CN106367432B - The method of fermenting and producing L lysines and the bar bacterium of transformation - Google Patents
The method of fermenting and producing L lysines and the bar bacterium of transformation Download PDFInfo
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- CN106367432B CN106367432B CN201610800601.XA CN201610800601A CN106367432B CN 106367432 B CN106367432 B CN 106367432B CN 201610800601 A CN201610800601 A CN 201610800601A CN 106367432 B CN106367432 B CN 106367432B
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Abstract
The invention provides the method for fermenting and producing L lysines, it includes transforming the gene of coding NCBI reference sequences NP_601029.1 and/or NP_599350.1 on corynebacterium genus bacteria chromosome, makes NP_601029.1 and/or NP_599350.1 activity and/or expression quantity reduction；With, with obtained from transformation bacterial fermentation produce l lysines.In addition, present invention also offers the methods and applications as derived from this method, and the bacterium that can be used in these methods and applications etc..
Description
Technical field
The invention belongs to field of amino acid fermentation, specifically, the present invention relates to the method for fermentation production of L-lysine and
Using, and the bacterium that can be used in these methods and applications etc..
Background technology
By the bacterium for producing 1B（Such as, the bacillus of the Escherichia coli of Escherichia and Corynebacterium）Fermentation
Commercial application is had been obtained for produce 1B.These bacteriums, can be from nature separate bacterium or
Bacterium obtained by mutagenesis or genetic engineering transformation, or both haves both at the same time.
The bacterium of production 1B includes the bacterium of Corynebacterium.For example, Chinese patent CN1017906B discloses production
The method of 1B, is synthesized including the use of the synzyme of dipicolinic acid containing synthesizing dihydro and/or succinyl kaikiaine
The step of Corynebacterium sp. bacteria of the recombinant DNA of enzyme is to ferment.
The method that Chinese patent application CN1187539A discloses production 1B, including the use of containing codes for aspartate
The step of corynebacterium genus bacteria of the recombinant DNA of kinases and coding diaminapimelate decarboxylase is to ferment.
The method that Chinese patent application CN1310234A discloses production 1B, including the use of containing α-ketoglutaric acid
The step of corynebacterium genus bacteria of the gene of dehydrogenase is to ferment.
The method that Chinese patent application CN1890372A discloses production 1B, including the use of the phosphorus of fructose -1,6- bis-
Phytase activity increases the step of Corynebacterium glutamicum is to ferment.
Chinese patent application CN101065484A discloses the rod campylobacter bacteria with the ability for producing l-amino acid, its
It is modified so that acetyl coenzyme A hydrolase activity is reduced.
Chinese patent application CN101855357A disclose production 1B method, including the use of encoding fructose-
There is the step of Corynebacterium glutamicum of mutation is to ferment on the ptsF genes of PTS enzymes.
The method that Chinese patent application CN104245921A discloses production 1B, it is different including the use of the xylose containing coding
The step of corynebacterium genus bacteria of the gene of structure enzyme and Xylulokinase is to ferment.
The document of above-mentioned production 1B is all based on the enzyme or gene of known biological function.
In the full-length genome for the Corynebacterium glutamicum ATCC 13032 that completion has been sequenced（Reference can be made to NCBI reference sequences：
NC_003450.3）In, there are about 3057 genes.However, in these genes, in addition to the clear and definite gene of biological function, having
About 1196 gene code biological functions are unclear " assuming that protein ".In these " assuming that protein ", have about
The protein of 236 gene codes by being annotated by bioinformatics method to show that they are similar to some protein or enzyme,
Or include some domains.However, the biological function of the protein of still old about 960 gene codes is not biological by enlightenment
Function is learned, their effectiveness in fermentation production of L-lysine are not known yet.
The present inventor experienced numerous failures, relied on some fortune, chanced on by studying for a long period of time and putting into practice
Raising 1B can aid in the transformation of the gene of two codings " assuming that protein " in the chromosome of bar bacterium
Yield.This method does not conflict with the chromosome transformation site of the bacterium of a large amount of high-yield L-lysines of existing transformation, can fold
Plus the effect improved, so as to can be used for various bacteria fermentation production of L-lysine in practice.
The content of the invention
The technical problem to be solved in the present invention is the method for providing new fermentation production of L-lysine and its related side
Method, including relative to the method that bacterium improves the fermenting and producing amount of 1B is not transformed, the bacterium of transformation is in fermenting and producing L-
Application in lysine, the bacterium of transformation relative to not transforming the application that bacterium improves the fermenting and producing amount of 1B, and/
Or, method of transformation bacterium etc..
Specifically, in a first aspect, the invention provides the method for fermentation production of L-lysine, it includes：
（1）Transform coding NCBI reference sequences NP_601029.1 and/or NP_ on corynebacterium genus bacteria chromosome
599350.1 gene, makes NP_601029.1 and/or NP_599350.1 activity and/or expression quantity reduction；With,
（2）Use step（1）Bacterial fermentation obtained from transformation produces 1B.
Herein, term " transformation " refers to it being that the object being accordingly modified changes, so as to reach certain effect
Really.The means that transformation is located at the gene on chromosome include but is not limited to, and mutagenesis, rite-directed mutagenesis, and/or homologous recombination are excellent
Choosing is both rear.The gene that transformation is located on chromosome make it that the nucleotide sequence of the gene is added, lacks or replaced one
Or multiple nucleotides, for example nonsense codon can be inserted in the gene, the gene can also be knocked out.Transformation can also be passed through
The regulating and controlling sequence of the gene transforms the gene indirectly, so that activity and/or the expression quantity reduction of the protein of its coding.
These technological means are recorded in molecular biology and microbiology document extensively, there is many even commercializations
., can be using the commercialization of Addgene companies according to the principle of homologous recombination in the embodiment of the present invention
PKOV pUC pUCs are transformed, it would however also be possible to employ pK18mobsacBPUC pUC is transformed.Therefore, herein,
Transform the transformation carried out preferably through homologous recombination, the knockout more preferably carried out by homologous recombination.
Herein, NCBI reference sequences NP_601029.1（Referred to as NP_601029.1）Be it is a kind of " assuming that albumen
Matter ", its amino acid sequence such as SEQ ID NO：Shown in 1（Also website http can be obtained from://www.ncbi.nlm.nih.gov,
NP_601029.1）.Encode NP_601029.1 gene（It is complementary）Nucleotide sequence such as SEQ ID NO：Shown in 2（Also it can obtain
It is derived from website http://www.ncbi.nlm.nih.gov,NCgl1751）.Although NP_601029.1 concrete activity is also not
Know, but in the embodiment of the present invention,NCgl1751After gene is knocked（That is, its activity and/or expression quantity disappears
Lose）, the output increased of lysine.Therefore, herein, NP_601029.1 activity and/or expression quantity preferably disappears.
Herein, NCBI reference sequences NP_599350.1（Referred to as NP_599350.1）Be it is a kind of " assuming that albumen
Matter ", its amino acid sequence such as SEQ ID NO：Shown in 3（Also website http can be obtained from://www.ncbi.nlm.nih.gov,
NP_599350.1）.Encode NP_599350.1 gene（It is complementary）Nucleotide sequence such as SEQ ID NO：Shown in 4（Also it can obtain
It is derived from website http://www.ncbi.nlm.nih.gov,NCgl0097）.Although NP_599350.1 concrete activity is also not
Know, but in the embodiment of the present invention,NCgl0097After gene is knocked（That is, its activity and/or expression quantity disappears
Lose）, the output increased of lysine.Therefore, herein, NP_599350.1 activity and/or expression quantity preferably disappears.
Correspondingly, present invention also offers others application or method.For example, in second aspect, the invention provides carry
The method of the amount of fermentation of high 1B, it includes：
（1）Transform coding NCBI reference sequences NP_601029.1 and/or NP_ on corynebacterium genus bacteria chromosome
599350.1 gene, makes NP_601029.1 and/or NP_599350.1 activity and/or expression quantity reduction, preferably disappears；
With,
（2）Use step（1）Bacterial fermentation obtained from transformation produces 1B.
And for example, in the third aspect, the invention provides application of the bacterium of transformation acquisition in fermentation production of L-lysine,
Wherein, it is described transformation acquisition be transformation corynebacterium genus bacteria chromosome on coding NCBI reference sequences NP_601029.1 and/or
NP_599350.1 gene, makes NP_601029.1 and/or NP_599350.1 activity and/or expression quantity reduction, preferably disappears
Lose.
Also such as, in fourth aspect, the invention provides the bacterium of transformation acquisition in the amount of fermentation for improving 1B
Using, wherein, the transformation acquisition is coding NCBI reference sequences NP_601029.1 on transformation corynebacterium genus bacteria chromosome
And/or NP_599350.1 gene, make NP_601029.1 and/or NP_599350.1 activity and/or expression quantity reduction, it is excellent
Choosing disappears.
Herein, such as it is not particularly limited（As do not limited with " transformation is obtained "）, term " bacterium " or " Corynebacterium is thin
Bacterium " is the bacterium or corynebacterium genus bacteria before not transforming or transform, and its chromosome has the coding NCBI reference sequences of wild type
NP_601029.1 and/or NP_599350.1 gene.
1B is as the important metabolite of bacterium, and most of corynebacterium genus bacterias can more or less ferment production
Raw a certain amount of 1B.Prior art did not pay close attention to coding NCBI reference sequences NP_ in lysine production/fermentation
601029.1 and/or NP_599350.1 gene, therefore the corynebacterium genus bacteria of production 1B of the prior art is usual all
The gene of coding NCBI reference sequences NP_601029.1 and/or NP_599350.1 with wild type are substantially all and can used
The method of the present invention is transformed, and improves the amount of fermentation of 1B.Herein, corynebacterium genus bacteria includes glutamic acid bar
Bacterium or Beijing corynebacterium, preferably Corynebacterium glutamicum.
More constitutionally, at the 5th aspect, the invention provides the method for transformation bacterium, it includes transforming corynebacterium genus bacteria
Method, it includes coding NCBI reference sequences NP_601029.1 and/or NP_ on transformation corynebacterium genus bacteria chromosome
599350.1 gene, makes NP_601029.1 and/or NP_599350.1 activity and/or expression quantity reduction, preferably disappears.
The bacterium that the method for fifth aspect present invention is transformed and obtained can be used in fermenting and producing or produce l- lysines.Cause
This, at the 6th aspect, the bacterium obtained the invention provides the transformation of the method for fifth aspect present invention.Sixth aspect present invention
Bacterium be corynebacterium genus bacteria, encode NCBI reference sequences NP_601029.1's and/or NP_599350.1 on its chromosome
The nucleotide sequence of gene locus is different from coding NCBI reference sequences NP_601029.1 and/or NP_599350.1 gene
NCBI reference sequences NP_601029.1 and/or NP_599350.1 gene quilt are encoded on nucleotide sequence, preferably its chromosome
Knock out.
The 7th aspect, the invention provides NCBI reference sequences NP_601029.1 and/or NP_599350.1 and/or its
Application of the encoding gene in corynebacterium genus bacteria fermentation production of L-lysine.Although NCBI reference sequences NP_ may be increased
601029.1 and/or NP_599350.1 activity and/or expression quantity can be used in reduction corynebacterium genus bacteria fermenting and producing L- and rely
The yield of propylhomoserin, it is preferred that the application for NCBI reference sequences NP_601029.1 and/or NP_599350.1 activity and/
Or expression quantity reduction（It is preferred that disappearing, such as its encoding gene is knocked out）Application, for improving corynebacterium genus bacteria fermenting and producing
The yield of 1B.Wherein, NCBI reference sequences NP_601029.1 amino acid sequence such as SEQ ID NO：1, it encodes base
Cause（It is complementary）Nucleotide sequence such as SEQ ID NO：Shown in 2；NCBI reference sequences NP_599350.1 amino acid sequence is such as
SEQ ID NO：Shown in 3, its encoding gene（It is complementary）Nucleotide sequence such as SEQ ID NO：Shown in 4.
In eighth aspect, the invention provides screening on the influential gene of corynebacterium genus bacteria fermentation production of L-lysine
Method, it includes：
（1）The gene of coding " assuming that protein " on corynebacterium genus bacteria chromosome is transformed, is made " assuming that protein "
Activity and/or expression quantity rise or reduce；
（2）Use step（1）Bacterial fermentation obtained from transformation produces 1B；With,
（3）By step（2）Obtained 1B yield is entered with the 1B yield for the corynebacterium genus bacteria do not transformed
Row compares.
It is preferred that in the method for eighth aspect present invention, influence is enhancing, and make " assuming that protein " activity and/
Or expression quantity reduction, preferably disappear, will such as encode the gene knockout of " assuming that protein ".
The beneficial effects of the present invention are open up and facts have proved the side of the amount of fermentation of new raising 1B
Formula, and do not conflict with the chromosome transformation site of the corynebacterium genus bacteria of a large amount of high-yield L-lysines of existing transformation, from
And can be used for the yield of further raising 1B in practice.
In order to make it easy to understand, the present invention will be described in detail by specific embodiment below.Need to refer in particular to
Go out, the description that these descriptions are merely exemplary, and be not meant to limit the scope of the invention.Opinion according to this specification
State, many changes of the invention, change and will be apparent from for one of ordinary skill in the art.
In addition, the present invention refer to open source literature, these documents are their full text in order to more clearly describe the present invention
Content is included and referred to herein, just looks like that repeated description herein has been excessively for their full text.
Embodiment
Present disclosure is further illustrated by the following examples.As do not specialized, technology used in embodiment
Conventional meanses that means are well known to those skilled in the art and commercially available common instrument, reagent, reference can be made to《Molecular Cloning: A Laboratory
Guide（3rd edition）》（Science Press）、《Microbiology Experiment（4th edition）》（Higher Education Publishing House）And corresponding instrument and
Manufacturers instruction of reagent etc. is referred to.
Embodiment 1 NCgl1751Down regulation of gene expression is tested
According to the NCBI Corynebacterium glutamicum ATCC13032 announced genome sequence, two pairs of amplifications are synthesizedNCgl1751
The primer of gene coding region two ends fragment, is used as upstream and downstream homology arm fragment.Design of primers is as follows（The handsome company's synthesis in Shanghai）：
P1:5' CCCAAGCTTCGACAGGGCTTGGATTG 3'（Hind3）
P2:5' ATGGAGAAAT ACGTCAAGGT TTTTCCTGCT CTTTAACACC 3'
P3:5'GGTGTTAAAG AGCAGGAAAA ACCTTGACGT ATTTCTCCAT 3'
P4:5' CGGGATCCCGGTGGGTTTGTTGATGT 3' （BamH1）
Using Corynebacterium glutamicum ATCC13032 as template, respectively with primer P1/P2 and P3/P4, enter performing PCR amplification, obtain
Upstream homology arm fragment 660bp and downstream homology arm fragment 780bp carry out OVER PCR with primer P1/P4 and obtain whole homologous again
Arm pieces section 1440bp, Hind3 and BamH1 restriction enzyme sites are contained at two ends respectively.After PCR reactions terminate, the product of amplification is carried out
Electrophoresis is reclaimed, the DNA fragmentation of the 1400bp required for being reclaimed using pillar DNA gel QIAquick Gel Extraction Kit, and passes through digestion
Connection is reclaimed, is connected with shuttle plasmid pk18mobsacB plasmids, obtains and knocks out plasmid.It is anti-containing kanamycins on the plasmid
Property mark.
Plasmid electricity will be knocked out and be transformed into lysine production patented strain YP97136（Its construction method can be found in
WO2014121669A1；Confirm to remain with wild type on the strain chromosome through sequencingNCgl1751Gene）, culture is produced
Single bacterium colony pass through following primer respectively（The handsome company's synthesis in Shanghai）Enter performing PCR identification：
P5:5' GGTAGTCCCACATCATCTCT 3'
P6:5' ATGCCCTGGTTGGTTCT 3'
The bacterial strain that above-mentioned PCR amplifies size 1000bp and 740bp band is positive strain, only amplifies 1000bp bars
The bacterial strain of band is opportunistic pathogen.Positive strain respectively on the culture medium containing kanamycins and without kanamycins cultivate, without
Grown on the culture medium of kanamycins, and the bacterial strain not grown on the culture medium containing kanamycins is further drawn using P5/P6
Thing enters performing PCR identification, and it is the genetic engineering that Ncgl1751 gene coding regions are knocked to amplify the bacterial strain that size is 740bp bands
Bacterial strain, it is named as YPL-1-001.
Embodiment 2NCgl0097Down regulation of gene expression is tested
According to the NCBI Corynebacterium glutamicum ATCC13032 announced genome sequence, two pairs of amplifications are synthesizedNCgl0097
The primer of gene coding region two ends fragment, is used as upstream and downstream homology arm fragment.Design of primers is as follows（The handsome company's synthesis in Shanghai）：
P11:5' CCCAAGCTTCGCAGCAGGTATGTAGTCAC 3'（Hind3）
P12:5' CACTTCATAG GGTTGAATAC AGCACGCGCA CGGAAAGCCA 3'
P13:5' TGGCTTTCCG TGCGCGTGCT GTATTCAACC CTATGAAGTG 3'
P14:5' GCTCTAGAGCGGGCATCCACAATCAT 3' （Xba1）
Using Corynebacterium glutamicum ATCC13032 as template, respectively with primer P11/P12 and P13/P14, enter performing PCR amplification,
Upstream homology arm fragment 740bp and downstream homology arm fragment 640bp is obtained to carry out OVER PCR with primer P11/P14 again and obtain whole
Contain Hind3 and BamH1 restriction enzyme sites respectively in individual homology arm fragment 1380bp, two ends.After PCR reactions terminate, the production to amplification
Thing carries out electrophoresis recovery, the DNA fragmentation of the 1380bp required for being reclaimed using pillar DNA gel QIAquick Gel Extraction Kit, and leads to
Cross digestion and reclaim connection, be connected with shuttle plasmid pk18mobsacB plasmids, obtain and knock out plasmid.Contain Ka Na on the plasmid
Resistance marker, can screen the recon obtained on plasmid integration to genome by Ka Na.
Plasmid electricity will be knocked out and be transformed into lysine production patented strain YP97136（Confirm through sequencing on the strain chromosome
Remain with wild typeNCgl0097Gene）, following primer is passed through respectively to the single bacterium colony that culture is produced（The handsome company in Shanghai closes
Into）Enter performing PCR identification：
P15:5' AACTGGGCTCTTGTTACTG 3'
P16:5' CGCTGCCGCTTCACGAT 3'
The bacterial strain that above-mentioned PCR amplifies size 1195bp and 645bp band is positive strain, only amplifies 1195bp bars
The bacterial strain of band is opportunistic pathogen.Positive strain respectively on the culture medium containing kanamycins and without kanamycins cultivate, without
Grown on the culture medium of kanamycins, and the bacterial strain not grown on the culture medium containing kanamycins further uses P15/P16
Primer enters performing PCR identification, and it is the genetic engineering bacterium that Ncgl0097 gene coding regions are knocked to amplify the bacterium that size is 645bp
Strain, it is named as YPL-1-002.
Embodiment 3NCgl1751 and NCgl0097Gene double expression lowers experiment
Based on YPL-1-001 bacterial strains, the Ncgl0097 gene coding regions on genome are knocked out, detailed process
It is identical with the knockout of above-mentioned Ncgl0097 gene coding regions, performing PCR is entered to bacterial strain by using identification primer P5/P6, P11/P12 and tested
Card, obtains the fragment that size is 740bp and 645bp respectively（Original strain PCR sizes are respectively 1000bp and 1195bp）,
The engineering strain that NCgl1751 and NCgl0097 gene coding regions are knocked, it is named as YPL-1-003, and it is paddy ammonia
Sour bar bacterium（Corynebacterium glutamicum）, Chinese microorganism strain preservation is preserved within 16th in August in 2016
Administration committee's common micro-organisms center（Address：The institute 3 of Chaoyang District Beijing North Star West Road 1）, preserving number is CGMCC No.
12856。
The fermenting lysine of embodiment 4 is tested
By the embodiment 1-3 bacterial strains built and original strain BLBIO-5GC-4-H models fermentation tank（Purchased from Shanghai hundred
Logical sequence bio tech ltd）In with shown in the culture medium shown in table 1 and table 2 process carry out fermenting experiment.Each bacterial strain weight
It is multiple three times, as a result as shown in table 3.
The fermentative medium formula of table 1
| The name of an article | Proportioning |
| Amylum hydrolysate of the sugar | 30g/l |
| Ammonium sulfate | 12g/L |
| Magnesium sulfate | 0.87g/l |
| Molasses | 20g/l |
| It is acidified corn steep liquor | 3ml/l |
| Phosphoric acid | 0.4ml/l |
| Potassium chloride | 0.53g/l |
| Defoamer（2% bubble enemy） | 4ml/L |
| Ferrous sulfate | 120mg/l |
| Manganese sulfate | 120mg/l |
| Niacinamide | 42mg/l |
| Calcium pantothenate | 6.3mg/l |
| VB1 | 6.3mg/l |
| Copper, zinc solution | 0.6g/L |
| Biotin | 0.88mg/L |
The fermentation process of table 2
The fermenting lysine experimental result of table 3
| Strain | Lysine production（%） | Conversion ratio（%） |
| YPL-1-001 | 20.0 | 64.9 |
| YPL-1-001 | 20.1 | 64.8 |
| YPL-1-001 | 20.2 | 65.1 |
| Average | 20.1 | 64.9 |
| YPL-1-002 | 20.5 | 65.2 |
| YPL-1-002 | 20.6 | 65.3 |
| YPL-1-002 | 20.8 | 65.5 |
| Average | 20.6 | 65.3 |
| YPL-1-003 | 21.8 | 66.7 |
| YPL-1-003 | 22.2 | 66.9 |
| YPL-1-003 | 22.1 | 66.9 |
| Average | 22.0 | 66.8 |
| Control | 18.8 | 64.1 |
| Control | 18.6 | 63.9 |
| Control | 18.8 | 63.7 |
| Average | 18.8 | 63.9 |
As a result as shown in table 3, the expression of NCgl1751 and/or NCgl0097 genes is lowered in bar bacterium, is both contributed to
The raising of 1B yield, wherein lowering the expression of NCgl1751 and NCgl0097 genes simultaneously, 1B yield is obtained
Maximum raising.
<110>Ningbo Eppen Biotech Co., Ltd.
<120>The method of fermentation production of L-lysine and the bar bacterium of transformation
<130> CN
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 84
<212> PRT
<213> Corynebacterium glutamicum
<400> 1
Met Ser Gln Ala Arg Arg Tyr Leu Val Gln Asp Arg Gly Val Ser Leu
1 5 10 15
Ser Asp Ala Asp Gly Val Leu Val Asp Leu Asn Phe Thr Cys Thr Gln
20 25 30
Val Asn Glu Ser Asn Asp Thr Asp Asp Leu Ser Val Phe Cys Ser Thr
35 40 45
Ala Ile Ala Gly Lys Asp Pro Ser Glu Leu Arg Lys Glu Leu Glu Ala
50 55 60
Glu Phe Tyr Phe Leu Pro Asp Gly Ala Asp Asp Ala Asp Asp Ala Asp
65 70 75 80
Asp Ala Met Gly
<210> 2
<211> 255
<212> DNA
<213> Corynebacterium glutamicum
<400> 2
ctagcccatt gcatcgtctg catcgtctgc atcgtctgca ccgtccggca aaaaatagaa 60
ttctgcttct agctctttcc gtaattcgga cggatctttt ccagcgatgg cggtactaca 120
gaaaacagat agatcatctg tgtcgttaga ctcattaacc tgggtgcatg tgaaattgag 180
atccactaaa acaccatcag catcgctgag ggaaacccct cgatcctgga caaggtatct 240
gcgcgcttga gacat 255
<210> 3
<211> 183
<212> PRT
<213> Corynebacterium glutamicum
<400> 3
Met Lys Asn Ala Lys Leu Phe Leu Ala Leu Ile Ser Ala Pro Leu Ile
1 5 10 15
Leu Ala Gly Cys Ser Ser Thr Asp Thr Gly Thr Ala Glu Ser Thr Ile
20 25 30
Ser Ser Glu Thr Ala Ser Ala Val Asp Ala Thr Thr Ser Thr Ser Ser
35 40 45
Ser Thr Ala Thr Ser Ala Val Ile Asp Asp Asp Pro Val Phe Asp Ile
50 55 60
Ile Asp Ile Val Leu Ala Gln Tyr Pro Asp Arg Ile Ile Thr Asp Ile
65 70 75 80
Asp Arg Glu Asp Ser Ser Asp Gln Tyr Glu Val Asp Val Val Val Gly
85 90 95
Gln Glu Val Leu Glu Leu Asp Val Thr Thr Ser Gly Gln Ile His Thr
100 105 110
Asp Asp Arg Asp Asn Asp Asp Asp Asp Asp Ile Arg Glu Ala His Ala
115 120 125
Ala Thr Val Thr Ala Ala Gln Ala Ile Gly Leu Ala Leu Asp Gln Tyr
130 135 140
Pro Asp Gly Ile Ile Asp Ser Val Glu Leu Asp Glu Asp Asp Gly Gln
145 150 155 160
Leu Lys Trp Lys Ile Asp Leu Asp Asp Thr Ser Gly Asn Asp Leu Ala
165 170 175
Asp Val Glu Ile Ala Ala Val
180
<210> 4
<211> 552
<212> DNA
<213> Corynebacterium glutamicum
<400> 4
ttaaactgct gcgatttcaa cgtcagcaag atcattgccg gaagtgtcat cgaggtctat 60
tttccatttc agctggccgt cgtcttcgtc taattcaaca gaatcaataa ttccgtctgg 120
gtattgatcc agcgctaggc caatggcttg agctgcggtg actgtggctg cgtgagcttc 180
gcggatgtcg tcatcatcat cgttgtcgcg gtcgtcggta tggatctggc cactggtggt 240
gacatcaagt tcaaggactt cttggccaac cacaacatcg acttcgtatt gatcggagga 300
gtcttcgcgg tcaatgtcgg tgatgatcct gtcggggtat tgggcaagga cgatgtcgat 360
gatgtcgaat accggatcgt catcaatcac ggcagaggtg gcggtacttg aggaggtaga 420
agtggtggca tctactgcag aagcagtttc gctggaaatg gtggattctg ctgttccagt 480
atcggtggag ctgcagccag cgaggataag aggagcggat atgagcgcga ggaaaagttt 540
tgcgttcttc at 552
Claims (22)
1. the method for the method of fermentation production of L-lysine or the amount of fermentation of raising 1B, it includes：
（1）Transform coding NCBI reference sequences NP_601029.1 and/or NP_599350.1 on corynebacterium genus bacteria chromosome
Gene, makes NP_601029.1 and/or NP_599350.1 activity and/or expression quantity reduction；With,
（2）Use step（1）Bacterial fermentation obtained from transformation produces 1B.
2. the method described in claim 1, wherein, make NP_601029.1 and/or NP_599350.1 activity and/or expression quantity
Disappear.
3. the method described in claim 1, wherein, coding NCBI reference sequences NP_ on transformation corynebacterium genus bacteria chromosome
601029.1 and/or NP_599350.1 gene is to be added, lack or replace one to the nucleotide sequence of the gene
Or multiple nucleotides.
4. the method described in claim 3, wherein, coding NCBI reference sequences NP_ on transformation corynebacterium genus bacteria chromosome
601029.1 and/or NP_599350.1 gene is that the nucleotide sequence of the gene is knocked out.
5. the method described in claim 1, wherein, the corynebacterium genus bacteria is Corynebacterium glutamicum.
6. application of the bacterium obtained in the amount of fermentation of fermentation production of L-lysine or raising 1B is transformed, wherein,
The transformation acquisition is coding NCBI reference sequences NP_601029.1 and/or NP_ on transformation corynebacterium genus bacteria chromosome
599350.1 gene, makes NP_601029.1 and/or NP_599350.1 activity and/or expression quantity reduction.
7. the application described in claim 6, wherein, make NP_601029.1 and/or NP_599350.1 activity and/or expression quantity
Disappear.
8. the application described in claim 6, wherein, coding NCBI reference sequences NP_ on transformation corynebacterium genus bacteria chromosome
601029.1 and/or NP_599350.1 gene is to be added, lack or replace one to the nucleotide sequence of the gene
Or multiple nucleotides.
9. the application described in claim 8, wherein, coding NCBI reference sequences NP_ on transformation corynebacterium genus bacteria chromosome
601029.1 and/or NP_599350.1 gene is that the nucleotide sequence of the gene is knocked out.
10. the application described in claim 6, wherein, the corynebacterium genus bacteria is Corynebacterium glutamicum.
11. transforming the method for corynebacterium genus bacteria, it includes encoding NCBI reference sequences on transformation corynebacterium genus bacteria chromosome
NP_601029.1 and/or NP_599350.1 gene, makes NP_601029.1 and/or NP_599350.1 activity and/or table
Up to amount reduction.
12. the method described in claim 11, wherein, make NP_601029.1 and/or NP_599350.1 activity and/or expression
Amount disappears.
13. the method described in claim 11, wherein, coding NCBI reference sequences NP_ on transformation corynebacterium genus bacteria chromosome
601029.1 and/or NP_599350.1 gene is to be added, lack or replace one to the nucleotide sequence of the gene
Or multiple nucleotides.
14. the method described in claim 13, wherein, coding NCBI reference sequences NP_ on transformation corynebacterium genus bacteria chromosome
601029.1 and/or NP_599350.1 gene is that the nucleotide sequence of the gene is knocked out.
15. the method described in claim 11, wherein, the corynebacterium genus bacteria is Corynebacterium glutamicum.
16. corynebacterium genus bacteria obtained from claim 11-15 any described method transformation.
17. the corynebacterium genus bacteria described in claim 16, wherein, the corynebacterium genus bacteria is Corynebacterium glutamicum.
18. NCBI reference sequences NP_601029.1 and/or NP_599350.1 activity and/or expression quantity are reduced in bar bacterium
Belong to the application in bacterial fermentation production 1B.
19. NCBI reference sequences NP_601029.1 and/or NP_599350.1 activity and/or expression quantity disappear in bar bacterium
Belong to the application in bacterial fermentation production 1B.
20. the method that screening has enhanced gene to corynebacterium genus bacteria fermentation production of L-lysine, it includes：
（1）The gene of coding " assuming that protein " on corynebacterium genus bacteria chromosome is transformed, makes the work of " assuming that protein "
Property and/or expression quantity reduction, the biological function of the protein of the gene code is also unclear not by enlightenment biological function
Effectiveness of the protein of Chu Suoshu gene codes in fermentation production of L-lysine；
（2）Use step（1）Bacterial fermentation obtained from transformation produces 1B；With,
（3）By step（2）Obtained 1B yield is compared with the 1B yield for the corynebacterium genus bacteria do not transformed
Compared with.
21. the method for claim 20, wherein, the activity and/or expression quantity of " assuming that protein " is disappeared.
22. the method for claim 20, wherein, the gene is NCBI reference sequences：In NC_003450.3.
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| CN201610800601.XA CN106367432B (en) | 2016-09-01 | 2016-09-01 | The method of fermenting and producing L lysines and the bar bacterium of transformation |
| ES17844780T ES2939979T3 (en) | 2016-09-01 | 2017-01-09 | Corynebacterium to produce L-lysine by fermentation |
| CA3035466A CA3035466C (en) | 2016-09-01 | 2017-01-09 | Corynebacterium for producing l-lysine by fermentation |
| EP17844780.1A EP3533875B1 (en) | 2016-09-01 | 2017-01-09 | Corynebacterium for producing l-lysine by fermentation |
| PL17844780.1T PL3533875T3 (en) | 2016-09-01 | 2017-01-09 | Corynebacterium for producing l-lysine by fermentation |
| DK17844780.1T DK3533875T5 (en) | 2016-09-01 | 2017-01-09 | CORYNEBACTERIUM FOR THE PRODUCTION OF L-LYSINE BY FERMENTATION |
| US16/329,765 US11242545B2 (en) | 2016-09-01 | 2017-01-09 | Corynebacterium for producing L-lysine by fermentation |
| PCT/CN2017/070629 WO2018040469A1 (en) | 2016-09-01 | 2017-01-09 | Corynebacterium for producing l-lysine by fermentation |
| JP2019533265A JP6739651B2 (en) | 2016-09-01 | 2017-01-09 | Corynebacterium that fermentatively produces L-lysine |
| US17/558,582 US11905540B2 (en) | 2016-09-01 | 2021-12-21 | Corynebacterium for producing L-lysine by fermentation |
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| ES2939979T3 (en) * | 2016-09-01 | 2023-04-28 | Ningxia Eppen Biotech Co Ltd | Corynebacterium to produce L-lysine by fermentation |
| CN109929887A (en) * | 2017-12-15 | 2019-06-25 | 宁夏伊品生物科技股份有限公司 | The method of improved fermentation production of L-lysine |
| CN109929888B (en) * | 2017-12-15 | 2023-07-28 | 宁夏伊品生物科技股份有限公司 | Improved fermentation production method of L-lysine |
| CN107974473B (en) * | 2017-12-15 | 2020-06-19 | 内蒙古伊品生物科技有限公司 | Fermentation production and post-treatment of glutamic acid |
| CN110607313B (en) * | 2019-09-27 | 2021-06-22 | 内蒙古伊品生物科技有限公司 | Recombinant strain for high yield of L-lysine and construction method and application thereof |
| CN111088202B (en) * | 2019-12-25 | 2022-01-04 | 南京工业大学 | Recombinant corynebacterium glutamicum for producing lysine through biological film-forming continuous fermentation and construction method thereof |
| CN111363711B (en) * | 2020-03-13 | 2022-02-11 | 南京工业大学 | Method for producing lysine by adsorption immobilized fermentation of recombinant corynebacterium glutamicum |
| CN111850010B (en) * | 2020-06-08 | 2021-04-09 | 黑龙江伊品生物科技有限公司 | dapB gene modified recombinant strain and construction method and application thereof |
| CN111979165B (en) * | 2020-08-07 | 2021-05-07 | 黑龙江伊品生物科技有限公司 | Recombinant strain for producing L-lysine and construction method and application thereof |
| KR20230042224A (en) | 2020-06-08 | 2023-03-28 | 이너 몽골리아, 에펜 바이오테크 컴퍼니 리미티드 | Recombinant strains producing L-amino acids and their construction methods and applications |
| JP2023527951A (en) | 2020-06-08 | 2023-07-03 | 黒竜江伊品生物科技有限公司 | L-lysine-producing recombinant strain, construction method and use thereof |
| EP4194545A1 (en) | 2020-08-07 | 2023-06-14 | Ningxia Eppen Biotech Co. Ltd | Recombinant strain for producing l-amino acid, and construction method therefor and use thereof |
| CN111979164B (en) * | 2020-08-07 | 2021-11-12 | 宁夏伊品生物科技股份有限公司 | Recombinant strain for producing L-lysine and construction method and application thereof |
| CN112063571B (en) * | 2020-08-14 | 2022-05-06 | 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 |
| CN112980867B (en) * | 2021-03-09 | 2023-04-11 | 中国科学院深圳先进技术研究院 | Recombinant strain for modifying corynebacterium glutamicum promoter, construction method thereof and application of recombinant strain for producing L-amino acid |
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