WO2019022375A1 - L-라이신을 생산하는 미생물 및 이를 이용한 l-라이신의 생산 방법 - Google Patents
L-라이신을 생산하는 미생물 및 이를 이용한 l-라이신의 생산 방법 Download PDFInfo
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- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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- 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
Definitions
- the present application relates to a microorganism producing L-lysine transformed by introducing a polynucleotide containing an L-lysine inducible promoter, and a method for producing L-lysine using the microorganism.
- DHAP dihydroxyacetone phosphate
- hdpA dihydroxyacetone phosphate
- gpp glycerol phosphatase gene
- poxB pyruvate dehydrogenase gene
- thrB homoserine kinase gene
- IlvBN branched chain amino acid
- pyruvate dehydrogenase subunit E1 gene involved in the conversion of pyruvate to acetylcopherol
- the homoserine dehydrogenase gene homo
- alanine aminotransferase gene inhibits the growth of microorganisms when deficient in the Corynebacterium sp. (J.Marienhagen et al., AEM, p7457-7462, Dec. 2008).
- the object of the present invention is to provide a microorganism transformed so that transcription of a target gene is inhibited by using an L-lysine inducible promoter and a method for producing L-lysine using the microorganism.
- One aspect of the present application provides a microorganism producing L-lysine, wherein the polynucleotide comprising the L-lysine inducible promoter is located at the lower end codon of the target gene.
- the term " promoter" refers to a nucleic acid sequence that is compared to the upstream of the coding region, including the binding site for the polymerase and having a transcription initiation activity of the promoter sub-gene into the mRNA. That is, the promoter is a DNA region that binds to a polymerase to initiate transcription of a gene, and may be located at the 5 'site of the mRNA transcription initiation site.
- L-lysine inducible promoter in the present application means a promoter whose expression level is regulated by lysine concentration, and specifically refers to a promoter whose expression inducing activity is increased when the concentration of L-lysine is increased .
- Lysine-inducible promoter of the present application can be used as long as it is a promoter in which the expression activity of the gene is increased by L-lysine.
- the L-lysine inducible promoter may be a promoter of the NCgl1182 gene or a promoter of the NCgl1504 gene.
- 'NCgl1182' and 'NCgl1504' are expressed in Corynebacterium glutamicum and their functions have not yet been clarified yet. However, 'NCgl1182' is presumed to function as an electron transfer flavoprotein beta-subunit and 'NCgl1504' is involved in the regulation of transcription of iron metabolism related genes .
- polynucleotide refers to a polymer of nucleotides in which a nucleotide monomer is linked in a long chain by covalent bonds.
- a DNA having a certain length or more of deoxyribonucleic acid or RNA (ribonucleic acid) it means.
- polynucleotide comprising an L-lysine inducible promoter in the present application means a polynucleotide consisting of the nucleotide sequence of the L-lysine inducible promoter, or a polynucleotide comprising the polynucleotide.
- the polynucleotide comprising the L-lysine inducible promoter may include not only the nucleotide sequence of the promoter but also a part of the downstream sequence of the promoter.
- a portion of the upstream sequence of the open reading frame operably linked to the promoter may be included.
- a portion of the upstream sequence of the open reading frame may be a sequence of 100 or fewer nucleotides from the N-terminus of the open reading frame. For example, no more than 90 nucleotide sequences, no more than 80 nucleotide sequences, no more than 70 nucleotide sequences, no more than 60 nucleotide sequences, no more than 50 nucleotide sequences, no more than 40 nucleotide sequences, or no more than 30 nucleotides Lt; / RTI >
- the polynucleotide comprising the L-lysine inducible promoter specifically has at least 75%, at least 80%, more preferably at least 90%, more particularly at least 95%, of the nucleotide sequence of any one of SEQ ID NOS: , More specifically more than 99% sequence homology, and may have increased expression-inducing activity when the concentration of L-lysine is increased.
- the present invention is not limited thereto and may include any nucleotide sequence that reverses the introduction of the target gene and weakens the expression of the target gene when the concentration of L-lysine increases, as in the present application.
- homology means the degree to which a given polypeptide sequence or polynucleotide sequence corresponds and may be expressed as a percentage. In the present specification, its homologous sequence having the same or similar activity as a given polynucleotide sequence is indicated as "% homology ". For example, standard software for calculating parameters such as score, identity and similarity, specifically BLAST 2.0, or by sequential hybridization experiments under defined stringent conditions, , And the appropriate hybridization conditions to be defined can be determined by methods well known to those skilled in the art.
- stop codon in the present application is a codon that acts as a signal to indicate that the protein synthesis process has ended without designating the amino acid in the codon on the mRNA.
- UAA, UAG and UGA is used as a general termination codon.
- the term “upper (or upstream)” refers to the 5 'direction, and the term “lower (downstream)” refers to the 3' direction.
- the lower end of the termination codon may refer to a genomic sequence present in the 3 'direction of the termination codon of the target gene.
- the lower termini of the termination codon may be between the termination codon of the target gene and another gene most closely present in the 3 'direction of the target gene.
- the lower end of the termination codon may be between the termination codon of the target gene and the upper end of the transcription terminator. More specifically, immediately after the stop codon of the target gene.
- the termination codon can be one of three DNA sequences (TAG, TAA, TGA), and the promoter insertion position at the lower end of the termination codon can be selected by an ordinary technician at an appropriate position corresponding to the termination codon of the target gene.
- transcription terminator refers to an inverted repeat sequence in which GC bases are abundant, and terminates transcription of the gene by forming a hairpin loop.
- an ordinary skilled artisan can select an appropriate position that weakens the expression of a target gene as a lower end of a termination codon for introducing a polynucleotide including an L-lysine inducible promoter, but does not affect the expression of another gene.
- the microorganism producing the L-lysine in which the polynucleotide comprising the L-lysine inducible promoter is located at the lower end codon of the target gene is, specifically, a polynucleotide comprising the inducible promoter, May be introduced and transformed.
- transformed in the present application means introducing a vector comprising a polynucleotide encoding the target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell.
- the transformed polynucleotides include all of them, whether inserted into the chromosome of the host cell or located outside the chromosome, so long as they can be expressed in the host cell.
- the method of transforming the vector of the present application into the cell includes any method of introducing a base into a cell and can be carried out by selecting a suitable standard technique as known in the art. (Eg, electroporation, calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran, cationic liposome, And the like, but are not limited thereto.
- the microorganism may be one in which the L-lysine inducible promoter is introduced to operate in the reverse direction of the transcription direction of the target gene in the genome. That is, when the polynucleotide comprising the L-lysine inducible promoter is transformed to be introduced at the lower end codon of the target gene in the chromosome of the microorganism, the transcription of the promoter may be introduced so as to operate in a direction opposite to the transcription direction of the target gene have.
- the polynucleotide comprising the L-lysine inducible promoter is introduced into the chromosome and initiates or mediates transcription in a direction opposite to the transcription direction of the target gene to weaken the expression.
- the concentration of L-lysine is increased, the RNA polymerase complex which proceeds in the direction of transcription of the target gene and the RNA polymerase complex derived from the L-lysine inducible promoter of the present application are reversed
- the target gene expression may be weakened as they collide with each other during the transcription process.
- Introduction of the polynucleotide comprising the L-lysine inducible promoter into the genome can be carried out using known gene recombination techniques known in the art.
- the term " target gene " may be a gene located at a branch point of a pathway for biosynthesis of L-lysine from a carbon source or a gene which induces the production of by-products. More specifically, it may be a gene located at a branching point of a carbon flow in a pathway for biosynthesis of lysine, or a gene of a pathway for biosynthesizing a by-product other than lysine.
- a gene encoding an enzyme related to the position of a branch at which a carbon stream is divided in a pathway where a carbon source is supplied to generate glucose, and a pathway through which the lysine is finally produced through the corresponding process and the TCA circuit pathway A gene encoding an enzyme involved in the biosynthesis of a by-product, and a gene encoding an enzyme involved in biosynthesis of a by-product.
- the present invention is not limited thereto.
- the target gene may be homoserine dehydrogenase (DHP), dihydroxy acetone phosphate phosphatase (hdpA), glycerol phosphatase (gpp), lactate dehydrogenase lactate dehydrogenase (ldh), pyruvate dehydrogenase (poxB), alanine aminotransferase (alaT), pyruvate dehydrogenase subunit E1 (aceE) (UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase: murE) or And genes coding for acetohydroxyacid synthase (ilvBN).
- DHP homoserine dehydrogenase
- hdpA dihydroxy acetone phosphate phosphatase
- gpp glycerol phosphatase
- microorganism in the present application includes both wild-type microorganisms and microorganisms in which natural or artificially genetically modified microorganisms have been introduced, and a specific mechanism is weakened due to the insertion of an external gene, Or enhanced microorganism.
- the microorganisms of the present application are, for example, those of the genus Escherichia, Erwinia, Serratia, Providencia, Corynebacterium or Breivibacterium, Brevibacterium, Arthrobacter spp. And Microbacterium spp.
- the microorganism may specifically be a microorganism belonging to the genus Corynebacterium.
- the microorganism of the genus Corynebacterium is selected from the group consisting of Corynebacterium glutamicum, Corynebacterium thermoaminogenes, Brevibacterium flavum, Brevibacterium lactofermentum, Mutants or strains produced therefrom, and the like.
- the microorganism may be a microorganism belonging to the genus Escherichia or Corynebacterium, and most specifically may be Corynebacterium glutamicum, but is not limited thereto.
- the present invention relates to a microorganism producing L-lysine of the present application as a microorganism which has been introduced into a natural strain or a gene associated with an external lysine production mechanism or has enhanced lysine production ability by enhancing or weakening an intrinsic gene activity do.
- Another aspect of the present application is a method for producing L-lysine, comprising: culturing a microorganism having L-lysine producing ability, wherein a polynucleotide comprising an L-lysine inducible promoter is located at a lower end codon of a target gene; And recovering L-lysine from the microorganism and the medium.
- the microorganism having L-lysine producing ability wherein the polynucleotide comprising the L-lysine inducible promoter is located at the lower end codon of the target gene, is as described above.
- the cultivation of the microorganism may be carried out according to a conventional method known in the art.
- the medium used for the above-mentioned culture is, for example, a medium as described in Manual of Methods for General Bacteriology by the American Society for Bacteriology (Washington D.C., USA, 1981).
- saccharide examples include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, , Fatty acids such as stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, either individually or as a mixture.
- sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch and cellulose
- oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, , Fatty acids such as stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, either individually or as a mixture.
- the medium may be, for example, a nitrogen source such as peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate Or mixtures thereof, but are not limited thereto.
- the medium may include, for example, potassium dihydrogenphosphate, dipotassium hydrogenphosphate or the corresponding sodium-containing salts as a source, but is not limited thereto.
- the medium may, for example, include, but is not limited to, metal salts such as magnesium sulfate or iron sulfate necessary for growth.
- Essential growth materials such as amino acids and vitamins or suitable precursors may also be added to the culture in the culture. The material may be added to the culture in a suitable manner, for example, batchwise or continuously, during the course of the culture.
- compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the microorganism culture medium in an appropriate manner to adjust the pH of the culture medium.
- bubble formation can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture.
- oxygen or an oxygen-containing gas e.g., air
- the temperature of the culture medium may be usually 20 to 45 ⁇ , for example, 25 to 40 ⁇ .
- the incubation period may be continued until an amount of the desired target substance is obtained, for example, 10 to 160 hours.
- the culture may be carried out continuously or batchwise, such as a batch process, an injection batch and a repeated batch batch process.
- a culturing method is well known in the art, and any method can be used.
- the term " cultivation step" may include both the medium preparation time and the culturing time.
- the method of the present application comprises the step of recovering L-lysine from microorganisms and media.
- the method for recovering L-lysine from the microorganism and the medium may be a suitable method known in the art depending on the culture method of the microorganism of the present application, for example, batch, continuous or fed-batch culture.
- the step of recovering L-lysine from the microorganism and the medium may further comprise a purification method.
- a protein precipitating agent salting method
- centrifugation centrifugation
- extraction ultrasonic disruption
- ultrafiltration dialysis
- molecular sieve chromatography gel filtration
- adsorption chromatography ion exchange chromatography
- affinity chromatography affinity chromatography
- Another aspect of the present application provides a method wherein a polynucleotide comprising an L-lysine inducible promoter is located at the lower end codon of a target gene to attenuate expression of the gene of interest.
- attenuation of gene expression in the present application means that the expression of the gene is not expressed at all in comparison with the strain of the wild-type strain or the strain before transformation, the expression is partially or completely absent, And the activity is weakened.
- the L-lysine inducible promoter introduced into the recombinant microorganism of the present application can autonomously regulate the expression of the target gene according to the concentration of L-lysine. Accordingly, the recombinant microorganism of the present application can increase the amount of the cell by efficiently utilizing the carbon source for growth until the L-lysine accumulates at a high concentration, and utilize the carbon source for increasing the L-lysine production as the L-lysine concentration increases And thus can be effectively used for mass production of L-lysine.
- FIG. 1 is a schematic diagram showing that the lysine-inducible promoter of the present invention is introduced between the termination codon of the target gene and the upper end of the transcription terminator so as to operate in a direction opposite to the direction of transcription of the target gene, thereby inhibiting the expression of the target gene.
- Figure 2 shows the lysine biosynthetic pathway of coryneform microorganisms.
- 3 is a graph showing the comparison of promoter intensities of respective genes according to lysine concentration.
- Example 1 Screening and selection of lysine inducible promoters
- KCCM11016P In order to search for a promoter whose expression level is regulated by lysine concentration, KCCM11016P (the above microorganism was disclosed as KFCC10881, re-deposited with the international depository institution under the Budapest Treaty and deposited with KCCM11016P, Korean Patent No. 10-0159812 The strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the seed medium and incubated at 30 ° C for 20 hours with shaking at 200 rpm.
- a probe of about 3,000 genes was prepared using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template, which was purified using a Sephades G 50 column.
- the microarray chip was then fabricated by integrating the dried probe with a Robotic Pin spotter (GeneMahines) instrument on the slide.
- CDNA was prepared from RNA of Sample 1 and Sample 2 (3 sets of lysine concentration) prepared in Example 1.1 using a cDNA synthesis kit (PhileKorea). Next, the cDNA of Sample 1 was labeled with Cy3 and the cDNA of Sample 2 was labeled with Cy5 using a Bioprime labeling Kit (Invitrogen). Cy3 or Cy5-labeled cDNA was mixed with 80 ⁇ l of a hybridization solution (5 ⁇ SSC, 0.1% SDS, 50% formamide, 10 ⁇ g salmon sperm DNA) and reacted at 100 ° C. for 3 minutes. The reaction product was applied to the prepared microarray chip and hybridization was carried out at 42 ° C for 16 hours. Hybridization-completed microarray chips were cleaned (1X SSC / 0.2% SDS 4 min, 0.1X SSC / 0.2% SDS 5 min, and 0.1X SSC) and immediately dried.
- a hybridization solution 5 ⁇ SSC, 0.1% SDS, 50% formamide, 10
- Hybridization photographs of microarray slides were taken using an Axon GenePix 4000B scanner (Axon Instruments) and analyzed using GenePix Pro 6.0 program (Axon Instruments).
- Example 1.2 By analyzing hybridization images of the microarray slides of Example 1.2, 92 genes having a value two times or more higher than that of Sample 2 (lysine-containing experimental medium) were selected as compared to Sample 1 (seed medium). Among the 92 species, the target gene to be inhibited was searched, and two genes having the highest signal intensity among the genes having higher signal intensity than that of hom in the sample 2, based on the homologous gene, And finally selected.
- the two genes representing the selected high signal intensity were confirmed to be NCgl1504 and NCgl1182 respectively by the base sequence analysis, and it was found that the promoters of the selected genes NCgl1504 and NCgl1182 were promoted to increase expression by lysine concentration there was. As a result of confirming a region corresponding to the normal promoter region, it was confirmed that the nucleotide sequence of the corresponding promoter is SEQ ID NO: 1 and SEQ ID NO: 2.
- Example 2 Using lysine inducible promoter hdpA Production inhibition of gene expression and confirmation of production effect of lysine according to introduced promoter structure
- a pDZ vector (Korean Patent Registration No. 10-0924065) was used as a basic vector for inserting the NCgl1182 promoter or the NCgl1504 promoter at the bottom of the hdpA gene on the chromosome of the Corynebacterium strain. Based on the reported nucleotide sequence, primers (SEQ ID NOS: 5 to 8) for amplifying the hdpA downstream gene region were synthesized.
- the first PCR using the primers of SEQ ID NOS: 5 and 6 and the second PCR using the primers of SEQ ID NOS: 7 and 8 were carried out,
- the lower end of NCgl1356, the last gene in the open reading frame, was amplified to about 600 bp and a SpeI recognition site was inserted in the middle of the amplified region.
- the PCR was performed at 95 ° C for 5 minutes, followed by denaturation at 95 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, and polymerization at 72 ° C for 60 seconds.
- the PCR was carried out at 72 ° C for 7 minutes.
- Both ends of the gene fragment amplified by PCR were treated with restriction enzyme SalI to obtain a DNA fragment, which was inserted into a pDZ vector for introduction of chromosome with restriction enzyme SalI terminus.
- This was introduced into Escherichia coli DH5a and then plated on LB solid medium containing 25 mg / L kanamycin. Colonies transformed with the vector into which the desired gene was inserted were selected through PCR using SEQ ID NO: 5 and SEQ ID NO: 8.
- the plasmid was then obtained using a conventionally known plasmid extraction method, and the obtained plasmid was named pDZ-hdpA-downstream.
- NCgl1504 promoter and the open reading frame top 39 nucleotides were amplified by PCR using the chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template by using these primers (SEQ ID NOS: 9 to 14) .
- the PCR was performed at 95 ° C for 5 minutes, denatured at 95 ° C for 30 seconds, annealed at 56 ° C for 30 seconds, and polymerized at 72 ° C for 30 seconds, and then polymerized at 72 ° C for 7 minutes.
- the polynucleotides amplified by PCR were individually treated with restriction enzyme SpeI to obtain respective DNA fragments, which were then inserted into a chromosome-introduced pDZ-hdpA-downstream vector treated with restriction enzyme SpeI. This was then used to transform E. coli DH5a and plated on LB solid medium containing 25 mg / L kanamycin.
- the obtained plasmids were designated as pDZ-hdpA-LiCTS1-1, pDZ-hdpA-LiCTS1-2, pDZ-hdpA-LiCTS2-1 and pDZ-hdpA-LiCTS2-2, respectively.
- the plasmid was constructed in such a manner that a portion of the hclpA gene, the NCgl1182 promoter, the open reading frame portion, the NCgl1504 promoter, the NCgl1504 promoter and the open reading frame were partially introduced into the hdpA gene so as to operate in the reverse direction of the hdpA gene transfer direction.
- the four plasmids were labeled with L - lysine producing strain, Corynebacterium glutamicum KCCM11016P. Thereafter, a total of four recombinant strains were obtained through a second-order cross-over process in which a DNA fragment containing the NCgl1182 promoter or the NCgl1504 promoter was introduced at the bottom of the chromosomal hdpA gene.
- the strains into which LiCTS1-1 or LiCTS1-2 was introduced and the strains into which the primers of SEQ ID Nos. 15 and 9, LiCTS2-1 or LiCTS2-2, respectively, were selectively isolated by PCR using the primers of SEQ ID NOs: 15 and 12 And these were designated as KCCM11016P :: hdpA-LiCTS1-1, KCCM11016P :: hdpA-LiCTS1-2, KCCM11016P :: hdpA-LiCTS2-1 and KCCM11016P :: hdpA-LiCTS2-2, respectively.
- KCCM11016P hdpA-LiCTS1-1
- KCCM11016P hdpA-LiCTS1-2
- KCCM11016P :: hdpA-LiCTS2-1
- KCCM11016P :: hdpA-LiCTS2-2
- the lysine production ability and the culture liquid component were analyzed.
- the composition of the seed medium and the production medium used for the cultivation is as follows.
- L-lysine production and by-products of each strain were measured by HPLC after completion of culture.
- the results of measurement of L-lysine and byproducts in the cultures of KCCM11016P and the above four strains are shown in Table 1 below.
- NCgl1182 promoter or “NCgl1504 promoter” described in the following examples are each referred to as "a polynucleotide comprising the promoter of NCgl1182 and the top 57 sequences at the top of the open reading frame" and the "NCgl1504 promoter” Polynucleotide comprising 39 sequences on the top of the frame ".
- Example 3 Production of strains inhibiting the expression of various genes using a lysine-inducible promoter and its production ability
- primers SEQ ID NOS: 16 to 19
- the chromosome of Corynebacterium glutamicum ATCC 13032 as a template in the same manner as in Example 2.1
- the lower end of the gpp gene was amplified to about 800 bp.
- Both ends of the gene fragment amplified by PCR were treated with restriction enzyme SalI to obtain a DNA fragment, which was then ligated to a pDZ vector for chromosome introduction having a restriction enzyme SalI terminus to transform E.
- a plasmid was obtained by a conventional plasmid extraction method after selecting colonies transformed with a vector having the desired gene inserted therein through PCR using SEQ ID NO: 16 and SEQ ID NO: 19, and this plasmid was introduced into pDZ-gpp-downstream Lt; / RTI >
- NCgl1182 promoter or NCgl1504 promoter was inserted into the pDZ-gpp-downstream vector as in Example 2.1, transformed into E. coli DH5 ⁇ , and plated on LB solid medium containing 25 mg / L of kanamycin.
- the plasmid was obtained by selecting the colonies transformed with the above vector through PCR (SEQ ID NO: 16 and SEQ ID NO: 9 and SEQ ID NO: 16 and SEQ ID NO: 12), and these plasmids were designated pDZ-gpp-LiCTS1 and pDZ- LiCTS2 < / RTI > The plasmid is introduced in such a manner that the NCgl1182 promoter and the NCgl1504 promoter are respectively inserted in the lower end of the gpp gene so as to operate in the direction opposite to the transcription direction of the gpp gene.
- the pDZ vector was used as the basic vector. Based on the reported nucleotide sequence, primers (SEQ ID NOS: 20 to 23) for amplifying the ldh downstream gene region were synthesized, and the chromosome of Corynebacterium glutamicum ATCC 13032 was used as a template to perform primary PCR and secondary PCR was performed to amplify the lower end of the ldh gene to about 800 bp.
- the plasmid was named pDZ-ldh-downstream.
- NCgl1182 promoter or the NCgl1504 promoter was inserted into the pDZ-ldh-downstream vector as in Example 2.1, transformed into E. coli DH5 ⁇ , and plated on LB solid medium containing 25 mg / L of kanamycin.
- the plasmids were obtained by selecting the colonies transformed with the above vector through PCR (SEQ ID NO: 20 and SEQ ID NO: 9 and SEQ ID NO: 20 and SEQ ID NO: 12), and these plasmids were transformed into pDZ-ldh-LiCTS1 and pDZ-ldh-LiCTS2 Lt; / RTI >
- the plasmid is introduced in such a manner that the NCgl1182 promoter and the NCgl1504 promoter are respectively inserted in the lower end of the ldh gene so as to operate in the reverse direction of the ldh gene transcription direction.
- a primer for amplifying the poxB lower gene site was synthesized based on the reported nucleotide sequence.
- the first PCR and the second PCR were performed using the above primers with the chromosome of Corynebacterium glutamicum ATCC 13032 as a template and the lower part of the poxB gene was amplified to about 600 bp.
- the gene fragment amplified by PCR was treated with restriction enzyme SalI to obtain a DNA fragment, which was then ligated to a pDZ vector for chromosomal introduction having a restriction enzyme SalI terminus, transformed into E. coli DH5 ⁇ , and 25 mg / L of kanamycin Lt; / RTI > solid medium.
- the colonies transformed with the vector into which the desired gene was inserted were selected through PCR (SEQ ID NOS: 24 and 27), and a plasmid was obtained using a conventionally known plasmid extraction method.
- the plasmid was named pDZ-poxB-downstream.
- the NCgl1182 promoter or the NCgl1504 promoter was inserted into the pDZ-poxB-downstream vector, transformed into E. coli DH5 ⁇ , and plated on LB solid medium containing 25 mg / L of kanamycin.
- the plasmid was obtained by selecting the colonies transformed with the above vector through PCR (SEQ ID NO: 24 and SEQ ID NO: 9 and SEQ ID NO: 24 and SEQ ID NO: 12), and the plasmid was inserted into pDZ-poxB-LiCTS1 and pDZ-poxB-LiCTS2 Lt; / RTI >
- the plasmid is a form in which the NCgl1182 promoter and the NCgl1504 promoter are introduced in the reverse direction of the transcription direction of the poxB gene at the lower end of the poxB gene, respectively.
- a primer for amplifying the alaT bottom gene region was synthesized based on the reported nucleotide sequence.
- Primary PCR and secondary PCR were performed using the above primers with the chromosome of Corynebacterium glutamicum ATCC 13032 as a template, and the lower end of the alaT gene was amplified to about 700 bp.
- the gene fragment amplified by PCR was treated with restriction enzyme SalI to obtain a DNA fragment, which was then ligated to a pDZ vector for chromosomal introduction having a restriction enzyme SalI terminus, transformed into E. coli DH5 ⁇ , and 25 mg / L of kanamycin Lt; / RTI > solid medium.
- the colonies transformed with the vector into which the desired alaT gene had been inserted were selected through PCR (SEQ ID NOS: 28 and 31), and plasmids were obtained using a conventionally known plasmid extraction method. This plasmid was named pDZ-alaT-downstream .
- the NCgl1182 promoter or the NCgl1504 promoter was inserted into the pDZ-alaT-downstream vector as in Example 2.1, transformed into E. coli DH5 ⁇ , and plated on LB solid medium containing 25 mg / L kanamycin.
- the plasmids transformed with the vector were selected through PCR (SEQ ID NO: 28 and SEQ ID NO: 9, and SEQ ID NO: 28 and SEQ ID NO: 12), and plasmids were obtained from the plasmids.
- the plasmids were transformed into pDZ-alaT-LiCTS1 and pDZ-alaT -LiCTS2 < / RTI >
- the plasmid is introduced in such a manner that the NCgl1182 promoter and the NCgl1504 promoter are respectively inserted in the lower end of the alaT gene so as to function in the direction opposite to the direction of transfer of the alaT gene.
- primers for amplifying the aceE bottom gene region were synthesized based on the reported nucleotide sequences.
- the first PCR and the second PCR were performed using the above primers with the chromosome of Corynebacterium glutamicum ATCC 13032 as a template and the lower end of the aceE gene was amplified to about 700 bp.
- the gene fragment amplified by PCR was treated with restriction enzyme SalI to obtain a DNA fragment, which was then ligated to a pDZ vector for chromosomal introduction having a restriction enzyme SalI terminus, transformed into E. coli DH5 ⁇ , and 25 mg / L of kanamycin Lt; / RTI > solid medium.
- Colonies transformed with the vector into which the desired aceE gene was inserted were selected through PCR (SEQ ID NOS: 32 and 35), and the plasmid was obtained.
- the plasmid was named pDZ-aceE-downstream.
- the NCgl1182 promoter or the NCgl1504 promoter was inserted into the pDZ-aceE-downstream vector, transformed into E. coli DH5 ⁇ , and plated on LB solid medium containing 25 mg / L of kanamycin.
- a plasmid was obtained by selecting the colonies transformed with the vector through PCR (SEQ ID NO: 32 and SEQ ID NO: 9 and SEQ ID NO: 32 and SEQ ID NO: 12), and the plasmid was inserted into pDZ-aceE-LiCTS1 and pDZ-aceE-LiCTS2 Lt; / RTI >
- the above plasmid is a form in which the NCgl1182 promoter and NCgl1504 promoter are introduced at the lower end of the aceE gene so as to operate in the reverse direction of the transcription direction of the aceE gene.
- primers for amplifying the thrB downstream gene site were synthesized based on the reported nucleotide sequence.
- the first PCR and the second PCR were performed using the above primers with the chromosome of Corynebacterium glutamicum ATCC 13032 as a template and the lower end of the thrB gene was amplified to about 700 bp.
- the gene fragment amplified by PCR was treated with restriction enzyme EcoRI to obtain a DNA fragment, which was then ligated to a pDZ vector for introducing a chromosome having an EcoRI terminus and transformed into E. coli DH5 ⁇ , and LB containing 25 mg / L of kanamycin And plated on solid medium.
- the plasmids were named pDZ-thrB-downstream by selecting plasmids transformed with the vector in which the thrB gene was inserted through PCR (SEQ ID NOS: 36 and 39) and using a conventionally known plasmid extraction method.
- the NCgl1182 promoter or NCgl1504 promoter was inserted into the pDZ-thrB-downstream vector as in Example 2.1, transformed into E. coli DH5 ⁇ , and plated on LB solid medium containing 25 mg / L kanamycin.
- the plasmid was obtained by selecting the colonies transformed with the above vector through PCR (SEQ ID NO: 36 and SEQ ID NO: 9, SEQ ID NO: 36 and SEQ ID NO: 12), and the plasmid was inserted into pDZ-thrB-LiCTS1 and pDZ-thrB-LiCTS2 Respectively.
- the plasmid is a form in which the NCgl1182 promoter and the NCgl1504 promoter are introduced at the lower end of the thrB gene so as to operate in the reverse direction of the transcription direction of the gene.
- a primer for amplifying the murE bottom gene site was synthesized based on the reported nucleotide sequence.
- the first PCR and the second PCR were performed using the above primers with the chromosome of Corynebacterium glutamicum ATCC 13032 as a template and the lower end of the murE gene was amplified to about 800 bp.
- the gene fragment amplified by PCR was treated with restriction enzyme XbaI to obtain a DNA fragment, which was then ligated to a pDZ vector for introducing a chromosome having a restriction endonuclease XbaI end, transformed into E. coli DH5 ⁇ , and contained 25 mg / L of kanamycin Lt; / RTI > solid medium.
- the colonies transformed with the vector into which the murE gene gene was inserted were selected through PCR (SEQ ID NOs: 40 and 43), and plasmids were obtained using a conventionally known plasmid extraction method. This plasmid was named pDZ-murE-downstream.
- the NCgl1182 promoter or the NCgl1504 promoter was ligated to the pDZ-murE-downstream vector, transformed into E. coli DH5 ⁇ , and plated on LB solid medium containing 25 mg / L kanamycin.
- the plasmid was obtained by selecting the colonies transformed with the vector through PCR (SEQ ID NO: 40 and SEQ ID NO: 9 and SEQ ID NO: 40 and SEQ ID NO: 12), and the plasmid was inserted into pDZ-murE-LiCTS1 and pDZ-murE-LiCTS2 Lt; / RTI >
- the plasmid is introduced in such a manner that the NCgl1182 promoter and the NCgl1504 promoter are introduced at the lower end of the murE gene so as to operate in the reverse direction of the transcription direction of the gene.
- a primer for amplifying the ilvBN bottom gene site was synthesized based on the reported nucleotide sequence.
- the first PCR and the second PCR were performed using the above primers with the chromosome of Corynebacterium glutamicum ATCC 13032 as a template and the lower part of the ilvBN gene was amplified to about 800 bp.
- the gene fragment amplified by PCR was digested with restriction enzyme EcoRI to obtain a DNA fragment, which was then ligated to a pDZ vector for introduction of a chromosome having a restriction endonuclease EcoRI end, transformed into E. coli DH5 ⁇ , and 25 mg / L of kanamycin Lt; / RTI > solid medium.
- the plasmids were obtained by selecting the colonies transformed with the vector into which the ilvBN gene was inserted through PCR (SEQ ID NOS: 44 and 47), and this plasmid was named pDZ-ilvBN-downstream.
- NCgl1182 promoter or NCgl1504 promoter was ligated to the pDZ-ilvBN-downstream vector as in Example 2.1, transformed into E. coli DH5 ⁇ , and plated on LB solid medium containing 25 mg / L kanamycin.
- the plasmid was obtained by selecting the colonies transformed with the above vector through PCR (SEQ ID NO: 44 and SEQ ID NO: 9 and SEQ ID NO: 44 and SEQ ID NO: 12), and the plasmid was inserted into pDZ-ilvBN-LiCTS1 and pDZ-ilvBN-LiCTS2 Lt; / RTI >
- the plasmid is a form in which the NCgl1182 promoter and the NCgl1504 promoter are introduced in the reverse direction of the transcription direction of the gene at the lower end of the ilvBN gene, respectively.
- KCCM11016P selectively isolating and isolating the isolated strains as KCCM11016P :: gpp-LiCTS1, KCCM11016P :: gpp-LiCTS2, KCCM11016P :: ldh-LiCTS1, KCCM11016P :: ldh- LiCTS2, KCCM11016P :: poxB- LiCTS1, KCCM11016P :: poxB- LiCTS2, KCCM11016P :: thrB-LiCTS1, KCCM11016P :: thrB-LiCTS2, KCCM11016P :: alEt-LiCTS2, , KCCM11016P :: murE-LiCTS2, KCCM11016P :: murE-LiCTS2, KCCM11016P ::: ilvBN-LiCTS1, and KCCM11016P :: ilv
- KCCM11016P :: ldh-LiCTS1, KCCM11016P :: ldh-LiCTS2, KCCM11016P :: poxB-LiCTS1, KCCM11016P :: gpp-LiCTS2, KCCM11016P :: poxB-LiCTS2, KCCM11016P :: alaT-LiCTS1, KCCM11016P :: alaT-LiCTS2, KCCM11016P :: aceE-LiCTS1, KCCM11016P :: aceE-LiCTS2, KCCM11016P :: thrB- LiCTS1, KCCM11016P :: thrB- LiCTS2, KCCM11016P The strains were cultured in the same manner as in Example 2.3 to confirm lysine production ability of :: murE-LiCTS1, KCCM11016P :: murE-L
- Example 4 Production of integrated strains inhibiting the expression of target genes using lysine-inducible promoter from various lysine producing strains and confirming lysine production ability
- KCCM11016P KCCM10770P (Korean Patent No. 10-0924065)
- KCCM11347P the above microorganism was disclosed as KFCC10750, was re-deposited to the international depository under the Budapest Treaty, and received KCCM11347P.
- (PDZ-hdpA-LiCTS2-2, pDZ-gpp-LiCTS2, pDZ-gpp-LiCTS2) prepared in Examples 2 to 10 were added to CJ3P (Binder et al.
- the primers (SEQ ID NO: 15 and SEQ ID NOs: 48 to 55) used in Example 2 and Example 3 and the primers of SEQ ID NO: 12 were used for PCR in which colonies in which the NCgl1504 promoter was introduced at the bottom of nine target genes on the chromosome of the strain Respectively.
- 9C-LiCTS2, KCCM10770P :: 9g-LiCTS2, and KCCM11347P :: KCCM11016P :: 9g-LiCTS2 prepared in the above Example 4.1 were mixed with the control strain (parent strains: KCCM11016P, KCCM10770P, KCCM11347P and CJ3P) in the same manner as in Example 3.10.
- 9g-LiCTS2, and CJ3P :: 9g-LiCTS2 were inoculated and cultured, respectively. After the incubation, the amount of L-lysine produced was measured by an amino acid analyzer. The results are shown in Table 3 below.
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Abstract
Description
균주 | 부산물 | 라이신 | |
목록 | 평균*(g/L) | 평균*(g/L) | |
KCCM11016P | DHA | 0.40 | 43.1 |
KCCM11016P::hdpA-LiCTS1-1 | 0.38 | 43.2 | |
KCCM11016P::hdpA-LiCTS1-2 | 0.35 | 43.4 | |
KCCM11016P::hdpA-LiCTS2-1 | 0.32 | 43.5 | |
KCCM11016P::hdpA-LiCTS2-2 | 0.30 | 43.5 |
균주 | 부산물 | 라이신 | |
목록 | 평균*(g/L) | 평균*(g/L) | |
KCCM11016P | 글리세롤 | 0.50 | 43.1 |
KCCM11016P::gpp-LiCTS1 | 0.40 | 43.5 | |
KCCM11016P::gpp-LiCTS2 | 0.30 | 43.6 | |
KCCM11016P | 락테이트 | 1.80 | 43.1 |
KCCM11016P::ldh-LiCTS1 | 1.20 | 43.9 | |
KCCM11016P::ldh-LiCTS2 | 1.20 | 44.0 | |
KCCM11016P | 아세테이트 | 1.50 | 43.1 |
KCCM11016P::poxB-LiCTS1 | 0.80 | 43.8 | |
KCCM11016P::poxB-LiCTS2 | 0.70 | 43.9 | |
KCCM11016P | 알라닌 | 2.00 | 43.1 |
KCCM11016P::alaT-LiCTS1 | 0.80 | 44.5 | |
KCCM11016P::alaT-LiCTS2 | 0.41 | 44.7 | |
KCCM11016P | 아세틸-CoA | 0.50 | 43.1 |
KCCM11016P::aceE-LiCTS1 | 0.30 | 43.8 | |
KCCM11016P::aceE-LiCTS2 | 0.20 | 44.0 | |
KCCM11016P | 쓰레오닌 | 1.00 | 43.1 |
KCCM11016P::thrB-LiCTS1 | 0.80 | 43.8 | |
KCCM11016P::thrB-LiCTS2 | 0.45 | 44.1 | |
KCCM11016P | 무람산(Muramic acid) | 0.30 | 43.1 |
KCCM11016P::murE-LiCTS1 | 0.10 | 43.8 | |
KCCM11016P::murE-LiCTS2 | 0.10 | 44.0 | |
KCCM11016P | 발린 | 0.40 | 43.1 |
KCCM11016P::ilvBN-LiCTS1 | 0.22 | 43.5 | |
KCCM11016P::ilvBN-LiCTS2 | 0.15 | 43.8 |
Claims (16)
- L-라이신 유도성 프로모터를 포함하는 폴리뉴클레오티드가 목적 유전자의 종결코돈 하단에 위치하는, L-라이신을 생산하는 미생물.
- 제1항에 있어서, 상기 L-라이신 유도성 프로모터는 NCgl1182 유전자의 프로모터 또는 NCgl1504 유전자의 프로모터인 것인, L-라이신을 생산하는 미생물.
- 제1항에 있어서, 상기 L-라이신 유도성 프로모터를 포함하는 폴리뉴클레오티드는 서열번호 1 내지 4 중 어느 하나의 뉴클레오티드 서열로 이루어진 것인, L-라이신을 생산하는 미생물.
- 제1항에 있어서, 상기 L-라이신 유도성 프로모터가 목적 유전자의 전사방향과 역방향으로 작동하도록 도입된 것인, L-라이신을 생산하는 미생물.
- 제1항에 있어서, 상기 종결코돈 하단은 상기 목적 유전자의 종결코돈과 전사종결자 상단의 사이인 것인, L-라이신을 생산하는 미생물.
- 제1항에 있어서, 상기 목적 유전자는 라이신을 생합성하는 경로에서 탄소흐름이 분할되는 분지점에 위치하는 유전자 또는 라이신 이외의 부산물을 생합성하는 경로의 유전자인, L-라이신 생산능을 가진 재조합 미생물
- 제1항에 있어서, 상기 목적 유전자는 hom, hdpA, gpp, ldh, poxB, alaT, aceE, thrB, murE, 및 ilvBN으로 이루어진 군으로부터 선택되는 하나 이상인 것인, L-라이신을 생산하는 미생물.
- 제1항에 있어서, 상기 미생물은 코리네움박테리움 속인 것인, L-라이신을 생산하는 미생물.
- 제7항에 있어서, 상기 미생물은 코리네박테리움 글루타미쿰인 것인, L-라이신을 생산하는 미생물.
- 청구항 1 내지 9 중 어느 한 항의 미생물을 배지에서 배양하는 단계; 및상기 미생물 및 배지로부터 L-라이신을 회수하는 단계를 포함하는 L-라이신의 생산방법.
- L-라이신 유도성 프로모터를 포함하는 폴리뉴클레오티드를 목적 유전자의 종결코돈 하단에 도입하여, 목적유전자의 발현을 약화하는 방법.
- 제11항에 있어서, 상기 L-라이신 유도성 프로모터는 서열번호 1 내지 4 중 어느 하나의 뉴클레오티드 서열로 이루어진 것인, 목적유전자의 발현을 약화하는 방법.
- 제11항에 있어서, 상기 L-라이신 유도성 프로모터가 목적 유전자의 전사방향과 역방향으로 작동하도록 도입된 것인, 목적유전자의 발현을 약화하는 방법.
- 제11항에 있어서, 상기 종결코돈 하단은 상기 목적 유전자의 종결코돈과 전사종결자 상단의 사이인 것인, 목적유전자의 발현을 약화하는 방법.
- 제11항에 있어서, 상기 목적 유전자는 라이신을 생합성하는 경로에서 탄소흐름이 분할되는 분지점에 위치하는 유전자 또는 라이신 이외의 부산물을 생합성하는 경로의 유전자인, 목적유전자의 발현을 약화하는 방법.
- 제11항에 있어서, 상기 목적 유전자는 hom, hdpA, gpp, ldh, poxB, alaT, aceE, thrB, murE, 및 ilvBN으로 이루어진 군으로부터 선택되는 하나 이상인 것인, 목적유전자의 발현을 약화하는 방법.
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WO2009096690A2 (ko) * | 2008-01-31 | 2009-08-06 | Cj Cheiljedang Corporation | 개량된 프로모터 및 이를 이용한 l-라이신의 생산 방법 |
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