WO2014121669A1 - 用改变乌头酸酶基因和/或其调控元件的细菌发酵生产l-赖氨酸的方法 - Google Patents
用改变乌头酸酶基因和/或其调控元件的细菌发酵生产l-赖氨酸的方法 Download PDFInfo
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- WO2014121669A1 WO2014121669A1 PCT/CN2014/070228 CN2014070228W WO2014121669A1 WO 2014121669 A1 WO2014121669 A1 WO 2014121669A1 CN 2014070228 W CN2014070228 W CN 2014070228W WO 2014121669 A1 WO2014121669 A1 WO 2014121669A1
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- Prior art keywords
- nucleotide sequence
- aconitase
- gene
- lysine
- bacteria
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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 124
- 108010009924 Aconitate hydratase Proteins 0.000 title claims abstract description 92
- 241000894006 Bacteria Species 0.000 title claims abstract description 79
- 239000004472 Lysine Substances 0.000 title claims abstract description 65
- 235000019766 L-Lysine Nutrition 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 51
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- 102000009836 Aconitate hydratase Human genes 0.000 claims abstract description 34
- 238000000855 fermentation Methods 0.000 claims abstract description 32
- 230000004151 fermentation Effects 0.000 claims abstract description 32
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 13
- 230000002829 reductive effect Effects 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims description 68
- 125000003729 nucleotide group Chemical group 0.000 claims description 68
- 241000186216 Corynebacterium Species 0.000 claims description 34
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- 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 15
- 108091033319 polynucleotide Proteins 0.000 claims description 14
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- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
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- 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 2
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
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- 241000173529 Aconitum napellus Species 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
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- 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
- 101710118630 Homocitrate dehydratase, mitochondrial Proteins 0.000 description 1
- FFEARJCKVFRZRR-UHFFFAOYSA-N L-Methionine Natural products CSCCC(N)C(O)=O FFEARJCKVFRZRR-UHFFFAOYSA-N 0.000 description 1
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- 229930195722 L-methionine Natural products 0.000 description 1
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 1
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- 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
- ODBLHEXUDAPZAU-UHFFFAOYSA-N isocitric acid Chemical compound OC(=O)C(O)C(C(O)=O)CC(O)=O ODBLHEXUDAPZAU-UHFFFAOYSA-N 0.000 description 1
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 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
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/01003—Aconitate hydratase (4.2.1.3)
Definitions
- the present invention is in the field of amino acid fermentation, and in particular, the present invention relates to a method for fermentative production of L-lysine, a method and application thereof, and bacteria and the like which can be used in such methods and applications.
- Background technique
- L-lysine-producing bacteria e.g., Escherichia coli and coryneform bacteria of the genus Corynebacterium
- L-lysine-producing bacteria e.g., Escherichia coli and coryneform bacteria of the genus Corynebacterium
- These bacteria may be bacteria isolated from nature, bacteria obtained by mutagenesis or genetic engineering, or both.
- the focus of genetic engineering is mainly on genes such as pnt, dap, and ppc.
- No aconitase is reported for L-lysine production (eg, aconite in E. coli).
- Aconitase is an enzyme in the Krebs cycle that catalyzes a two-step chemical reaction, converting citric acid to aconitic acid and aconitic acid to isocitrate. It is currently known that in Escherichia, the acnA gene (the nucleotide sequence of which is shown in SEQ ID No: 1) encodes aconitase A, but may be due to its metabolism away from the final L-lysine product. Far, the intermediate metabolic branch is too much and complicated, but it has not attracted people's attention in L-lysine fermentation.
- lysine-fermenting bacteria such as Corynebacterium glutamicum
- aconitase enzymes mentioned in documents such as Chinese patents CN1289368 A and CN101631871 A are related to the fermentation of L-glutamic acid and do not teach the effect of aconitase on L-lysine fermentation.
- the prior art either introduces a beneficial enzyme gene whose expression amount and/or enzyme activity is increased by increasing copying and/or site-directed mutagenesis, or by knocking out an unfavorable gene to cause the enzyme activity and/or expression amount to disappear.
- the inventors have found that the aconitase gene and its regulatory elements cannot be simply increased or eliminated.
- the non-expression of the acnA gene after knockout makes the growth of the bacteria difficult, and it is difficult to practically apply, so development A new method for engineering the aconitase gene and/or its regulatory elements to increase L-lysine production.
- the technical problem to be solved by the present invention is to provide a new method for fermentative production of L-lysine and related methods, including a method for increasing the fermentation yield of L-lysine relative to unmodified bacteria, and the modified bacteria are fermented.
- the present invention also provides polynucleotides, vectors, and/or bacteria and the like which can be used in the above methods.
- the present invention provides a method of fermentatively producing L-lysine, which comprises:
- the term "reconstruction” refers to a change in the corresponding modified object to achieve a certain effect.
- Means for engineering genes or regulatory elements located on a chromosome include, but are not limited to, mutagenesis, site-directed mutagenesis, and/or homologous recombination, preferably the latter two.
- the transformation of a gene or regulatory element located on a chromosome allows the nucleotide sequence of the gene or regulatory element to be added, deleted or replaced with one or more nucleotides.
- the gene can be modified by Addgene's commercial pKOV plasmid system, or the pK18mob SaC B plasmid system can be used for transformation, and the unmodified bacterial chromosome
- the acidase gene and/or its regulatory element modified into a new aconitase gene and/or its regulatory element capable of reducing the expression amount and/or enzymatic activity of the aconitase of the bacteria obtained by the transformation but not disappearing . Therefore, in the present context, it is preferred that the transformation is a modification by homologous recombination.
- the inventors have found through long-term research that in the bacteria such as Escherichia coli and Corynebacterium, the expression of aconitase disappears. For example, by means of gene knockout, the growth of the bacteria itself is difficult, and even the acid cannot be produced. Therefore, the "reconstruction" of the present invention reduces the expression of aconitase of the bacteria obtained by the transformation with respect to the unmodified bacteria, but does not disappear, and preferably reduces the expression of aconitase A of the bacteria obtained by the transformation. 20% ⁇ 95%, more preferably 50 ⁇ 90, such as 65%, 70% or 80%.
- the present invention also provides other applications or methods.
- the present invention provides an improvement
- a method of fermenting an amount of L-lysine which comprises:
- L-lysine is an important metabolite of bacteria, and most bacteria are more or less capable of fermenting a certain amount of L-lysine.
- L-lysine-producing bacteria are not suitable for the economical production of L-lysine, The method of Ming can still increase the fermentation of L-lysine, and it can still be used in places that are not sensitive to economic benefits.
- the bacterium is a high-yield L-lysine-producing bacterium. The yield can be further increased by the method of the present invention.
- aconitase gene on the bacterial chromosome and/or its regulatory elements in addition to engineering the aconitase gene on the bacterial chromosome and/or its regulatory elements, no further modifications may be made, such as even after modification of the regulatory elements of the aconitase gene.
- the aconitase gene on the bacterial chromosome may not be engineered, and vice versa.
- only one of the aconitase gene on the bacterial chromosome and its regulatory elements can be engineered.
- the present invention provides the use of the bacterium obtained by the transformation in the fermentation production of L-lysine, wherein the transformation obtains an aconitase gene and/or a regulatory element thereof on the chromosomal chromosome of the bacterium.
- the enzymatic activity and/or expression amount of the aconitase of the bacteria obtained by the transformation is reduced but does not disappear.
- the modified bacteria can be used alone in the fermentation production of L-lysine, or can be mixed with other L-lysine-producing bacteria to produce L-lysine, or used in other ways to produce L-lysine.
- bacteria is an unmodified or pre-engineered bacterium, and the aconitase gene of the chromosome and the regulatory elements before and after the locus of the gene are, unless otherwise defined (as defined by "reconstructed”). There is no aconitase gene and regulatory elements that reduce the enzymatic activity and/or expression of aconitase, such as the aconitase gene and regulatory elements in wild-type bacteria.
- the present invention provides the use of the bacterium obtained by the transformation in increasing the fermentation amount of L-lysine, wherein the transformation is obtained by modifying the aconitase gene on the bacterial chromosome and/or Obtained by the regulatory element, and the enzymatic activity and/or expression level of the aconitase of the bacteria obtained by the transformation is reduced but does not disappear.
- the bacterium may be an L-lysine-producing bacterium such as a bacterium belonging to the genus Escherichia or Corynebacterium.
- the bacterium may be a bacterium belonging to the genus Escherichia, more preferably an Escherichia coli, such as a subsequent strain of the E. coli K-12 strain, including a W3110-derived strain; in another preferred aspect, the bacterium may be a rod A bacterium belonging to the genus Bacillus, more preferably a Corynebacterium glutamicum or a Corynebacterium sinensis.
- the aconitase gene of the prior art L-lysine-producing bacteria especially bacteria of the genus Escherichia or Corynebacterium, such as Escherichia coli, Corynebacterium glutamicum or Corynebacterium sinensis
- the regulatory elements before or after it have not been reported to have been modified, so basically have the wild-type aconitase gene and its regulatory elements before and after, basically can be modified by the method of the present invention, improve L- Lai The amount of fermentation of the acid.
- the high-yield or low-yield L-lysine-producing bacteria can be modified by the method of the present invention to increase the fermentation amount of L-lysine.
- the present invention provides a method of engineering a bacterium comprising modifying an aconitase gene on the bacterial chromosome and/or a regulatory element thereof, and modifying the aconitase enzyme of the obtained bacterium The activity and/or expression is reduced but does not disappear.
- the bacteria obtained by the modification of the method of the fifth aspect of the invention can be used for fermentation production or production of L-lysine.
- the present invention provides a bacterium obtained by the modification of the method of the fifth aspect of the invention.
- the nucleotide sequence of many aconitase genes (acnA) in Escherichia bacteria is shown in SEQ ID No: 1
- Corynebacterium bacteria e.g.
- the nucleotide sequence of many aconitase genes in Corynebacterium glutamicum or Corynebacterium sinensis is shown in SEQ ID No: 2
- the aconitase gene is modified to make the obtained bacteria
- the enzymatic activity and/or expression level of the acid is reduced but does not disappear, and the amount of fermentation of L-lysine can be increased.
- the nucleotide sequence of the aconitase gene is represented by SEQ ID No: 1 or 2.
- the nucleotide sequence of the aconitase gene in the bacteria can also be obtained by means of sequencing and sequence identity comparison, thereby modifying according to the method of the present invention.
- the preferred aconitase gene on the modified bacterial chromosome is to add, delete or replace one or more nucleotides to the nucleotide sequence of the aconitase gene, as long as the transformation can be obtained.
- the enzymatic activity and/or expression level of the aconitase of the bacteria is reduced but does not disappear.
- a nucleotide sequence outside the enzyme domain can be mutated.
- a preferred engineered bacterial chromosome aconitase gene may be one or more nucleotides substituted for the nucleotide sequence of the aconitase gene.
- the substitution comprises replacing the start codon of the aconitase gene, preferably with GTG.
- a preferred augmented bacterial chromosome aconitase gene may also be one or more nucleotides deleted from the nucleotide sequence of the aconitase gene.
- the deletion comprises deleting the nucleotide sequence of the aconitase gene, preferably deleting 1-120 nucleotides, more preferably deleting 1-90 nucleotides, most preferably deleting 90 nucleotides,
- 90 nucleotides are deleted before the stop codon of the nucleotide sequence of the aconitase gene. This can be carried out for Escherichia coli, Corynebacterium glutamicum, and Corynebacterium Beijing.
- a regulatory element refers to a polynucleotide located upstream or downstream of a gene (eg, an aconitase gene) and capable of regulating the transcription and/or expression of the gene, thereby affecting the expression of the gene, including non-coding sequences. And coding sequences.
- the regulatory element can be a promoter, enhancer, repressor or other polynucleotide associated with transcription and/or expression regulation.
- a preferred regulatory element can be a promoter.
- the nucleotide sequence of the promoter is as shown in SEQ ID No: 4 or 6.
- Preferred regulatory elements may also be repressor sequences, such as transcriptional repressor proteins.
- the nucleotide sequence of the transcription repressor protein is set forth in SEQ ID No: 7.
- a preferred regulatory element for the aconitase gene on the bacterial chromosome is the aconitase group.
- the nucleotide sequence of the regulatory element is added, deleted or replaced with one or more nucleotides as long as the enzymatic activity and/or expression amount of the aconitase of the bacterium obtained by the transformation can be lowered without disappearing.
- nucleotide sequences can be added to increase the distance between the promoter and the start codon of the gene, or to replace the originally existing promoter with a weak transcriptional promoter.
- a preferred regulatory element for modifying the aconitase gene on the bacterial chromosome may be to replace one or more nucleotides of the promoter of the aconitase gene, such as a weak transcriptional promoter, More preferably, it is replaced with a nucleotide sequence as shown in SEQ ID No: 3 or 5.
- a preferred regulatory element for modifying the aconitase gene on the bacterial chromosome may also be a nucleotide sequence of a transcriptional repressor protein of the aconitase gene, such as adding a new transcriptional repression
- a transcriptional repressor protein can be added to a promoter (especially a strong transcriptional promoter) to increase its expression level, thereby more effectively inhibiting the transcription of the aconitase gene.
- a nucleotide sequence as shown in SEQ ID Nos: 8 and 7 is added in series.
- the present invention also provides an intermediate product which can be used in the above method, such as a polynucleotide and/or a carrier, and the like, and the like.
- an intermediate product which can be used in the above method, such as a polynucleotide and/or a carrier, and the like, and the like.
- the present invention provides a polynucleotide having a nucleotide sequence selected from
- deletion of the nucleotide sequence as shown in SEQ ID No: 1 or 2 preferably deleting 1-120 nucleotides, more preferably deleting 1-90 nucleotides, most preferably deleting 90 nucleosides a nucleotide sequence obtained after the acid, such as a nucleotide sequence obtained by deleting 90 nucleotides before the stop codon of the nucleotide sequence shown as SEQ ID No: 1 or 2;
- the invention provides a vector comprising the polynucleotide of the seventh aspect of the invention.
- the invention provides the use of a polynucleotide of the seventh aspect of the invention and/or a vector of the eighth aspect of the invention in a method or use of the first, second, third and/or fourth aspects of the invention. That is, in the method or application of the first, second, third and/or four aspects of the invention, the present invention provides the polynucleotide of the seventh aspect of the invention and/or the vector of the eighth aspect of the invention.
- the present invention provides the use of the polynucleotide of the seventh aspect of the invention and/or the vector of the eighth aspect of the invention for the preparation of the bacterium of the fifth aspect of the invention. That is, in the preparation of the bacterium of the fifth aspect of the invention, the use of the present The invention provides a polynucleotide of the seventh aspect of the invention and/or a vector of the eighth aspect of the invention.
- the beneficial effects of the present invention are that it has opened up and practiced a new way of increasing the fermentation amount of L-lysine, which is applicable to both high-yield and low-yield L-lysine-producing bacteria, and a large number of high-yield L--
- the chromosomal engineering sites of lysine bacteria have no conflicts, and the effect of increasing the yield can be observed, so that it can be used in the practice of bacterial fermentation to produce L-lysine, which is convenient for popularization and application.
- the present invention will be described in detail below by way of specific examples. It is to be understood that the description is not intended to be limiting of the scope of the invention. Many variations and modifications of the invention will be apparent to those skilled in the ⁇ RTIgt;
- coli K12 W3110 strain (available from NITE Biological Resource Center, NBRC)
- the genomic chromosome was used as a template, and PCR amplification was performed with primers P1 and P2, P3 and P4, respectively, and two DNA fragments of 510 bp and 620 bp in length (named Upl and Downl fragments, respectively) were obtained.
- PCR was carried out as follows: denaturation at 94 °C for 30 s (seconds), 52. C is annealed for 30 s (seconds), and extended at 72 °C for 30 s (seconds) (30 cycles).
- the primer sequence is as follows: P1: 5 '-CGCGGATCCGGAGTCGTCACCATTATGCC-S '
- PCR was carried out as follows: denaturation at 94 °C for 30 s (seconds), annealing at 52 °C for 30 s (seconds), and extension at 72 °C for 60 s (seconds) (30 cycles).
- the Up-Downl and pKOV plasmids (purchased from Addgene), which were separated and purified by agarose gel electrophoresis, were double-digested with Bam Hi/Not I, separated and purified by agarose gel electrophoresis, and then ligated for obtaining.
- the vector pKOV-Up-Downl, and the vector pKOV-Up-Downl was sent to the sequencing company for sequencing and identification, indicating that it contains the correct point mutation (AG) acnA gene fragment, and stored for later use.
- the constructed pKOV-Up-Downl plasmid was separately electroporated into the L. lysine-low E. coli NRRL B-12185 strain (available from the Agricultural Research Service Culture Collection (NRRL); For the construction method, see US4346170A) and the high-yield L-lysine E. coli K12 W3110 ⁇ 3 strain (available from the Institute of Microbiology, Chinese Academy of Sciences, which is a lysine production engineering strain obtained by mutation of E.
- coli K12 W3110) (Sequence confirmed that the wild type acnA gene (ie, 1333855 to 1336530 in Genbank accession number U00096.2 and its upstream and downstream elements) was retained on the chromosomes of the two strains, and cultured at LB at 30 °C, 100 rpm. After 2 h of resuscitation in the basal phase, the homologous recombination-positive monoclonal was selected according to the product guide of Addgene's pKOV plasmid. The start codon of the wild-type acnA gene on the chromosome was confirmed to be agglutinated from GTG to GTG.
- Escherichia coli low/high-yield L-lysine mutated with acnA start codon, designated as YP-13633 and YP-13664, respectively.
- Aconitase enzyme expression decreased the amount of about 75 to about 85% (slight differences in different media).
- Aconitase activity reducing mutation aconitase 2
- E. coli acnA gene stops the first 90 bp base of the codon to reduce aconitase activity.
- the extracted wild-type E. coli K12 W3110 genomic chromosome was used as a template, and primers P5 and P6, P7 and P8 were used for PCR amplification to obtain two DNAs of 752 bp and 657 bp in length, respectively. Fragments (Up2 and Down2 clips).
- PCR was carried out as follows: denaturation at 94 °C for 30 s (seconds), annealing at 52 °C for 30 s (seconds), and extension at 72 °C for 30 s (seconds) (30 cycles).
- the primer sequence is as follows:
- PCR was carried out as follows: denaturation at 94 °C for 30 s (seconds), annealing at 52 °C for 30 s (seconds), and extension at 72 °C for 60 s (seconds) (30 cycles).
- the Up-Down2 and pKOV plasmids (purchased from Addgene), which were separated and purified by agarose gel electrophoresis, were digested with Bam Hi/Not I, separated and purified by agarose gel electrophoresis, and ligated to obtain for introduction.
- the vector P KOV-Up-Down2, and the vector pKOV-Up-Down2 was sent to the sequencing company for sequencing and identification, which indicated that it contained the acnA gene fragment of the 90 bp base deletion before the stop codon, and was stored for use.
- the constructed pKOV-Up-Down2 plasmid was electrotransformed into the low-yield L-lysine-containing E. coli NRRL B-12185 strain according to the product guide of Addgene's pKOV plasmid; the construction method can be found in US 4346170A) and the high yield L -E. coli K12 W3110 ⁇ 3 strain of lysine (sequence confirmed that both strains retain the wild-type acnA gene and its upstream and downstream elements on the chromosomes), and the homologous recombination-positive monoclonals were selected and confirmed by sequencing.
- the 90 bp base of the stop codon of the wild-type acnA gene on the chromosome was deleted, and the low-protein/low-producing L-lysine Escherichia coli was obtained, respectively, which were named YP-13675 and YP-13699, respectively. .
- the aconitase activity of the two strains obtained decreased by about 60-80% (slightly different in different media).
- Corynebacterium pekinense which is similar to Corynebacterium glutamicum, is sometimes mistaken for Corynebacterium glutamicum.
- CGMCC China General Microorganisms Collection and Management Center
- the following primers P9 ⁇ P12 correspond to the above primers P4 ⁇ P8, and the amplified Up2 fragment is 542 bp, and the Down2 fragment is 527 bp. , the Up-Down2 fragment is about 1069 bp, wherein the primer sequences used are as follows;
- the Up-Down2 fragment purified by agarose gel electrophoresis and the pK18mob SaC B plasmid (available from the American Type Culture Collection (ATCC)) were digested with Bam ffl/Sma1 and subjected to agarose gel electrophoresis. After isolation and purification, the recombinant vector pK18mobsacB-Up-P-Down lacking the 90 bp base before the stop codon of the acn gene was obtained. After sequencing and identification, the constructed pK18mobsacB-Up-Down2 plasmid was separately transformed into low-yield L. - Lysine of Corynebacterium sinensis AS1.299 strain, L-lysine-producing Corynebacterium glutamicum
- the extracted wild-type E. coli K12 W3110 genomic chromosome was used as a template, and primers P13 and P14, P15 and P16 were respectively subjected to PCR amplification to obtain two DNAs of 580 bp and 618 bp in length, respectively. Fragments (named Up3 and Down3, respectively).
- the plasmid containing the above weak transcription promoter was used as a template, and PCR amplification was carried out with P17 and P18 to obtain a weak transcription promoter fragment (designated P fragment) of 161 bp in length.
- PCR was carried out as follows: denaturation at 94 °C for 30 s (seconds), annealing at 52 °C for 30 s (seconds), and elongation at 72 °C for 30 s (seconds) (30 cycles).
- PCR was carried out as follows: denaturation at 94 °C for 30 s (seconds), annealing at 52 °C for 30 s (seconds), and extension at 72 °C for 60 s (seconds) (30 cycles).
- Up-P and Down3 fragments isolated and purified by agarose gel electrophoresis were used as templates, and P13 and P16 were used as primers to amplify a fragment of about 1334 bp (named Up-P-down fragment) by Overlap PCR.
- PCR was carried out as follows: denaturation at 94 °C for 30 s (seconds), annealing at 52 °C for 30 s (seconds), and extension at 72 °C for 60 s (seconds) (30 cycles).
- the constructed pKOV-Up-P-Down plasmid was electrotransformed into E. coli NRRL B-12185 strain with low L-lysine production and E. coli K12 W3110 ⁇ 3 strain with high L-lysine production (sequence confirmed by sequencing
- the wild type acnA gene and its upstream and downstream elements are retained on the chromosomes of these two strains, cultured at LB at 30 °C, 100 rpm.
- a homologous recombination-positive monoclonal was selected according to the product guide of Addgene's pKOV plasmid, and it was confirmed by sequencing that the upstream promoter of the wild-type acnA gene on the chromosome was replaced with a weak transcriptional promoter.
- the aconitase of the two strains was tested in different media. The expression levels all decreased by 65 ⁇ 80%.
- the genome of Corynebacterium sinensis AS1.299 (available from China General Microbial Culture Collection Management Center) was used as a template.
- the following primers P19 ⁇ P24 correspond to the above primers P13 ⁇ P18, and the amplified Up3 fragment is 573 bp, Down3.
- the fragment is 581 bp, the P fragment is 130 bp, and the Up-P-down fragment is about 1284 bp, wherein the primer sequences used are as follows;
- the Up-P-down fragment and the pK18mob SaC B plasmid separated and purified by agarose gel electrophoresis were double-digested with Bam ffl/Eco RI, separated and purified by agarose gel electrophoresis, and ligated to obtain a promoter.
- the recombinant vector pK18mobsacB-Up-P-Down was replaced and the sequence was correctly identified.
- pKl 8mobsacB-Up-P-Down plasmid was electrotransformed into L. lysii strain AS1.299 with low L-lysine production, C.
- Example 4 Adding the copy number of the aconitase gene transcription repressor-encoding gene acnR increases the copy number of the acn transcription repressor-encoding gene acnR to reduce the transcription level of the acn gene.
- PCR amplification was performed with primers P25 and P26, P27 and P28, respectively, and two DNA fragments of 715 bp and 797 bp in length were obtained (named Up4, respectively).
- PCR amplification with primers P29 and P30 gave a 567 bp acnR fragment (designated R fragment, the nucleotide sequence of which is shown in SEQ ID No: 7).
- R fragment the nucleotide sequence of which is shown in SEQ ID No: 7
- the expression plasmid pXMJ19 (available from Biovector Science Lab, Inc.) was used as a template, and PCR amplification was carried out by primers P31 and P32 to obtain a strong promoter P ⁇ of 164 bp in length (designated as P tac fragment, Its nucleotide sequence is shown as SEQ ID No: 8.
- PCR was carried out as follows: denaturation at 94 °C for 30 s (seconds), annealing at 52 °C for 30 s (seconds), and extension at 72 °C for 30 s (seconds) (30 cycles).
- the R fragment and the P tac fragment separated and purified by agarose gel electrophoresis were mixed into a template, and the fragments of about 731 13 ⁇ 4) were amplified by Overlap PCR using P31 and P30 as primers (designated as 1 ⁇ -1 fragment);
- the Up4 and P tac -R fragments isolated and purified by agarose gel electrophoresis were mixed into a template, and the fragment of about 1446 bp (named Up4-P tae -R fragment) was amplified by Overlap PCR using P25 and P30 as primers.
- Up4-P tac -R and Down 4 fragments separated and purified by agarose gel electrophoresis were mixed into a template, and P25 and P28 were used as primers, and the length was amplified by Overlap PCR.
- a fragment of approximately 2243 bp (designated as Up-P tac -R-Do W n fragment).
- PCR was carried out as follows: 94 ° degeneration for 30 s (seconds), annealing at 52 °C for 30 s (seconds), and extension at 72 °C for 60 s (seconds) (30 cycles).
- Up-P tae -RD 0wn and pK18mob SaC B plasmids separated and purified by agarose gel electrophoresis were double-digested with Bam HI/Eco RI, separated and purified by agarose gel electrophoresis, and then ligated.
- the non-coding region of the chromosome was inserted into the extra copy of the acnR recombinant vector pK18mobsacB-Up-P tac -R-Down, and the vector pKl 8mobsacB-Up-P tac -R-Down was sent to the sequencing company for sequencing identification, indicating that it contained P The tac- acnR gene fragment was saved for later use.
- the E. coli K12 W3110 A3 strain, the E. coli NRRL B-12185 strain and the E. coli strain constructed in Example 1-3 were respectively inoculated into 25 mL of the seed medium described in Table 1, 37. C, 220 rpm for 9 h. Then, a culture of 1 mL of the seed culture medium was inoculated into 25 mL of the fermentation medium described in Table 1, and cultured at 37 V, 220 rpm for 48 hours. When the culture was completed, the production of L-lysine was measured by HPLC.
- Corynebacterium AS1.299 strain, ATCC13032 strain and AS1.563 strain and Corynebacterium strains constructed in Examples 2-4 were inoculated separately in 30 mL of the seed medium described in Table 2 at 30 V. Incubate at 220 rpm for 8 h. Then, a culture of 1 mL of the seed culture medium was inoculated into 30 mL of the fermentation medium described in Table 2, and cultured at 30 ° C, 220 rpm for 48 hours. When the culture was completed, the production of L-lysine was measured by HPLC. Table 2 Corynebacterium medium formula
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
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US14/384,370 US20160002684A1 (en) | 2013-02-08 | 2014-01-07 | Method for Producing L-Lysine by Modifying Aconitase Gene and/or Regulatory Elements thereof |
EP14748825.8A EP2824186B1 (en) | 2013-02-08 | 2014-01-07 | L-lysine generation method by fermenting bacteria having modified aconitase gene and/or regulatory element |
ES14748825.8T ES2673582T3 (es) | 2013-02-08 | 2014-01-07 | Método de generación de L-lisina por medio de bacterias fermentadoras que poseen un gen de aconitasa y/o un elemento regulador modificado |
KR1020157024148A KR102127181B1 (ko) | 2013-02-08 | 2014-01-07 | 아코니트산수화 효소 유전자와(또는) 그 조절요소를 개조한 세균 발효를 통한 l-리신 생산방법 |
CA2900580A CA2900580C (en) | 2013-02-08 | 2014-01-07 | Method for producing l-lysine by modifying aconitase gene and/or regulatory elements thereof |
CN201480002118.6A CN104619852B (zh) | 2013-02-08 | 2014-01-07 | 用改变乌头酸酶基因和/或其调控元件的细菌发酵生产l-赖氨酸的方法 |
JP2015556383A JP6335196B2 (ja) | 2013-02-08 | 2014-01-07 | アコニターゼ遺伝子および/またはその調節エレメントの改変により、l−リジンを生産する方法 |
DK14748825.8T DK2824186T3 (en) | 2013-02-08 | 2014-01-07 | L-light generation method using fermentation bacteria with modified aconitic gene and / or regulatory element |
RU2015134995A RU2792116C2 (ru) | 2013-02-08 | 2014-01-07 | Способ получения L-лизина модифицированием гена аконитазы и/или его регуляторных элементов |
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CN201310050144.3A CN103146772B (zh) | 2013-02-08 | 2013-02-08 | 用乌头酸酶表达弱化和/或酶活性降低的细菌发酵生产l-赖氨酸的方法 |
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WO2018040469A1 (zh) | 2016-09-01 | 2018-03-08 | 宁夏伊品生物科技股份有限公司 | 发酵生产l-赖氨酸的棒杆菌 |
CN110607313A (zh) * | 2019-09-27 | 2019-12-24 | 内蒙古伊品生物科技有限公司 | 一种高产l-赖氨酸的重组菌株及其构建方法与应用 |
CN110846312A (zh) * | 2019-09-29 | 2020-02-28 | 黑龙江伊品生物科技有限公司 | 一种sdaA基因的启动子核酸序列、含有该核酸序列的重组菌株及其应用 |
CN111909944A (zh) * | 2020-06-08 | 2020-11-10 | 内蒙古伊品生物科技有限公司 | 一种lysC基因改造的重组菌株及其构建方法与应用 |
CN111979165A (zh) * | 2020-08-07 | 2020-11-24 | 黑龙江伊品生物科技有限公司 | 一种产l-赖氨酸的重组菌株及其构建方法与应用 |
WO2021248890A1 (zh) | 2020-06-08 | 2021-12-16 | 黑龙江伊品生物科技有限公司 | 产l-赖氨酸的重组菌株及其构建方法与应用 |
WO2021248902A1 (zh) | 2020-06-08 | 2021-12-16 | 内蒙古伊品生物科技有限公司 | 产l-氨基酸的重组菌株及其构建方法与应用 |
WO2022027924A1 (zh) | 2020-08-07 | 2022-02-10 | 宁夏伊品生物科技股份有限公司 | 一种产l-氨基酸的重组菌株及其构建方法与应用 |
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WO2018040469A1 (zh) | 2016-09-01 | 2018-03-08 | 宁夏伊品生物科技股份有限公司 | 发酵生产l-赖氨酸的棒杆菌 |
CN110607313A (zh) * | 2019-09-27 | 2019-12-24 | 内蒙古伊品生物科技有限公司 | 一种高产l-赖氨酸的重组菌株及其构建方法与应用 |
CN110607313B (zh) * | 2019-09-27 | 2021-06-22 | 内蒙古伊品生物科技有限公司 | 一种高产l-赖氨酸的重组菌株及其构建方法与应用 |
CN110846312A (zh) * | 2019-09-29 | 2020-02-28 | 黑龙江伊品生物科技有限公司 | 一种sdaA基因的启动子核酸序列、含有该核酸序列的重组菌株及其应用 |
CN110846312B (zh) * | 2019-09-29 | 2020-11-10 | 黑龙江伊品生物科技有限公司 | 一种sdaA基因的启动子核酸序列、含有该核酸序列的重组菌株及其应用 |
CN111909944A (zh) * | 2020-06-08 | 2020-11-10 | 内蒙古伊品生物科技有限公司 | 一种lysC基因改造的重组菌株及其构建方法与应用 |
WO2021248890A1 (zh) | 2020-06-08 | 2021-12-16 | 黑龙江伊品生物科技有限公司 | 产l-赖氨酸的重组菌株及其构建方法与应用 |
WO2021248902A1 (zh) | 2020-06-08 | 2021-12-16 | 内蒙古伊品生物科技有限公司 | 产l-氨基酸的重组菌株及其构建方法与应用 |
CN111979165A (zh) * | 2020-08-07 | 2020-11-24 | 黑龙江伊品生物科技有限公司 | 一种产l-赖氨酸的重组菌株及其构建方法与应用 |
CN111979165B (zh) * | 2020-08-07 | 2021-05-07 | 黑龙江伊品生物科技有限公司 | 一种产l-赖氨酸的重组菌株及其构建方法与应用 |
WO2022027924A1 (zh) | 2020-08-07 | 2022-02-10 | 宁夏伊品生物科技股份有限公司 | 一种产l-氨基酸的重组菌株及其构建方法与应用 |
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KR102127181B1 (ko) | 2020-06-30 |
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CA2900580A1 (en) | 2014-08-14 |
JP6335196B2 (ja) | 2018-05-30 |
DK2824186T3 (en) | 2018-07-23 |
US20160002684A1 (en) | 2016-01-07 |
RU2015134995A (ru) | 2019-03-28 |
CA2900580C (en) | 2022-05-31 |
ES2673582T3 (es) | 2018-06-22 |
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KR20150115009A (ko) | 2015-10-13 |
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