CN103981230B - The method of the bacterial fermentation production L-lysine of reduction and/or enzymatic activity reduction is expressed with aconitase - Google Patents
The method of the bacterial fermentation production L-lysine of reduction and/or enzymatic activity reduction is expressed with aconitase Download PDFInfo
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- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 title claims abstract description 86
- 239000004472 Lysine Substances 0.000 title claims abstract description 32
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 29
- 235000019766 L-Lysine Nutrition 0.000 title claims abstract description 26
- 108010009924 Aconitate hydratase Proteins 0.000 title claims abstract description 22
- 238000000855 fermentation Methods 0.000 title claims abstract description 21
- 230000004151 fermentation Effects 0.000 title claims abstract description 21
- 230000002255 enzymatic effect Effects 0.000 title claims abstract description 19
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 18
- 102100039868 Cytoplasmic aconitate hydratase Human genes 0.000 title abstract 2
- 230000010757 Reduction Activity Effects 0.000 title description 3
- 230000009467 reduction Effects 0.000 title description 3
- 241000894006 Bacteria Species 0.000 claims abstract description 51
- 230000009466 transformation Effects 0.000 claims abstract description 47
- 102000009836 Aconitate hydratase Human genes 0.000 claims description 20
- 241000588724 Escherichia coli Species 0.000 claims description 19
- 210000000349 chromosome Anatomy 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 15
- 125000003729 nucleotide group Chemical group 0.000 claims description 15
- 101150113917 acnA gene Proteins 0.000 claims description 8
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 claims description 6
- 108020004705 Codon Proteins 0.000 claims description 3
- 108091033319 polynucleotide Proteins 0.000 abstract description 12
- 239000002157 polynucleotide Substances 0.000 abstract description 12
- 102000040430 polynucleotide Human genes 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 description 30
- 230000000694 effects Effects 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 108091081024 Start codon Proteins 0.000 description 7
- 210000003578 bacterial chromosome Anatomy 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 6
- 238000006062 fragmentation reaction Methods 0.000 description 6
- 235000018977 lysine Nutrition 0.000 description 6
- 238000002703 mutagenesis Methods 0.000 description 6
- 231100000350 mutagenesis Toxicity 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 241000588722 Escherichia Species 0.000 description 5
- 108020005038 Terminator Codon Proteins 0.000 description 5
- 101150063416 add gene Proteins 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 238000010010 raising Methods 0.000 description 5
- 239000001124 (E)-prop-1-ene-1,2,3-tricarboxylic acid Substances 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 238000012408 PCR amplification Methods 0.000 description 4
- 229940091181 aconitic acid Drugs 0.000 description 4
- GTZCVFVGUGFEME-IWQZZHSRSA-N cis-aconitic acid Chemical compound OC(=O)C\C(C(O)=O)=C\C(O)=O GTZCVFVGUGFEME-IWQZZHSRSA-N 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- GTZCVFVGUGFEME-UHFFFAOYSA-N trans-aconitic acid Natural products OC(=O)CC(C(O)=O)=CC(O)=O GTZCVFVGUGFEME-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 150000008545 L-lysines Chemical class 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 101100054574 Corynebacterium diphtheriae (strain ATCC 700971 / NCTC 13129 / Biotype gravis) acn gene Proteins 0.000 description 1
- ODBLHEXUDAPZAU-ZAFYKAAXSA-N D-threo-isocitric acid Chemical compound OC(=O)[C@H](O)[C@@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-ZAFYKAAXSA-N 0.000 description 1
- 101100215150 Dictyostelium discoideum aco1 gene Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- ODBLHEXUDAPZAU-FONMRSAGSA-N Isocitric acid Natural products OC(=O)[C@@H](O)[C@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-FONMRSAGSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- ODBLHEXUDAPZAU-UHFFFAOYSA-N threo-D-isocitric acid Natural products OC(=O)C(O)C(C(O)=O)CC(O)=O ODBLHEXUDAPZAU-UHFFFAOYSA-N 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Abstract
The present invention provides the methods of fermentation production of L-lysine comprising transformation bacterium makes the expression quantity of its aconitase A and/or enzymatic activity reduce but not disappear；With produce L-lysine with the bacterial fermentation of transformation.In addition, the present invention also provides the methods and applications as derived from this method, and polynucleotides, carrier and the bacterium that can be used in these methods and applications.
Description
Technical field
The invention belongs to field of amino acid fermentation, specifically, the present invention relates to the method for fermenting and producing l- lysine and
Its derivative methods and applications, and polynucleotides, carrier and the bacterium that can be used in these methods and applications.
Background technique
By bacterium (e.g., the bacillus of the Escherichia coli of the Escherichia and Corynebacterium) fermentation for producing l- lysine
Industrial application has been obtained to produce l- lysine.These bacteriums can be the bacterium separated from nature, be also possible to
Have both at the same time by the bacterium, or both that mutagenesis or genetic engineering transformation obtain.In current document report, pass through gene work
The attention of journey transformation is concentrated mainly onpnt、dapAndppcOn equal genes, has no for L-lysine production and pay close attention to aconitic acid
Enzyme (e.g., aconitase A) and its encoding gene.
Aconitase is an enzyme in tricarboxylic acid cycle, and two step of enzymatic chemical reaction, respectively citric acid convert
Isocitric acid is converted into for aconitic acid and aconitic acid.It is currently known in Escherichia,acnA(its nucleotide sequence is such as gene
Shown in SEQ ID No:1) coding aconitase A, but may be since its metabolism is too far apart from final l- lysine product,
Intermediate supersession branch is too many and complicated, and does not attract people's attention always in l- fermenting lysine.
The present inventor has especially relied on some fortune, has chanced on by studying for a long period of time and practicingacnAThe transformation of gene
It can aid in the yield for improving l- lysine；However, the prior art otherwise by increase copy and/or rite-directed mutagenesis import table
The beneficial enzyme gene that is improved up to amount and/or enzymatic activity or by knocking out unfavorable gene to be allowed to enzymatic activity and/or expression
Amount disappears, but different, the inventors discovered that,acnAGene cannot be simply increased or be knocked out, and especially be knocked out
After make bacterial growth difficult, it is difficult to practical application, therefore develop new be directed toacnAThe method of gene regulation is mentioned with this
The yield of high l- lysine, and site is transformed in the chromosome of the bacterium of this method and a large amount of high-yield L-lysines of existing transformation
Do not conflict, the effect of raising can be superimposed, to can be used for bacterial fermentation production l- lysine in practice.
Summary of the invention
The technical problem to be solved in the present invention is that provide new fermenting and producing l- lysine method and its relevant side
Method, including the method relative to the fermenting and producing amount that bacterium raising l- lysine is not transformed, the bacterium of transformation is in fermenting and producing l-
Application in lysine, the bacterium of transformation relative to application bacterium is not transformed improved the fermenting and producing amount of l- lysine, and/
Or, the method etc. of transformation bacterium.In addition, the present invention also provides the polynucleotides that can be used in the above method, carrier and/or
Bacterium etc..
Specifically, in a first aspect, the present invention provides the methods of fermenting and producing l- lysine comprising:
(1) wild type on bacterial chromosome is transformedacnAGene, the table of the aconitase A for the bacterium for obtaining transformation
It reduces but does not disappear up to amount and/or enzymatic activity；With,
(2) bacterial fermentation obtained from step (1) transformation produces l- lysine.
Herein, term " transformation " refers to it being that the object being accordingly modified changes, to reach certain effect
Fruit.The means that transformation is located at the gene on chromosome include but is not limited to mutagenesis, rite-directed mutagenesis, and/or homologous recombination, excellent
Choosing is both rear.These technological means are recorded in extensively in molecular biology and microbiology document, and there are many even engaged in trade
Product.In a specific embodiment of the invention, according to the principle of homologous recombination, using the commercialization of Addgene company
PKOV pUC pUC is transformed, by the wild type not being transformed on bacterial chromosomeacnAGene, being transformed into can make to change
The expression quantity and/or enzymatic activity for making the aconitase A of the bacterium of acquisition are reduced but are not disappeared newacnAGene.Therefore, exist
Herein in text, preferably transformation is the transformation carried out by homologous recombination.
The present inventor passes through the discovery that studies for a long period of time, so thatacnAThe expression quantity of the aconitase A of coded by said gene disappears, or
So thatacnAThe enzymatic activity of the aconitase A of coded by said gene disappears, and will all cause bacterium itself to grow difficulty, or even can not give birth to
Length/breeding.Therefore, " transformation " of the invention relative to the bacterium not being transformed, will make the aconitase A's of the bacterium of transformation acquisition
Expression quantity and/or enzymatic activity are reduced but are not disappeared, the expression quantity and/or enzyme of the aconitase A for the bacterium for preferably obtaining transformation
Activity reduces by 20% ~ 95%, more preferably reduces by 50% ~ 90%, such as reduces by 65%, 70% or 80%.
Correspondingly, the present invention also provides other application or methods.For example, the present invention provides raisings in second aspect
The method of the amount of fermentation of l- lysine comprising:
(1) wild type on bacterial chromosome is transformedacnAGene, the table of the aconitase A for the bacterium for obtaining transformation
It reduces but does not disappear up to amount and/or enzymatic activity；With,
(2) bacterial fermentation obtained from step (1) transformation generates l- lysine.
Important metabolite of the l- lysine as bacterium, most of bacteriums can more or less ferment generate it is a certain amount of
L- lysine.Although the bacterium of low yield L-lysine is not suitable for producing l- lysine with having an economic benefit, by this hair
Bright method can still improve the amount of fermentation of l- lysine, can still use for the place insensitive to economic benefit.When
So, herein, preferred bacterium is the bacterium of high-yield L-lysine.By means of the invention it is also possible to further increase its production
Amount.In addition, in method or application of the invention, in addition to the wild type on transformation bacterial chromosomeacnAIt, can other than gene
No longer to carry out other transformations.For example, being only transformed on bacterial chromosome for particularly with the bacterium of high-yield L-lysine
Wild typeacnAGene.
For another example, in the third aspect, application of the bacterium obtained the present invention provides transformation in fermenting and producing l- lysine,
Wherein, it is the wild type being transformed on bacterial chromosome that the transformation, which obtains,acnAGene and obtain, and make transformation obtain
The expression quantity and/or enzymatic activity of the aconitase A of bacterium is reduced but is not disappeared.
The bacterium that transformation obtains can be applied individually to any in fermenting and producing l- lysine, can also produce L-lysine with other
Bacterium mixed fermentive produce l- lysine, or otherwise be applied to fermenting and producing l- lysine in.Herein, such as
It is not particularly limited (as do not limited with " transformation obtain "), term " bacterium " is the bacterium before not being transformed or be transformed, chromosome
'sacnAGene on gene locus is wild typeacnAGene.
Also such as, in fourth aspect, the present invention provides the bacteriums of transformation acquisition in the fermenting and producing amount for improving l- lysine
Application, wherein it is wild type on transformation bacterial chromosome that the transformation, which obtains,acnAGene and obtain, and make to be transformed
The expression quantity and/or enzymatic activity of the aconitase A of the bacterium of acquisition is reduced but is not disappeared.
Herein, bacterium is preferably Escherichia bacteria, more preferably Escherichia coli, such as e. coli k-12 bacterial strain
Subsequent bacterial strain, including bacterial strain derived from W3110.Since the prior art is paid close attention to almost without in l- lysine production/fermentation
Cross bacteriumacnAThe gene of gene, the chromosome of transformation is mostly focused onpnt、dapAndppcOn equal gene locis, thus it is existing
There is the bacterium (especially Escherichia bacteria, such as Escherichia coli) in technology not to be reported without wild typeacnABase
Cause.In a specific embodiment of the invention, no matter the bacterium of high yield or low yield L-lysine, as long as with wild typeacnAGene is transformed by means of the present invention, and the amount of fermentation of L-lysine can be made to be improved.
More constitutionally, at the 5th aspect, the present invention provides the methods of transformation bacterium comprising the Bacterial stain is transformed
Wild type on bodyacnAGene, make transformation obtain bacterium aconitase A expression quantity and/or enzymatic activity reduce but not
It disappears.
The bacterium that the method for fifth aspect present invention is transformed and is obtained can be used in fermenting and producing or generate L-lysine.Cause
This, in the 6th aspect, the bacterium obtained the present invention provides the transformation of the method for fifth aspect present invention.
Most of wild type in Escherichia bacteria (e.g., Escherichia coli)acnAThe nucleotide sequence of gene is such as
Shown in SEQ ID No:1, and a specific embodiment of the invention also demonstrates that band is just like SEQ ID on a variety of chromosomes
Wild type shown in No:1acnAImplement transformation of the invention on the bacterium of gene, the amount of fermentation of L-lysine can be improved.
It is advantageous to herein, the wild typeacnAThe nucleotide sequence of gene is as shown in SEQ ID No:1.
It studies and confirms by the present inventor, it is highly preferred that herein, the wild type on the transformation bacterial chromosome
'sacnAGene is by the wild type of the bacteriumacnAThe initiation codon of gene is mutated.For example, by wild typeacnA
The initiation codon ATG of gene is mutated, as by the wild type as shown in SEQ ID No:1acnAThe initiation codon of gene
ATG sports GTG.
It also passes through the present inventor's research and confirms, it is highly preferred that herein, it is wild on the transformation bacterial chromosome
TypeacnAGene is by the wild type of the bacteriumacnA1-120 nucleotide of gene delection preferably lacks 1-90 core
Thuja acid most preferably lacks 90 nucleotide, as in the wild typeacnA90 nucleosides are lacked before the terminator codon of gene
Acid.In a specific embodiment of the invention, the wild type as shown in SEQ ID No:1acnAThe terminator codon of gene
90 nucleotide of preceding missing.
In addition, the present invention also provides the substances such as the polynucleotides that can be used in the above method and/or carrier.For example,
At the 7th aspect, the present invention provides polynucleotides, the polynucleotides are selected from,
(a) wild typeacnAThe initiation codon of gene (preferably its nucleotide sequence is as shown in SEQ ID No:1) is prominent
The polynucleotides for becoming and obtaining, the preferably described mutation is that ATG is sported GTG；With,
(b) wild typeacnAGene (preferably its nucleotide sequence is as shown in SEQ ID No:1) lacks 1-120 nucleosides
Acid and the polynucleotides obtained, preferably wild typeacnA1-90 nucleotide of gene delection and the polynucleotides obtained, it is more excellent
Select wild typeacnA90 nucleotide of gene delection and the polynucleotides obtained, such as wild typeacnAThe termination of gene is close
The polynucleotides for lacking 90 nucleotide before numeral and obtaining.
For another example, in eighth aspect, the present invention provides carriers, and it includes the polynucleotides of seventh aspect present invention.
The beneficial effects of the present invention are open up and facts have proved the side of the amount of fermentation of new raising L-lysine
Formula is all suitable for the bacterium of high yield and low yield L-lysine, and the bacterium with a large amount of high-yield L-lysines of existing transformation
Chromosome transformation site do not conflict, observed can be superimposed the effect for improving yield, to can be used in practice thin
Bacterium fermenting and producing l- lysine, it is easy to promote and utilize.
In order to make it easy to understand, the present invention will be described in detail by specific embodiment below.It needs to refer in particular to
Out, these descriptions are only exemplary description, and are not meant to limit the scope of the invention.Opinion according to this specification
It states, many variations of the invention, change will be apparent from for those skilled in the art.
In addition, the present invention refers to open source literature, these documents are their full text in order to more clearly describe the present invention
Content is included in and is referred to herein, just looks like that repeated description herein has been excessively for their full text.
Specific embodiment
The contents of the present invention are further illustrated by the following examples.Such as not specified, technology used in embodiment
The conventional means and commercially available common instrument, reagent that means are well known to those skilled in the art, reference can be made to " Molecular Cloning: A Laboratory
Guide (the 3rd edition) " (Science Press), " Microbiology Experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and
The reference such as manufacturers instruction of reagent.
Embodiment 1 is constructed to replaceacnAInitiation codon ATG be GTG
With the wild-type e. coli of extractingE. coli K12 W3110 bacterial strain (is purchased from day this technology evaluation study institute
Biological Resource Center (NITE Biological Resource Center, NBRC)) genome chromosome be template, with primer
P1 and P2, P3 and P4 carry out PCR amplification respectively, obtain two DNA fragmentations that length is respectively 510 bp and 620 bp and (order respectively
Entitled Up1 and Down1 segment).Wherein, PCR is carried out as follows: 94 DEG C are denaturalized 30 s(seconds), 52 DEG C are annealed 30 s(seconds),
And 72 DEG C extend 30 s(second) (30 recycle).Wherein, primer sequence is as follows:
P1:5’-CGCGGATCC(underscore shows GGAGTCGTCACCATTATGCC-3 'BamHI restriction enzyme site)
P2:5’-TCTCGTAGGGTTGACGACACAGCTCCTCCTTAATGACAGG-3 ' (underscore shows point mutation)
P3:5’-CCTGTCATTAAGGAGGAGCTGTGTCGTCAACCCTACGAGA-3 ' (underscore shows point mutation)
P4:5’-ATTGCGGCCGC(underscore shows TCCATTCACCGTCCTGCAAT-3 'NotI restriction enzyme site)
By above-mentioned two DNA fragmentations after agarose gel electrophoresis isolates and purifies, then with above-mentioned two DNA fragmentations
It is mixed into template, using P1 and P4 as primer, Up-Down1 (is named as by the segment that Overlap PCR amplification is about 1200 bp
Segment).Wherein, PCR is carried out as follows: 94 DEG C are denaturalized 30 s(seconds), 52 DEG C are annealed 30 s(seconds) and 72 DEG C of extensions 60
S(seconds) (30 circulations).
Up-Down1 and pKOV plasmid (being purchased from Addgene company) after agarose gel electrophoresis is isolated and purified is respectively
It with BamHI/NotI double digestion, is connected after agarose gel electrophoresis isolates and purifies, obtains the carrier pKOV-Up- for importing
Down1, and send sequencing company to carry out sequencing identification carrier pKOV-Up-Down1, show that it contains correct point mutation (A-G)acnAGenetic fragment saves backup.
By the pKOV-Up-Down1 plasmid built, electrotransformation enters low yield L-lysine respectivelyE. coli NRRL B-
12185 bacterial strains (are purchased from american agriculture Culture Collection Center (Agricultural Research Service Culture
Collection；NRRL)；Its construction method can be found in US4346170A) and high-yield L-lysineE. coli K12
3 bacterial strain of W3110 △ (is purchased from institute of microbiology, the Chinese Academy of Sciences, for warpE. coli K12 W3110 mutagenesis is mutated
Lysine production engineering bacterium) (confirms through sequencing and remain with the acnA gene of wild type (i.e. on the two strain chromosomes
1333855 to 1336530 in the record such as Genbank U00096.2) and its upstream and downstream element), in 30 DEG C, 100 rpm,
It is recovered after 2 h in LB culture medium, according to the commodity guide of the pKOV plasmid of Addgene company, picks out the homologous recombination positive
Monoclonal confirms the wild type on its chromosome through sequencingacnAThe initiation codon of gene sports GTG by ATG, respectively
It arrivesacnA(production L-lysine low/high) Escherichia coli of initiation codon mutation.Through detecting, the aconitic acid of two bacterial strains of acquisition
Expression of enzymes amount has dropped about 75 ~ 85%(and slightly has difference in different medium).
Construct embodiment 2acnAGene order mutation reduces aconitase activity
90bp base is before missing terminator codon to reduce aconitase activity.Specifically, the wild type with extracting is big
EnterobacteriaE. coli K12 W3110 genome chromosome is template, carries out PCR amplification respectively with primer P5 and P6, P7 and P8,
Obtain two DNA fragmentations (Up3 and Down3 segment) that length is respectively 752 bp and 657 bp.Wherein, PCR is as follows
Carry out: 94 DEG C are denaturalized 30 s(second), 52 DEG C are annealed 30 s(second) and 72 DEG C of extensions 30 s(seconds) (30 recycle).Wherein, draw
Object sequence is as follows:
P5:5’-CGCGGATCC(underscore shows CGTCACACGATCCGATACCT-3 'BamHI restriction enzyme site)
(underscore shows a little to dash forward P6:5 '-CGGCAAGCAAATAGTTGTTATACGACTTCCTGGCTACCAT -3 '
Become)
P7:5 '-ATGGTAGCCAGGAAGTCGTATAACAACTATTTGCTTGCCG-3 ' (underscore shows point mutation)
P8:5’-ATTGCGGCCGC(underscore shows CATGGGGCGATTTCCTGATG-3 'NotI restriction enzyme site)
By above-mentioned two DNA fragmentations after agarose gel electrophoresis isolates and purifies, then with above-mentioned two DNA fragmentations
It is mixed into template, using P5 and P8 as primer, Up-Down2 (is named as by the segment that Overlap PCR amplification is about 1400 bp
Segment).Wherein, PCR is carried out as follows: 94 DEG C are denaturalized 30 s(seconds), 52 DEG C are annealed 30 s(seconds) and 72 DEG C of extensions 60
S(seconds) (30 circulations).
Up-Down2 and pKOV plasmid (being purchased from Addgene company) after agarose gel electrophoresis is isolated and purified is respectively
It with BamHI/NotI double digestion, is connected after agarose gel electrophoresis isolates and purifies, obtains the carrier pKOV-Up- for importing
Down2, and send sequencing company to carry out sequencing identification carrier pKOV-Up-Down2 shows 90bp before it contains terminator codon
Base deletionacnAGenetic fragment saves backup.
It is according to the commodity guide of the pKOV plasmid of Addgene company, the pKOV-Up-Down2 plasmid built is electric respectively
It is transformed into low yield L-lysineE. coli NRRL B-12185 bacterial strain (is purchased from american agriculture Culture Collection Center
(Agricultural Research Service Culture Collection；NRRL)；Its construction method can be found in
) and high-yield L-lysine US4346170AE. coli 3 bacterial strain of K12 W3110 △ (is purchased from Chinese Academy of Sciences's microbe research
Institute, for warpE. coli The lysine production engineering bacterium that K12 W3110 mutagenesis is mutated) (the two bacterium are confirmed through sequencing
Wild type is remained on strain chromosomeacnAGene and its upstream and downstream element), the monoclonal of the homologous recombination positive is picked out,
The wild type on its chromosome is confirmed through sequencingacnA90bp base deletion, respectively obtains before the terminator codon of geneacnAEnzyme
(production L-lysine low/high) Escherichia coli that activity reduces.Through detecting, the aconitase enzymatic activity of two bacterial strains of acquisition declines
About 60 ~ 80%(slightly has difference in different medium).
The experiment of effect example fermenting lysine
It willE. coli 3 bacterial strain of K12 W3110 △,E. coli NRRL B-12185 bacterial strain and Examples 1 and 2
Bacterial strain is seeded in respectively in seed culture medium described in 25mL table 1, cultivates 9 h in 37 DEG C, 220rpm.Then 1 mL seed is taken to train
The culture for supporting base is seeded in fermentation medium described in 25 mL tables 1, in 37 DEG C, 48 h of 220rpm culture culture.Work as culture
When completion, the generation of l- lysine is measured by HPLC.
1 culture medium prescription of table
| Seed culture based formulas | Fermentative medium formula | |
| (ingredient) | (g/L) | (g/L) |
| Glucose | 15 | 40 |
| Ammonium sulfate | 4 | 10 |
| Potassium dihydrogen phosphate | 3 | 1.6 |
| Epsom salt | 0.4 | 1 |
| Ferrous sulfate heptahydrate | 0.01 | 0.03 |
| Manganese sulfate monohydrate | 0.01 | 0.03 |
| Yeast extract | 2.0 | 4.0 |
| Calcium carbonate | 25 | |
| KOH | pH 7.0 | pH 7.0 |
| L- tyrosine | 0.1 | |
| L- methionine | 0.5 | |
| L- threonine | 0.1 | |
| L- isoleucine | 0.05 |
As a result,E. coli The l- of the bacterial strain of 3 bacterial strain of K12 W3110 △ and the high yield l- lysine of Examples 1 and 2
Lysine production is respectively 10.2 g/L, 16 .1 g/L and 12.5 g/L；E. coli NRRL B-12185 bacterial strain and implementation
The l- lysine production of the bacterial strain of the low yield l- lysine of example 1 and 2 is respectively 1.5 g/L, 2.1 g/L and 1.8 g/L, it is seen then that
No matter for high yield or low yield L-lysine original strain,acnAThe initiation codon of gene is mutated or its enzymatic activity reduces
Escherichia coli both contribute to the raising of l- lysine production.
<110>Ningbo Eppen Biotech Co., Ltd.
<120>method of the bacterial fermentation production L-lysine of reduction and/or enzymatic activity reduction is expressed with aconitase
<130> CN
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 2676
<212> DNA
<213>Escherichia coli
<400> 1
atgtcgtcaa ccctacgaga agccagtaag gacacgttgc aggccaaaga taaaacttac 60
cactactaca gcctgccgct tgctgctaaa tcactgggcg atatcacccg tctacccaag 120
tcactcaaag ttttgctcga aaacctgctg cgctggcagg atggtaactc ggttaccgaa 180
gaggatatcc acgcgctggc aggatggctg aaaaatgccc atgctgaccg tgaaattgcc 240
taccgcccgg caagggtgct gatgcaggac tttaccggcg tacctgccgt tgttgatctg 300
gcggcaatgc gcgaagcggt taaacgcctc ggcggcgata ctgcaaaggt taacccgctc 360
tcaccggtcg acctggtcat tgaccactcg gtgaccgtcg atcgttttgg tgatgatgag 420
gcatttgaag aaaacgtacg cctggaaatg gagcgcaacc acgaacgtta tgtgttcctg 480
aaatggggaa agcaagcgtt cagtcggttt agcgtcgtgc cgccaggcac aggcatttgc 540
catcaggtta acctcgaata tctcggcaaa gcagtgtgga gtgaattgca ggacggtgaa 600
tggattgctt atccggatac actcgttggt actgactcgc acaccaccat gatcaacggc 660
cttggcgtgc tggggtgggg cgttggtggg atcgaagcag aagccgcaat gttaggccag 720
ccggtttcca tgcttatccc ggatgtagtg ggcttcaaac ttaccggaaa attacgtgaa 780
ggtattaccg ccacagacct ggttctcact gttacccaaa tgctgcgcaa acatggcgtg 840
gtggggaaat tcgtcgaatt ttatggtgat ggtctggatt cactaccgtt ggcggatcgc 900
gccaccattg ccaatatgtc gccagaatat ggtgccacct gtggcttctt cccaatcgat 960
gctgtaaccc tcgattacat gcgtttaagc gggcgcagcg aagatcaggt cgagttggtc 1020
gaaaaatatg ccaaagcgca gggcatgtgg cgtaacccgg gcgatgaacc aatttttacc 1080
agtacgttag aactggatat gaatgacgtt gaagcgagcc tggcagggcc taaacgccca 1140
caggatcgcg ttgcactgcc cgatgtacca aaagcatttg ccgccagtaa cgaactggaa 1200
gtgaatgcca cgcataaaga tcgccagccg gtcgattatg ttatgaacgg acatcagtat 1260
cagttacctg atggcgctgt ggtcattgct gcgataacct cgtgcaccaa cacctctaac 1320
ccaagtgtgc tgatggccgc aggcttgctg gcgaaaaaag ccgtaactct gggcctcaag 1380
cggcaaccat gggtcaaagc gtcgctggca ccgggttcga aagtcgtttc tgattatctg 1440
gcaaaagcga aactgacacc gtatctcgac gaactggggt ttaaccttgt gggatacggt 1500
tgtaccacct gtattggtaa ctctgggccg ctgcccgatc ctatcgaaac ggcaatcaaa 1560
aaaagcgatt taaccgtcgg tgcggtgctg tccggcaacc gtaactttga aggccgtatc 1620
catccgctgg ttaaaactaa ctggctggcc tcgccgccgc tggtggttgc ctatgcgctg 1680
gcgggaaata tgaatatcaa cctggcttct gagcctatcg gccatgatcg caaaggcgat 1740
ccggtttatc tgaaagatat ctggccatcg gcacaagaaa ttgcccgtgc ggtagaacaa 1800
gtctccacag aaatgttccg caaagagtac gcagaagttt ttgaaggcac agcagagtgg 1860
aagggaatta acgtcacacg atccgatacc tacggttggc aggaggactc aacctatatt 1920
cgcttatcgc ctttctttga tgaaatgcag gcaacaccag caccagtgga agatattcac 1980
ggtgcgcgga tcctcgcaat gctgggggat tcagtcacca ctgaccatat ctctccggcg 2040
ggcagtatta agcccgacag cccagcgggt cgatatctac aaggtcgggg tgttgagcga 2100
aaagacttta actcctacgg ttcgcggcgt ggtaaccatg aagtgatgat gcgcggcacc 2160
ttcgccaata ttcgcatccg taatgaaatg gtgcctggcg ttgaaggggg gatgacgcgg 2220
catttacctg acagcgacgt agtctctatt tatgatgctg cgatgcgcta taagcaggag 2280
caaacgccgc tggcggtgat tgccgggaaa gagtatggat caggctccag tcgtgactgg 2340
gcggcaaaag gtccgcgtct gcttggtatt cgtgtggtga ttgccgaatc gtttgaacga 2400
attcaccgtt cgaatttaat tggcatgggc atcctgccgc tggaatttcc gcaaggcgta 2460
acgcgtaaaa cgttagggct aaccggggaa gagaagattg atattggcga tctgcaaaac 2520
ctacaacccg gcgcgacggt tccggtgacg cttacgcgcg cggatggtag ccaggaagtc 2580
gtaccctgcc gttgtcgtat cgacaccgcg acggagttga cctactacca gaacgacggc 2640
attttgcatt atgtcattcg taatatgttg aagtaa 2676
Claims (2)
1. the method for fermentation production of L-lysine comprising:
(1) transformation produces the acnA gene of the wild type on the escherichia coli chromosome of L-lysine, the bacterium for obtaining transformation
The expression quantity and/or enzymatic activity of aconitase A is reduced but is not disappeared；With,
(2) bacterial fermentation obtained from step (1) transformation produces L-lysine,
Wherein, the acnA gene of the wild type on the escherichia coli chromosome of the transformation production L-lysine includes:
Nucleotide sequence is produced to the end of the acnA gene of the wild type of the Escherichia coli of L-lysine as shown in SEQ ID No:1
90 nucleotide are only lacked before codon.
2. the method for improving the amount of fermentation of L-lysine comprising:
(1) transformation produces the acnA gene of the wild type on the escherichia coli chromosome of L-lysine, the bacterium for obtaining transformation
The expression quantity and/or enzymatic activity of aconitase A is reduced but is not disappeared；With,
(2) bacterial fermentation obtained from step (1) transformation produces L-lysine,
Wherein, the acnA gene of the wild type on the escherichia coli chromosome of the transformation production L-lysine includes:
Nucleotide sequence is produced to the end of the acnA gene of the wild type of the Escherichia coli of L-lysine as shown in SEQ ID No:1
90 nucleotide are only lacked before codon.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006116400A2 (en) * | 2005-04-27 | 2006-11-02 | Massachusetts Institute Of Technology | Promoter engineering and genetic control |
| CN101631871A (en) * | 2007-02-22 | 2010-01-20 | 味之素株式会社 | Method of producing L-amino acid |
| CN102191289A (en) * | 2011-03-18 | 2011-09-21 | 宁夏伊品生物科技股份有限公司 | Fermentation preparation method of lysine |
| CN102471790A (en) * | 2009-07-29 | 2012-05-23 | 味之素株式会社 | Method for producing l-amino acid |
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2013
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006116400A2 (en) * | 2005-04-27 | 2006-11-02 | Massachusetts Institute Of Technology | Promoter engineering and genetic control |
| CN101631871A (en) * | 2007-02-22 | 2010-01-20 | 味之素株式会社 | Method of producing L-amino acid |
| CN102471790A (en) * | 2009-07-29 | 2012-05-23 | 味之素株式会社 | Method for producing l-amino acid |
| CN102191289A (en) * | 2011-03-18 | 2011-09-21 | 宁夏伊品生物科技股份有限公司 | Fermentation preparation method of lysine |
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