amplification program of over-lap PCR is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 58 DEG C of annealing 30sec, 72 DEG C of extension 4min, 30 recycle；72 DEG C of extensions 10min, 4 DEG C of preservations.
Preferred according to the present invention, specific step is as follows for the step (4):
(i) picking corynebacterium glutamicum (Corynebacterium glutamicum) single colonie, it is trained in seed culture medium It supports to cell concentration OD600It is 0.7~0.9, is placed in cooled on ice, is centrifuged after cooling, turns buffer washing thalline with the electricity of pre-cooling 3~5 times, electricity turns buffer and thallus is resuspended, and competent cell is made；
(ii) single endonuclease digestion, 28~32 DEG C of 2~5h of digestion, electroporation to step are carried out to fusion made from step (3) In competent cell made from rapid (i), move into liquid resuscitation culture medium, after 28~32 DEG C of 3~4h of culture, screen to get.
According to the present invention it is further preferred that single endonuclease digestion system is as follows in the step (ii), total system is 40 μ l:
10×K Buffer 4μl
Restriction enzymeBamH I 4μl
Tnp1-Cmr(fusion) 20 μ l
ddH2O 12μl。
According to the present invention it is further preferred that in the step (i), seed culture medium, every liter of component is as follows:
8~12g of peptone, 4~6g of yeast powder, 8~12g of sodium chloride, 85~96 g of sorbierite.
According to the present invention it is further preferred that in the step (i), electricity turns buffer, and every liter of component is as follows:
85~96 g of sorbierite, 85~96 g of mannitol, 95~105 mL of glycerol.
According to the present invention it is further preferred that in the step (i), the condition of electroporation are as follows: 2100 V electric shock 5ms。
According to the present invention it is further preferred that liquid resuscitation culture medium, every liter of component are as follows in the step (ii):
8~12g of peptone, 4~6g of yeast powder, 8~12g of sodium chloride, 85~96 g of sorbierite, 65~73g of mannitol.
The corynebacterium glutamicum engineering bacteria of high-yield L-lysine made from above-mentioned construction method is in production L-lysine Application.
Construct principle
For insetion sequence under the mediation of transposase, swivel base is inserted into gene coding region, generates mutation or frameshit may cause gene Inactivation.In addition, insetion sequence can also swivel base to upstream region of gene, hybridize starting by own promoter or with downstream gene formation Son, to adjust the expression of neighboring gene.The transposition as caused by transposase mostly occurs when strain is exposed to poor environment, and The change for usually causing strain genome leads to spawn degeneration, reduces the ability for even losing production purpose product.Inventor is logical After crossing the knockout of research discovery corynebacterium glutamicum endogenous gene Tnp7a, the stability in strain fermentation latter stage can be improved, increase Add L-lysine yield.
Beneficial effect
The present invention provides a kind of construction method of corynebacterium glutamicum for improving L-lysine yield and stability, and building is somebody's turn to do Bacterial strain is since endogenous gene Tnp7a inactivation causes to reduce swivel base effect, and compared with wild type, which synthesizes end in L-lysine The stability of phase has certain enhancing, and then improves L-lysine yield, reduces production cost.
Specific embodiment
Technical solution of the present invention is further elaborated below with reference to embodiment, but institute's protection scope of the present invention is not limited to This.
Embodiment 1: gene knockout segment building
1) (i) extract corynebacterium glutamicum (Corynebacterium glutamicum) 23604 genomic DNAs, with the base Because group DNA is template, PCR amplification is carried out, homology arm is obtainedTnp1；
The PCR primer sequence is as follows:
F1: CGGGATCCCTGGGGGATCTACCAGCGGA；
R1: CATGTGAGCAAAAGGCCAGCAAAAGAATCCGCCGGAACTCG；
The PCR amplification system are as follows:
Reagent Volume (μ L)
2×HiFi-PCR master 25
F1(10 μm of ol/L) 2
R1(10 μm of ol/L) 2
Template 2
dd H2O 19
The PCR amplification program is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 30sec, 30 recycle；72 DEG C are prolonged Stretch 10min, 4 DEG C of preservations；
Agarose gel electrophoresis examine PCR product, length is about 350bp, using SanPrep pillar DNA plastic recovery kit (on Glue recycling Hai Shenggong) is carried out, -20 DEG C of recovery product preservations are spare；
(ii) extract shuttle plasmid pHT01(be purchased from Hangzhou Bao Sai Biotechnology Co., Ltd) DNA, using DNA as template, carry out PCR amplification obtains CmrSegment；
The PCR primer sequence is as follows:
F2: CGAGTTCCGGCGGATTCTTTTGCTGGCCTTTTGCTCACATG；
R2: CGGGATCCTAGTGACTGGCGATGCT；
The PCR amplification system are as follows:
Reagent Volume (μ L)
2×HiFi-PCR master 25
F2(10 μm of ol/L) 2
R2(10 μm of ol/L) 2
Template 2
dd H2O 19
The PCR amplification program is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 3min, 30 recycle；72 DEG C of extensions 10min, 4 DEG C of preservations；
Agarose gel electrophoresis examines PCR product, and length is about 1300bp, uses SanPrep pillar DNA plastic recovery kit (the raw work in Shanghai) carries out glue recycling, and -20 DEG C of recovery product preservations are spare；
It (iii) will be made from step (i)Tnp1Cm made from segment and step (ii)rSegment carries out over-lap PCR, is madeTnp1- CmrSegment；
The amplification system of the over-lap PCR are as follows:
Reagent Volume (μ L)
(1) 2×HiFi-PCR master 12.5
Tnp1 2
Cmr 2
dd H2O 8.5
(2) product after first stage amplification 25
2×HiFi-PCR master 12.5
F1(10 μm of ol/L) 2
R2(10 μm of ol/L) 2
dd H2O 8.5
The first stage amplification program of the over-lap PCR is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 2min, 5 recycle；72 DEG C of extensions 10min；
The second stage amplification program of the over-lap PCR is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 58 DEG C of annealing 30sec, 72 DEG C of extension 4min, 30 recycle；72 DEG C of extensions 10min, 4 DEG C of preservations；
Agarose gel electrophoresis examine PCR product, length 1572bp, using SanPrep pillar DNA plastic recovery kit (on Glue recycling Hai Shenggong) is carried out, -20 DEG C of recovery product preservations are spare；
Embodiment 2: preparation bacillus licheniformis competence
(i) picking corynebacterium glutamicum (Corynebacterium glutamicum) 23604 single colonies, it is inoculated in 10mL In seed culture medium, 37 DEG C, 220r/min are incubated overnight；
Seed culture medium, component are as follows:
Peptone 10g, yeast powder 5g, sodium chloride 10g, 91 g of sorbierite.
(ii) take the above-mentioned bacterium solution of 1mL to be transferred in 100mL seed culture medium, 37 DEG C, 220r/min cultivates to OD600=0.9；
(iii) bacterium solution is transferred to 100mL centrifuge tube, ice bath 15-20min makes thallus stop growing；
4 DEG C after (iv) ice bath, 5000g, 5min centrifugation, collect thallus；
(v) the electricity of the thallus pre-cooling after being centrifuged turns buffer (ETM) and washs 3 times；
Electricity turns buffer, and every liter of component is as follows:
91 g of sorbierite, 91 g of mannitol, 100 mL of glycerol.
(vi) after washing, turn buffer using 1000 μ L electricity and thallus is resuspended；
(vii) the competent cell prepared is dispensed into the 100 every pipes of μ L, -80 DEG C of preservations are spare.
Embodiment 3:Tnp1-CmrSegment electrotransformation corynebacterium glutamicum (Corynebacterium glutamicum) 23604
(i) willTnp1-CmrSegment restriction enzymeBamH I, 30 DEG C of digestion 3h；
Digestion system (40 μ L) is as follows:
Reagent Volume (μ L)
10×K Buffer 4
BamH I 4
Tnp1-Cmr 20
ddH2O 12
(ii) digestion products are concentrated and purified
(1) 1/10 volume 3M sodium acetate and 2.5 times of volume dehydrated alcohols are added, are placed in -20 DEG C of refrigerator 20min；
(2) 12000r/min, centrifugation 5min must be precipitated；
Precipitating is resuspended in the ethyl alcohol that (3) 300 μ L percents by volume are 75%；
(4) 12000r/min, centrifugation 5min, removing ethyl alcohol, 37 DEG C of air-dried 30min,
(5) 15~18 μ L ddH are added2DNA is resuspended in O, is placed in -20 DEG C of preservations.
(iii) electrotransformation
It is measured first with nucleic acid ultramicrospectrophotometerTnp1-CmrFragment concentrations reach 2100 V after 300 μ g/mL concentration Shock by electricity 5ms, carries out electrotransformation and takes 100 μ L to be coated on after obtained cell is using 30 DEG C of recovery culture 1h of recovery medium and contain It on the LB solid medium of 200 μ g/mL chloramphenicol, is cultivated 2 days at 37 DEG C, screens the transformant with chlorampenicol resistant.
Liquid resuscitation culture medium, every liter of component are as follows:
Peptone 10g, yeast powder 5g, sodium chloride 10g, sorbierite 91g, mannitol 69.4g.
Embodiment 4: the culture and identification of positive restructuring bacterium
The above-mentioned positive restructuring bacterium colony of picking is inoculated into the LB liquid medium containing 200 μ g/mL chlorampenicol resistants and cultivated for 37 DEG C Night after the completion of culture, extracts recombinant bacterium DNA using the kit that Shanghai bioengineering Co., Ltd provides, and with the base of acquisition Because group is template, F1And R2PCR amplification is carried out for primer, amplified production is verified using agarose gel electrophoresis；
The PCR primer sequence is as follows:
F1: CGGGATCCCTGGGGGATCTACCAGCGGA；
R2: CGGGATCCTAGTGACTGGCGATGCT；
Wherein, underscore mark is restriction enzyme site
The PCR amplification system is 20 μ l:
Reagent Volume (μ L)
2×HiFi-PCR master 10
F1(10 μm of ol/L) 1
R2(10 μm of ol/L) 1
Template 1
dd H2O 7
The PCR amplification program is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 57 DEG C of annealing 30sec, 72 DEG C of extension 4min, 30 recycle；72 DEG C of extensions 10min, 4 DEG C of preservations；
Agarose gel electrophoresis examines PCR product, the results show that using primers F1And R2A specific gene can be amplified Band, size are about 1600b, close with theoretical value 1572bp, show that target gene is successfully integrated into corynebacterium glutamicum On genome, the corynebacterium glutamicum engineering bacteria of high-yield L-lysine is made.
The test of embodiment 4:L- fermenting lysine
The corynebacterium glutamicum engineering 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 seed culture 20h is carried out at 30 DEG C of 220rpm, thereafter 100 mL fermentation mediums (glucose 100g/L, peptone 20g/L, corn pulp are seeded to by 2% inoculum concentration of percent by volume 30 mL, urea 5 g/L, (NH4)2SO425g/L, L-Leu 0.34g/L, KH2PO42g/L, MgSO4·7H2O 1.5g/ L, biotin 0.001g/L) fermented and cultured 48h, it is sampled every 12h, and pass through the Shandong Province bio-sensing analyzer SBA-40C( Academy of sciences's biological study is made) measurement fermentation liquid in L-lysine content, as a result as shown in Table 1.
The yield of table one different time corynebacterium glutamicum engineering bacteria and original bacteria L-lysine
Strain fermentation time 12h 24h 36h 48h
Engineering bacteria 3.9 g/L 16.6 g/L 42.1 g/L 46.8 g/L
Original bacteria 4.1 g/L 18.0 g/L 39.7 g/L 41.3 g/L
As the result is shown compared with original bacteria, after the 48h that ferments, L-lysine contains in corynebacterium glutamicum engineering bacterium fermentation liquid Amount reaches 46.8 g/L, yield when 36h compared with improve 4.7 g/L, and yield of the original bacteria in 48h substantially with 36h base Originally maintain an equal level, this can be by maintaining bacterial activity to improve the production in L-lysine fermentation later period after showing Tnp7a gene inactivation Sour water is flat.
Sequence table
<110>Zhucheng Dongxiao Biotechnology Co., Ltd.
<120>a kind of construction method for the corynebacterium glutamicum for improving L-lysine yield and stability
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 584
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GCAGTATGGATTAGCAGCAGCGTAAGAAGAAAAATCAATCAAGCTGTCTCAAAAACTTGA 120
CGAGCGCAACCTGGGGGATCTACCAGCGGATGATCGCGGCCTACCGCGAGAAGGACCGAT 180
CCCTCGGCCGCGCGGCGATGGAGGCGCTCATCGACGCCGTCAGCCAAGACGTCCCCGCCG 240
GGCTGGACGAGTTGCGCAAGCTCGGTCGGACCCTGAAGGCTCGCGCCACCGACGTGCTGG 300
CCTACTTCGAGCGGCCTGGCACCAGCAATGGCCCCACAGAGGCGATCAACGGACGCCTGG 360
AGCACCTGCGCGGCTCGGCCCTGGGCTTCCGCAACCTGACCAACTACATCGCCAGATCCC 420
TGCTCGAGTTCCGGCGGATTCAGACCTCAACTACACCCTCATCTGTGAAGAGCCGCTTTA 480
GACATCCCTCATCGTCACGGACCACTATGAACGATGTCCCGACTCACCTATGAACGATGT 540
CCTGAACCTACACACCTTCAGCTCCACCAATATGTTCAGGCAAT 584
<400> 1
<210> 2
<211> 138
<212> DNA
<213>corynebacterium glutamicum (Glutamate Bacillus)
<220>
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<223> TTGACGAGCGCAACCTGGGGGATCTACCAGCGGATGATCGCGGCCTACCGCGAGAAGGAC 60
CGATCCCTCGGCCGCGCGGCGATGGAGGCGCTCATCGACGCCGTCAGCCAAGACGTCCCC 120
GCCGGGCTGGACGAGTTGCGCAAGCTCGGTCGGACCCTGAAGGCTCGCGCCACCGACGTG 180
CTGGCCTACTTCGAGCGGCCTGGCACCAGCAATGGCCCCACAGAGGCGATCAACGGACGC 240
CTGGAGCACCTGCGCGGCTCGGCCCTGGGCTTCCGCAACCTGACCAACTACATCGCCAGA 300
TCCCTGCTCGAGTTCCGGCGGATTCAGACCTCAACTACACCCTCATCTGTGAAGAGCCGC 360
TTTAGACATCCCTCATCGTCACGGACCACTATGAACGATGTCCCGACTCACCTATGA 417
<400> 2
<210> 3
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<212> PRT
<213>amino acid (Glutamate Bacillus)
<220>
<221> PEPTIDE
<223> MTSATWGIYQRMIAAYREKDRSLGRAAMEALIDAVSQDVPAGLDELRKLGRTLKARATDV 60 LA YFERPGTSNGPTEAINGRLEHLRGSALGFRNLTNYIARSLLEFRRIQTSTTPSSVKSR 120 FRHPSSSRTTMNDVPTHL 138
<400> 3
Met Thr Ser Ala Thr Trp Gly Ile Tyr Gln Arg Met Ile Ala Ala Tyr
1 5 10 15
Arg Glu Lys Asp Arg Ser Leu Gly Arg Ala Ala Met Glu Ala Leu Ile
20 25 30
Asp Ala Val Ser Gln Asp Val Pro Ala Gly Leu Asp Glu Leu Arg Lys
35 40 45
Leu Gly Arg Thr Leu Lys Ala Arg Ala Thr Asp Val Leu Ala Tyr Phe
50 55 60
Glu Arg Pro Gly Thr Ser Asn Gly Pro Thr Glu Ala Ile Asn Gly Arg
65 70 75 80
Leu Glu His Leu Arg Gly Ser Ala Leu Gly Phe Arg Asn Leu Thr Asn
85 90 95
Tyr Ile Ala Arg Ser Leu Leu Glu Phe Arg Arg Ile Gln Thr Ser Thr
100 105 110
Thr Pro Ser Ser Val Lys Ser Arg Phe Arg His Pro Ser Ser Ser Arg
115 120 125
Thr Thr Met Asn Asp Val Pro Thr His Leu
130 135

Claims (10)

1. a kind of corynebacterium glutamicum construction method for improving L-lysine yield and stability, which is characterized in that by paddy ammonia Sour corynebacteria (Corynebacterium glutamicum) Tnp7a gene inactivation after construct obtain, the core of Tnp7a gene Nucleotide sequence is as shown in SEQ ID NO. 1.
2. a kind of corynebacterium glutamicum construction method for improving L-lysine yield and stability as described in claim 1, It is characterized in that, the corynebacterium glutamicum (Corynebacterium glutamicum) derive from Chinese industrial microorganism Culture presevation administrative center, bacterium numbering CICC 23604.
3. a kind of corynebacterium glutamicum building side for improving L-lysine yield and stability as claimed in claim 1 or 2 Method, which is characterized in that steps are as follows:
(1) genetic fragment by a segment length in PCR amplification Tnp7a gene encoder block greater than 300bp is as homology arm sequence, The nucleotide sequence of Tnp7a gene encoder block is as shown in SEQ ID NO. 2, amino acid such as 3 institute of SEQ ID NO. of coding Show；
(2) pass through PCR amplification resistance label genetic fragment；
(3) by resistance label genetic fragment made from homology arm sequence made from step (1) and step (2) carry out over-lap PCR into Row connection, identical restriction enzyme digestion sites are contained in two ends that fusion segment is made, and the restriction enzyme site is not Quasi- knock out in gene and resistance label gene can be appeared in；
(4) corynebacterium glutamicum competent cell is prepared, fusion made from step (3) is converted into glutamic acid after digestion Corynebacteria competent cell to get.
4. a kind of corynebacterium glutamicum construction method for improving L-lysine yield and stability as claimed in claim 3, It is characterized in that, in the step (1), PCR amplification with corynebacterium glutamicum (Corynebacterium glutamicum) Genomic DNA be template, the nucleotide sequence of PCR amplification primer is as follows:
F1: CGGGATCCCTGGGGGATCTACCAGCGGA；
R1: CATGTGAGCAAAAGGCCAGCAAAAGAATCCGCCGGAACTCG；
The reaction system of PCR amplification is as follows, and total system is 50 μ l:
2×HiFi-PCR master 25μl
F1(10 μm of ol/L) 2 μ l
R1(10 μm of ol/L) 2 μ l
2 μ l of template
dd H2O 19μl；
PCR amplification program is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 54 DEG C of annealing 30sec, 72 DEG C of extension 30sec, 30 recycle；72 DEG C are prolonged Stretch 10min, 4 DEG C of preservations.
5. a kind of corynebacterium glutamicum construction method for improving L-lysine yield and stability as claimed in claim 3, It is characterized in that, PCR amplification template is the DNA of shuttle plasmid pHT01 in the step (2)；The nucleotide of PCR amplification primer Sequence is as follows:
F2: CGAGTTCCGGCGGATTCTTTTGCTGGCCTTTTGCTCACATG；
R2: CGGGATCCTAGTGACTGGCGATGCT；
The reaction system of PCR amplification is as follows, and total system is 50 μ l:
2×HiFi-PCR master 25μl
F2(10 μm of ol/L) 2 μ l
R2(10 μm of ol/L) 2 μ l
2 μ l of template
dd H2O 19μl；
The PCR amplification program is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 3min, 30 recycle；72 DEG C of extensions 10min, 4 DEG C of preservations.
6. a kind of corynebacterium glutamicum construction method for improving L-lysine yield and stability as claimed in claim 3, It is characterized in that, the first stage amplification system of over-lap PCR is as follows in the step (3), total system is 25 μ l:
2×HiFi-PCR master 12.5μl
Tnp1 2μl
Cmr 2μl
dd H2O 8.5μl
The first stage amplification program of over-lap PCR is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 2min, 5 recycle；72 DEG C of extensions 10min；
The second stage amplification system of over-lap PCR is overall for following ingredient is added on the basis of the product after amplification in the first stage System is 50 μ l:
2×HiFi-PCR master 12.5μl
F1(10 μm of ol/L) 2 μ l
R2(10 μm of ol/L) 2 μ l
dd H2O 8.5μl
The second stage amplification program of over-lap PCR is as follows:
95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 58 DEG C of annealing 30sec, 72 DEG C of extension 4min, 30 recycle；72 DEG C of extensions 10min, 4 DEG C of preservations.
7. a kind of corynebacterium glutamicum construction method for improving L-lysine yield and stability as claimed in claim 3, It is characterized in that, specific step is as follows for the step (4):
(i) picking corynebacterium glutamicum (Corynebacterium glutamicum) single colonie, it is trained in seed culture medium It supports to cell concentration OD600It is 0.7~0.9, is placed in cooled on ice, is centrifuged after cooling, turns buffer washing thalline with the electricity of pre-cooling 3~5 times, electricity turns buffer and thallus is resuspended, and competent cell is made；
(ii) single endonuclease digestion, 28~32 DEG C of 2~5h of digestion, electroporation to step are carried out to fusion made from step (3) In competent cell made from rapid (i), move into liquid resuscitation culture medium, after 28~32 DEG C of 3~4h of culture, screen to get.
8. a kind of corynebacterium glutamicum construction method for improving L-lysine yield and stability as claimed in claim 7, It is characterized in that, single endonuclease digestion system is as follows in the step (ii), total system is 40 μ l:
10×K Buffer 4μl
Restriction enzymeBamH I 4μl
Tnp1-Cmr 20μl
ddH2O 12μl。
9. a kind of corynebacterium glutamicum construction method for improving L-lysine yield and stability as claimed in claim 7, It is characterized in that, in the step (i), seed culture medium, every liter of component is as follows:
8~12g of peptone, 4~6g of yeast powder, 8~12g of sodium chloride, 85~96 g of sorbierite；
Preferably, in the step (i), electricity turns buffer, and every liter of component is as follows:
85~96 g of sorbierite, 85~96 g of mannitol, 95~105 mL of glycerol；
Preferably, in the step (i), the condition of electroporation are as follows: 2100 V electric shock 5ms；
Preferably, liquid resuscitation culture medium in the step (ii), every liter of component are as follows:
8~12g of peptone, 4~6g of yeast powder, 8~12g of sodium chloride, 85~96 g of sorbierite, 65~73g of mannitol.
10. a kind of corynebacterium glutamicum construction method system for improving L-lysine yield and stability described in claim 1 Application of the corynebacterium glutamicum engineering bacteria of the high-yield L-lysine obtained in production L-lysine.
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CN113278572A (en) * 2021-05-27 2021-08-20 齐鲁工业大学 Recombinant corynebacterium for modifying 5' -terminal sequence of HTS gene and application thereof
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