KR20150042692A - Method for producing L-amino acid - Google Patents

Abstract

The present invention relates to a method for producing L-amino acid by using recombinant coryneform microorganisms which weaken expression of a target gene by using a transcription inhibition method.

Description

Method for producing L-amino acid < RTI ID = 0.0 >

The present invention relates to a method for producing L-amino acid using a gene transcription inhibiting method.

In the coryneform microorganism, pyruvate produced from various carbon sources through Glycolysis is converted to aspartate via oxaloacetate. The aspartate is converted to amino acids such as threonine, methionine, isoleucine and lysine via each biosynthetic pathway (Fig. 1). Thus, by inhibiting the expression of the gene located at the branching point in the amino acid biosynthesis process, it is possible to reduce the by-product production and increase the target amino acid production.

In order to develop a microorganism capable of producing a target substance at a high frequency by using genetic engineering or metabolic engineering techniques, it is necessary to selectively control the expression of a gene involved in various metabolic processes in a microorganism. Recently, a technique called " artificial convergent transcription " has been reported to attenuate gene expression (Krylov et < RTI ID = 0.0 > al ., J Mol Microbiol Biotechnol , 18: 1-13, 2010). In the artificial convergence transcription technology, a promoter is inserted at the lower end of a transcription terminator of a target gene so as to operate in a direction opposite to the direction of transcription of the gene, so that RNA polymerase complexes derived from each promoter It is a technique that causes collision to weaken target gene expression.

Accordingly, the present inventors have developed a technique for selectively inhibiting the expression of a target gene under the condition that acetate is present by inserting an acetate inducible promoter to operate in a direction opposite to the transcription direction of the target gene, and expressing the gene located at the branch point It is possible to provide a coryneform microorganism having an increased productivity of L-amino acid. Thus, the present invention has been completed.

It is an object of the present invention to provide a method for producing L-amino acid by inhibiting gene transcription using an acetate inducible promoter.

In order to achieve the above object,

1) culturing a recombinant coryneform microorganism having an ability to produce L-amino acid, which is transformed by introducing an acetate inducible promoter into a lower end codon of a target gene in a chromosome; And

  2) enhancing L-amino acid producing ability of the recombinant coryneform microorganism by weakening expression of the target gene during culturing by adding acetate in the culturing step;

Amino acid. ≪ / RTI >

As described above, the present invention utilizes an acetate-inducible promoter to weaken the expression of a target gene by adding acetate in an appropriate time to produce a desired L-amino acid in a high yield, and thus can be effectively used for the production of L-amino acid .

Fig. 1 is a view showing a branch point of an amino acid biosynthesis process in a coryneform microorganism.
FIG. 2 (a) shows a case in which the promoter of the aceA gene is inserted between the termination codon of the aceE gene and the upper end of the transcription terminator so that the promoter of the aceA gene is operative in the reverse direction to the transcription direction of the aceE gene at the lower end of the transcription terminator to inhibit the expression of the aceE gene Fig.

Hereinafter, the present invention will be described in detail.

In one embodiment,

1) culturing a recombinant coryneform microorganism transformed with L-amino acid producing ability by introducing an acetate inducible promoter into a lower end codon of a target gene in a chromosome; And

2) enhancing L-amino acid producing ability of the recombinant coryneform microorganism by weakening expression of the target gene during culturing by adding acetate in the culturing step;

Amino acid. ≪ / RTI >

In the present invention, the term "acetate-inducible promoter" refers to a promoter having an expression-inducing activity under the presence of acetate.

In the coryneform microorganism, the acetate was replaced by acetate kinase (ackA, NCgl2656) and phosphotransacetylase (pta, NCgl2657) or by succinyl-CoA: acetate is converted to acetyl CoA by acetyl CoA-transferase, actA, NCgl2480, and then metabolized through the action of isocitrate lyase (aceA, NCgl2248) in the glyoxylic acid circuit. Acetyl-CoA synthetase (acs, b4069) converts acetyl to acetylcohols in Escherichia coli (Gerstmeir et al., J Biotechnol, 104, 99-122, 2003). The above genes involved in acetate metabolism are induced in the presence of acetate. Therefore, when the promoters of these genes are used, gene expression can be induced specifically in the presence of acetate.

Thus, a promoter of a gene (aceA, NCgl2248) encoding isocitrate lyase or a gene encoding a acetate kinase (ackA, NCgl2656) and phosphotransacetylase phosphotransacetylase) (pta, NCgl2657) promoter of the operon, that is, the upper promoter of the pta gene. More specifically, among the acetate inducible promoters described above, the promoter of the aceA gene has the nucleotide sequence shown in SEQ ID NO: 1 and has 486 base pairs at the top of the aceA gene and an open reading frame (ORF) And contains 36 base pairs from the N-terminus.

The upper promoter of the pta gene, another acetate inducible promoter, has the nucleotide sequence shown in SEQ ID NO: 2 and contains 340 base pairs at the top of the pta gene.

Also, it is obvious that the present invention is within the scope of the present invention if it can induce the expression of the target gene by acetate. For example, it includes the nucleotide sequence of SEQ ID NO: 1 or 2 or the conserved sequence of the nucleotide sequence of SEQ ID NO: 1 or 2, and includes one or more amino acid residues at one or more positions And more preferably 2 to 10, and more preferably 2 to 5) bases are substituted, deleted, inserted, added, or inverted base sequence (s) As long as it can maintain or enhance the function of the inducible promoter, at least 80%, preferably at least 90%, more preferably at least 95%, of the nucleotide sequence of SEQ ID NO: 1 or 2 , Particularly preferably 97% or more homology, and substitution, deletion, insertion, addition, or inversion of the base may be naturally occurring as long as it retains the inducible promoter function Generated can include even the mutant sequence or an artificial mutant sequence.

The term "homology" of the present invention means identity between two different base sequences, using BLAST 2.0, which calculates parameters such as score, identity and similarity , And may be determined by methods well known to those skilled in the art, but are not particularly limited thereto.

In the present invention, unless otherwise stated, the term "upstream" refers to the 5 'direction and the term "downstream" refers to the 3' direction.

In the present invention, the target gene in the chromosome is a gene involved in the biosynthesis process of amino acids such as threonine, methionine, isoleucine and lysine from various carbon sources, It can be a gene located at a branch.

For example, in lysine, pyruvate dehydrogenase subunit E1 (aceE, NCgl2167) gene involved in converting pyruvate to acetyl CoA, homoserin from aspartate Homoserine dehydrogenase (hom, NCgl1136) gene, and meso-2,6-diaminopimelate, which is a precursor of lysine, (UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase, murE, NCgl2083) gene is biosynthesized It is located at the minute point of the path.

In threonine, dihydrodipicolinate synthase (dapA, NCgl1896) gene, which is involved in lysine production from aspartate, and methionine, homoserine kinase, which is involved in threonine production from homoserine, thrB, NCgl1137) gene is the branch point. In the case of alanine (alanine) and valine (pyruvate-derived amino acids), the pyruvate dehydrogenase subunit E1 (aceE, NCgl2167) gene involved in converting pyruvate to acetyl CoA Is located at the branch point.

As a specific example of the target gene, a gene encoding a pyruvate dehydrogenase subunit E1 (aceE, NCgl12167), a gene encoding homoserine dehydrogenase (hom, NCgl1136), yipipin-acetylmuramoyl (DgA, NCgl1896) coding for the gene (murE, NCgl2083) or the dihydrodipicolinate synthase coding for the N-di-glutamate-2,6-diaminopimera ligationase , But is not limited thereto.

Specifically, pyruvate dehydrogenase subunit E1 (aceE, NCgl2167) is a pyruvate dehydrogenase complex (PDHC) involved in introducing pyruvate, which is an end metabolite of the process, into a TCA circuit (tricarboxylic acid cycle) Lt; / RTI > protein. Thus, by weakening the expression level of the aceE gene, it is possible to increase the lysine production by weakening the entry of the carbon source into the TCA circuit and increasing the entry of the carbon source toward lysine biosynthesis.

Homoserine dehydrogenase (hom, NCgl1136) is an enzyme that synthesizes homoserine from aspartate semialdehyde. Since aspartate semialdehyde is one of the intermediate precursors in the lysine biosynthetic pathway, it may weaken the activity of the hom gene, thereby weakening the entry of the carbon source into the homoserine synthesis pathway and increasing the carbon source entry towards lysine biosynthesis, thereby increasing lysine production.

(UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase, murE, NCgl2083) was synthesized as a mycelium- Meso-2,6-Diaminopimelate is used for the synthesis of microbial cells. Meso-2,6-diamino pimarate is also used as a precursor for lysine biosynthesis, which weakens the activity of the murE gene, weakening the entry of the carbon source into the cell synthesis, and increasing the entry of the carbon source into the lysine biosynthetic pathway, have.

Dihydrodipicolinate synthase (dapA, NCgl1896) is an enzyme involved in lysine production using aspartate semialdehyde. Since aspartate semialdehyde is one of the intermediate precursors in the threonine biosynthetic pathway, it weakens the activity of the dapA gene, thereby weakening the carbon source entry into the lysine synthesis pathway and increasing the source of carbon source toward threonine biosynthesis, thereby increasing threonine production .

In the present invention, the term " stop codon "is a codon that serves as a signal indicating that the protein synthesis process has been completed without designating the amino acid in the codon on the mRNA. Generally, three types of UAA, UAG and UGA are terminated It is used as a codon.

In the present invention, the term " transcription terminator "refers to an inverted repeat sequence in which a large number of GC bases exist, and terminates transcription of a gene by forming a hairpin loop.

In the present invention, for the purpose of attenuating the expression of the target gene as described above, an acetate inducible promoter may be introduced between the terminating codon of the target gene, preferably between the termination codon and the transcription terminator. The use of an acetate inducible promoter allows reverse transcription of the target gene using acetate at a time point when the expression of the target gene can be attenuated so that the RNA polymerase complex causes collision during the transcription process, Lt; / RTI >

The time point at which the expression of the target gene can be attenuated can be any time during the culturing period, more specifically, before or during culturing.

In the present invention, the term "transformation" means introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The introduced polynucleotide can be inserted into the chromosome of the host cell or located outside the chromosome as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as far as it is capable of being introduced into a host cell and expressed.

The coryneform microorganism having L-amino acid producing ability used in the present invention is preferably selected from the group consisting of Corynebacterium genus, Brevibacterium genus, Arthrobacter sp. And Microbacterium sp. ). ≪ / RTI > Such coryneform microorganisms include, but are not limited to, Corynebacterium glutamicum, Corynebacterium thermoaminogenes, Brevibacterium flavum, Brevibacterium lactofermentum, Amino acid production mutants or strains, and preferably Corynebacterium glutamicum. However, the present invention is not limited to these examples.

More specifically, in the present invention, as a coryneform microorganism, Corynebacterium glutamicum KCCM11016P strain (former Deposit No. KFCC10881, see Korean Patent No. 0159812), Corynebacterium glutamicum KCCM10770P strain (see Korean Patent No. 0924065 ), And Corynebacterium glutamicum KCCM11347P strain (former Accession No. KFCC10750, Korean Patent Registration No. 1994-0001307) was used.

Also, in the present invention, Corynebacterium glutamicum CJ3P strain was used as a coryneform microorganism, and according to the report of Binder et al., Corynebacterium glutamicum wild stock (ATCC13032) (Binder et al., Genome Biology 2012, 13: R40) by introducing mutations into three genes related to productivity ( pyc (P458S), hom (V59A), and lysC (T311I) .

As another coryneform microorganism, Corynebacterium glutamicum KCCM11222P strain was used, which is an L-threonine producing strain (see Korean Patent Registration No. 20131126) 2011-0127955).

In one embodiment of the present invention, the Coryneform microorganisms into which the promoter having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention introduced increased L-lysine or L-threonine production ability as compared with the parent strain.

In the method of the present invention, culturing of the coryneform microorganism may be carried out by any culture conditions and culture methods known in the art.

As a medium that can be used for culturing the coryneform strain, for example, there is a medium as disclosed in Manual of Methods for General Bacteriology by the American Society for Bacteriology (Washington D.C., USA, 1981).

Sugars which can be used in the medium 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 and coconut oil, palmitic acid, , Fatty acids such as linoleic acid, glycerol, alcohols such as ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture.

Examples of nitrogen sources that may be used include peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean wheat and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen source may also be used individually or as a mixture.

The number of people that can be used includes salts containing dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium. In addition, the culture medium must contain a metal salt such as magnesium sulfate or iron sulfate necessary for growth. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used. In addition, suitable precursors may be used in the culture medium. The above-mentioned raw materials can be added to the culture in a batch manner or in a continuous manner by an appropriate method.

During the culture of the microorganism, the pH of the culture can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, ammonia or an acid compound such as phosphoric acid or sulfuric acid in a suitable manner. In addition, bubble formation can be suppressed by using a defoaming agent such as a fatty acid polyglycol ester. An oxygen or oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The temperature of the culture is usually 20 ° C to 45 ° C, preferably 25 ° C to 40 ° C. The incubation time can be continued until the desired amount of L-amino acid is produced, but is preferably 10 to 160 hours.

In the method of the present invention, the culturing can be continuous or batch-wise, such as a batch process, an injection batch, and a repetitive injection batch process. Such a culturing method is well known in the art, and any method can be used.

In the present invention, the term "cultivation step" may include both a medium preparation time and a culture time.

The L-amino acid produced in the culturing step of the present invention may further include purification or recovery, and the purification or recovery method may be performed by a method of culturing the microorganism of the present invention, for example, a batch, The desired L-amino acid can be purified or recovered from the culture using a suitable method known in the art according to the culture method and the like.

In the present invention, the L-amino acid produced in the culturing step may be one selected from the group consisting of threonine, methionine, isoleucine, lysine, valine or alanine, preferably lysine or threonine.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

[ Example  ]

Example  1: Selection of acetate inducible promoter

Isocitrate lyase (aceA, NCgl2248) is the major enzyme of the glyoxylate cycle, and its gene is expressed in the presence of acetate. In addition, acetate kinase (ackA, NCgl2656) and phosphotransacetylase (pta, NCgl2657) are also involved in acetate metabolism and are operonized, and their expression is enhanced in the presence of acetate . The promoter regions of the aceA gene and the pta-ackA operon as described above are already known (Gerstmeir et < RTI ID = 0.0 > al ., J Biotechnol , 104, 99-122, 2003).

In this Example, the promoter of the aceA gene and the promoter of the pta-ack operon, that is, the upper promoter region of the pta gene were selected for the purpose of inhibiting the transcription of the target gene under the condition of acetate. Based on the nucleotide sequence of the aceA gene (NCBI Accession No. NCgl2248) registered with the National Institute of Health's NIH GenBank, 486 base pairs of the aceA gene and an open reading frame (ORF) And a base sequence (SEQ ID NO: 1) containing 36 base pairs from the N-terminal was obtained. Based on the nucleotide sequence of the pta gene (NCBI Accession No. NCgl2657) registered with the Gene Bank of the National Institutes of Health, a nucleotide sequence (SEQ ID NO: 2) containing 340 base pairs at the upper end of the pta gene was obtained.

Example  2 : aceE   Generation of vectors for gene expression inhibition

Pyruvate dehydrogenase subunit E1, aceE, NCgl2167) is a component of the pyruvate dehydrogenase complex (PDHC) involved in the introduction of pyruvate, the final metabolite of the process, into the tricarboxylic acid cycle Lt; / RTI > Thus, by weakening the expression level of the aceE gene, it is possible to weaken the entry of the carbon source into the TCA circuit and increase the uptake of the carbon source towards lysine biosynthesis, thereby increasing lysine production (Blombach B et al ., Appl . Microbiol Biotechnol . 2007 Sep; 76 (3): 615-23).

the aceA gene promoter was inserted into the lower part of the aceE gene so as to operate in the reverse direction of the transcription direction of the aceE gene, thereby selectively inhibiting the expression of the aceE gene under the presence of acetate (Fig. 2).

First, the transcription terminator of the aceE gene was predicted using the CLC main workbench software (CLC bio, Denmark). The transcription terminator is an inverted repeat sequence with a large number of GC bases, and forms a hairpin loop at this site to terminate gene transcription. As a result of prediction of the transcription terminator of the aceE gene, 36 base pairs from the stop codon of the aceE gene to the bottom of the 21st base to the 56th base form a hairpin loop structure, Was predicted. Based on this, in this example, two vectors capable of inserting the aceA gene promoter to operate in the reverse direction of the aceE gene transcription direction were constructed at the upper or lower end of the aceE gene transcription terminator.

<2-1> aceE  For inhibition of gene expression pDZ - aceE1 - PaceA  Vector production

A vector for inserting the aceA gene promoter between the termination codon of the aceE gene termination codon and the transcription termination terminator was constructed.

In order to obtain the aceE gene fragment derived from Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as a template, and a restriction enzyme XbaI recognition site and a restriction enzyme Primers (SEQ ID NOS: 3 and 4) designed to have a SpeI recognition site were synthesized. A DNA fragment containing 296 bp from the 2474th nucleotide to the 2769th nucleotide, which is the termination codon, was obtained from the aceE gene start codon through PCR using the synthesized primer. Also, primers (SEQ ID NOS: 5 and 6) designed to have a restriction enzyme SpeI recognition site and a restriction enzyme XbaI recognition site at the 3 'end were synthesized at the 5' end of the fragment, and 300 bp of the aceE gene end codon was included DNA fragments were obtained. PfuUltra ^TM high-confidence DNA polymerase (Stratagene) was used as the polymerase, and the PCR conditions were denaturation at 95 ° C for 30 seconds; Annealing 55 캜, 30 seconds; And polymerization was repeated 30 times at 72 캜 for 30 seconds, and then polymerization was carried out at 72 캜 for 7 minutes.

The pDZ-aceE1 vector was prepared by cloning the above two PCR amplification products with the In-fusion Cloning Kit (TAKARA, JP) using a pDZ vector for introducing a chromosome (see Korean Patent No. 0924065) prepared by digesting with restriction enzyme XbaI in advance Respectively.

SEQ ID NO: 3: aceE-P1F 5'- ccggggatcctctagacctccggcccatacgttgc -3 '

SEQ ID NO: 4: aceE-P1R 5'- ttgagactagttattcctcaggagcgtttg -3 '

SEQ ID NO: 5: aceE-P2F 5'-gaataactagtctcaagggacagataaatc -3 '

SEQ ID NO: 6: aceE-P2R 5'- gcaggtcgactctagagaccgaaaagatcgtggcag -3 '

In order to obtain a promoter fragment of the aceA gene derived from Corynebacterium glutamicum, a primer (SEQ ID NOS: 7 and 8) designed to have a Spe I restriction enzyme site at the 5 'end and the 3' end of the fragment was synthesized . Using the chromosomal DNA of Corynebacterium glutamicum KCCM11016P as a template, a promoter region of about 500 bp having the nucleotide sequence of SEQ ID NO: 1 was amplified by PCR using the synthesized primer. The DNA fragment obtained by treating the above PCR amplification product and the pDZ-aceE1 vector with a restriction enzyme SpeI was cloned using an In-fusion Cloning Kit (TAKARA, JP) to prepare a pDZ-aceE1-PaceA vector.

SEQ ID NO: 7: PaceA-P3F 5'-gtcccttgagactagtagcactctgactacctctg -3 '

SEQ ID NO: 8: PaceA-P3R 5'- ctgaggaataactagtttcctgtgcggtacgtggc -3 '

<2-2> aceE  For inhibition of gene expression pDZ - aceE2 - PaceA  Vector production

A vector for inserting the aceA gene promoter at the bottom of the aceE gene transcription terminator was constructed.

In order to obtain the aceE gene fragment derived from Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as a template, and a restriction enzyme XbaI recognition site and a restriction enzyme Primers (SEQ ID NOS: 9 and 10) designed to have a SpeI recognition site were used. A DNA fragment containing 294 bp was obtained from the 2538th base at the aceE gene initiation codon to the 62nd base at the bottom of the termination codon through PCR using the synthesized primer. The primers (SEQ ID NOS: 11 and 12) designed to have a restriction enzyme SpeI recognition site at the 5 'end of the fragment and a restriction enzyme XbaI recognition site at the 3' end were used and PCR was performed to amplify the 69th base To 362 &lt; th &gt; bases. PfuUltra ^TM high-confidence DNA polymerase (Stratagene) was used as the polymerase, and the PCR conditions were denaturation at 95 ° C for 30 seconds; Annealing 55 캜, 30 seconds; And polymerization was repeated 30 times at 72 캜 for 30 seconds, and then polymerization was carried out at 72 캜 for 7 minutes.

The pDZ-aceE2 vector was prepared by cloning the two PCR amplification products with the In-fusion Cloning Kit (TAKARA, JP) for chromosome introduction pDZ vector prepared by digesting with restriction enzyme XbaI in advance.

SEQ ID NO: 9: aceE-P4F 5'- ccggggatcctctagaggtcccaggcgactacacc -3 '

SEQ ID NO: 10: aceE-P4R 5'-gagctactagtacgacgaatcccgccgccagacta -3 '

SEQ ID NO: 11: aceE-P5F 5'- gtcgtactagtagctctttttagccgaggaacgcc -3 '

SEQ ID NO: 12: aceE-P5R 5'- gcaggtcgactctagacatgctgttggatgagcac -3 '

Primers (SEQ ID NOS: 13 and 14) were designed to have a restriction enzyme SpeI recognition site at the 5 'and 3' ends of the fragment, respectively, in order to secure a promoter fragment of the aceA gene derived from Corynebacterium glutamicum. Using the chromosomal DNA of Corynebacterium glutamicum KCCM11016P as a template, a promoter region of about 500 bp having the nucleotide sequence of SEQ ID NO: 1 was amplified by PCR using the synthesized primer. A pDZ-aceE2-PaceA vector was prepared by cloning the DNA fragment obtained by treating the above PCR amplification product and the pDZ-aceE2 vector with a restriction enzyme SpeI using an In-fusion Cloning Kit (TAKARA, JP).

SEQ ID NO: 13: PaceA-P6F 5'- aaaaagagctactagtagcactctgactacctctg -3 '

SEQ ID NO: 14: PaceA-P6R 5'-gattcgtcgtactagtttcctgtgcggtacgtggc -3 '

Example  3: aceE  Gene bottom aceA  Production of gene promoter insertion

The vector pDZ-aceE1-PaceA and pDZ-aceE2-PaceA prepared in Example 2 were transformed into L-lysine producing strain, Corynebacterium glutamicum KCCM11016P, by electric pulse method (Appl. Microbiol. Biotechnol. (1999) 52: 541-545), aceE gene at the chromosomal terminus The isolate inserted into the lower codon of the aceA gene promoter to act in the reverse direction of the transcription of the aceE gene was PCR- Lysine-producing strains were named Corynebacterium glutamicum KCCM11016P :: aceE1-PaceA and Corynebacterium glutamicum KCCM11016P :: aceE2-PaceA, respectively. Corynebacterium glutamicum KCCM11016P :: aceE1-PaceA is a Corynebacterium glutamicum CA01-2271, deposited with the Korean Society for Microbiological Research on June 12, 2013 under accession number KCCM11432P. The prepared strain was subjected to PCR using primers of SEQ ID NO: 3 and SEQ ID NO: 6 for KCCM11016P :: aceE1-PaceA and SEQ ID NO: 9 and SEQ ID NO: 12 for KCCM11016P :: aceE2-PaceA, And finally confirmed through sequence analysis.

Example  4: aceE  Gene bottom aceA  Insertion of gene promoter lysine Production capacity  compare

Corynebacterium glutamicum KCCM11016P strain used as parent strain and Corynebacterium glutamicum KCCM11016P :: aceE1-PaceA and KCCM11016P :: aceE2-PaceA, which are L-lysine producing strains prepared in Example 3, For production, the cells were cultured in the following manner.

KCCM11016P :: aceE1-PaceA and KCCM11016P :: aceE2-PaceA were inoculated into a 250 ml corn-baffle flask containing 25 ml of the following seed medium and cultured for 20 hours at 30 DEG C for 200 hours in a Corynebacterium glutamicum strain KCCM11016P, 0.0 &gt; rpm. &lt; / RTI &gt; A 250 ml corner-baffle flask containing 24 ml of the following production medium was inoculated with 1 ml of the seed culture and incubated at 30 ° C for 72 hours with shaking at 200 rpm. The composition of the seed medium and the production medium are as follows.

< Bell badge  ( pH  7.0)>

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea _{1.5 g, KH 2 PO 4 4} g, K 2 HPO 4 8 g, MgSO 4 7H 2 O 0.5 g, biotin 100 ㎍, thiamine HCl 1000 ㎍, calcium- Pantothenic acid 2000 占 퐂, nicotinamide 2000 占 퐂 (1 liter of distilled water)

< Production badge  ( pH  7.0)>

Glucose _{100 g, (NH 4) 2} SO 4 40 g, soy protein 2.5 g, corn steep solids (Corn Steep Solids) 5 g, urea _{3 g, KH 2 PO 4 1} g, MgSO 4 7H 2 O 0.5 g, biotin 1000 티 of thiamine hydrochloride, 2000 칼 of calcium-pantothenic acid, 3000 ㎍ of nicotinamide, 30 g of CaCO ₃ (based on 1 liter of distilled water).

After the incubation, the amount of L-lysine produced was measured by HPLC. L-lysine concentrations in the culture medium for Corynebacterium glutamicum KCCM11016P and KCCM11016P :: aceE1-PaceA and KCCM11016P :: aceE2-PaceA when acetate was not added were as shown in Table 1 below.

Changes in L-lysine production (no acetate added) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3   KCCM11016P 43.5 43.1 43.4 KCCM11016P ::
aceE1-PaceA 43.7 43.2 43.6 KCCM11016P ::
aceE2-PaceA 43.3 43.4 43.7

In addition, L-lysine producing ability was compared by culturing the strain in the same manner, except that 5 g / L of acetate was added to the production medium. The concentration of L-lysine in the culture was as shown in Table 2 below.

Changes in L-lysine production (added with 5 g / L acetate) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3   KCCM11016P 45.6 45.3 45.8 KCCM11016P ::
aceE1-PaceA 47.2 47.1 47.4 KCCM11016P ::
aceE2-PaceA 46.7 46.5 47.0

As shown in Table 1, it was confirmed that KCCM11016P :: aceE1-PaceA and KCCM11016P :: aceE2-PaceA did not change the parent strain KCCM11016P and lysine production ability under the absence of acetate.

However, as shown in the above Table 2, in the case of the presence of acetate, in the case of KCCM11016P :: aceE1-PaceA strain, the lysine producing ability was 3.6% or more and KCCM11016P :: aceE2-PaceA strain was higher than that of the parent strain KCCM11016P Showed that the lysine production capacity was increased by 2.5% or more as compared with the parent strain KCCM11016P.

When KCCM11016P :: aceE1-PaceA is compared with KCCM11016P :: aceE2-PaceA, the case of KCCM11016P :: aceE1-PaceA in which the aceA promoter is inserted between the top of the aceE gene transcription terminator, that is, the termination codon and the transcription terminator, It can be seen that the use of the lower end of the termination codon at the insertion site, that is, between the termination codon and the upper end of the transcription terminator, is more effective in inhibiting gene expression, as it is more effective in producing lysine.

Example  5: aceE   For inhibition of gene expression pDZ - aceE - Ppta  Vector production

Acetate kinase (ackA, NCgl2656) and phosphotransacetylase (pta, NCgl2657) are enzymes involved in the metabolism of acetate and form an operon, and in the presence of acetate in the same manner as isocitrate lyase Expression is enhanced. Therefore, when the promoters of these genes are used, gene expression can be induced specifically in the presence of acetate.

In this Example, a vector for using the promoter of the pta-ack operon, that is, the upper promoter region of the pta gene, was constructed for the purpose of inhibiting the expression of the aceE gene under the condition of acetate.

In order to inhibit the expression of the aceE gene, a vector was constructed which was inserted between the termination codon of the aceE gene and the top of the transcription termination codon, so that the pta gene promoter can be inserted to operate in the reverse direction of the aceE gene transcription direction.

In order to obtain aceE gene fragment derived from Corynebacterium glutamicum, a pDZ-aceE1 vector was constructed in the same manner as in Example 2, using the chromosomal DNA of Corynebacterium glutamicum KCCM11016P as a template.

Primers (SEQ ID NOs: 15 and 16) designed to have a restriction enzyme SpeI recognition site at the 5 'and 3' ends of the fragment were synthesized in order to secure a promoter fragment of the pTA gene derived from Corynebacterium glutamicum. Using the chromosomal DNA of Corynebacterium glutamicum KCCM11016P as a template, a promoter region of about 340 bp having the nucleotide sequence of SEQ ID NO: 2 was amplified by PCR using the synthesized primer. The DNA fragment obtained by treating the PCR amplification product and the pDZ-aceE1 vector with a restriction enzyme SpeI was cloned using an In-fusion Cloning Kit (TAKARA, JP) to construct a pDZ-aceE1-Ppta vector.

SEQ ID NO: 15: Ppta-P7F 5'-gtcccttgagactagtctttgctggggtcagatttg -3 '

SEQ ID NO: 16: Ppta-P7R 5'- Ctgaggaataactagtacatcgcctttctaatttc -3 '

Example  6: aceE  Gene bottom pta  Comparison of production and lysine production ability of gene promoter insertion

The vector pDZ-aceE1-Ppta prepared in Example 5 was transformed into L-lysine producing strain Corynebacterium glutamicum KCCM11016P in the same manner as in Example 3, and the pdA gene The strain in which the promoter was inserted so as to operate in the reverse direction of the aceE gene transcription direction was isolated by PCR to obtain L-lysine producing strain and named KCCM11016P :: aceE1-Ppta. The production strain KCCM11016P :: aceE1-Ppta was finally confirmed by sequencing the target site by performing PCR using SEQ ID NO: 3 and SEQ ID NO: 6 as primers.

The prepared strain was cultured in the same manner as in Example 4, and the concentration of the recovered L-lysine was measured. When no acetate was added, the L-lysine concentration in the culture was as shown in Table 3 below.

Changes in L-lysine production (no acetate added) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3   KCCM11016P 42.9 43.5 43.4 KCCM11016P ::
aceE1-Ppta 43.2 43.3 43.6

In addition, L-lysine producing ability was compared by culturing the strain in the same manner, except that 5 g / L of acetate was added to the production medium. L-lysine concentration in the culture was as shown in Table 4 below.

Changes in L-lysine production (added with 5 g / L acetate) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3   KCCM11016P 45.5 45.7 45.3 KCCM11016P ::
aceE1-Ppta 46.7 46.5 46.6

As shown in Table 3, it was confirmed that KCCM11016P :: aceE1-Ppta strain had no change in the productivity of the parent strain KCCM11016P and lysine in the absence of acetate.

However, as shown in the results of Table 4, it was confirmed that the KCCM11016P :: aceE1-Ppta strain increased the lysine production capacity by 2.4% or more as compared with the parent strain KCCM11016P under the condition of the presence of acetate.

In addition, considering that the KCCM11016P :: aceE1-PaceA strain has better lysine production ability than the KCCM11016P :: aceE1-Ppta strain, the use of the promoter of the aceA gene in the presence of acetate is superior to the use of the promoter of the pta gene It is possible to more effectively inhibit the expression of the target gene.

Example  7: aceE  Gene bottom aceA  Production of gene promoter insertion

The vector pDZ-aceE1-PaceA prepared in Example 2 was transformed into L-lysine-producing strains Corynebacterium glutamicum KFCC10750, KCCM10770P and CJ3P three strains, and chromosomal aceE AceE1-PaceA, aceE1-PaceA, and CJ3P :: producing a L-lysine by isolating a strain inserted at the bottom of the gene termination codon so that the aceA gene promoter operates in the reverse direction of the aceE gene transcription direction by PCR. AceE1-PaceA three strains were obtained. The prepared strains were subjected to PCR using SEQ ID NO: 3 and SEQ ID NO: 6 as primers and finally confirmed by sequencing of the target site.

The prepared strain was cultured in the same manner as in Example 4, and the concentration of the recovered L-lysine was measured. When acetate was not added, the L-lysine concentration in the culture was as shown in Table 5 below.

Changes in L-lysine production (no acetate added) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3 KFCC10750 38.3 38.0 38.4 KFCC10750 :: aceE1-PaceA 38.6 38.2 38.3 KCCM10770P 47.5 47.3 47.6 KCCM10770P :: aceE1-PaceA 47.3 47.7 47.5 CJ3P 8 8.4 8.3 CJ3P :: aceE1-PaceA 8.2 8.1 8.5

In addition, L-lysine producing ability was compared by culturing the strain in the same manner, except that 5 g / L of acetate was added to the production medium. L-lysine concentration in the culture was as shown in Table 6 below.

Changes in L-lysine production (added with 5 g / L acetate) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3 KFCC10750 39.3 39.5 39.2 KFCC10750 :: aceE1-PaceA 41.3 41.6 41.0 KCCM10770P 47.5 47.3 47.6 KCCM10770P :: aceE1-PaceA 49.0 48.6 48.8 CJ3P 8 8.4 8.3 CJ3P :: aceE1-PaceA 9.5 9.7 9.4

As shown in Table 5 above, the strains KFCC10750 :: aceE1-PaceA, KCCM10770 :: aceE1-PaceA and CJ3P :: aceE1-PaceA showed no change in the parent strain and lysine production ability in the absence of acetate Respectively.

However, as shown in the results of Table 6, in the presence of acetate, the productivity of lysine production was 5% as compared with the parent strain of KFCC10750 :: aceE1-PaceA, 2.8% as compared with the parent strain of KCCM10770P :: aceE1- The CJ3P :: aceE1-PaceA strain was increased by 15% compared to the parent strain.

Example  8 : hom  Generation of vectors for gene expression inhibition

Lysine biosynthetic pathway, thereby increasing the ability to produce L-lysine. A method of reducing the enzyme activity of homoserine dehydrogenase (hom, NCgl 1136) gene that produces homoserine from aspartate in order to weaken the L-threonine biosynthesis pathway.

In Corynebacterium glutamicum, the hom gene consists of the thrB gene and the hom-thrB operon, and the transcription terminator is located at the bottom of the thrB gene. It has been reported that a promoter is present at the top of the hom-thrB operon, that is, at the top of the hom gene, and that a second promoter is also present at the top of the thrB gene ORF (Mateos et al ., J Bacteriol , 176 (23), 7362-7371, 1994). Therefore, we inserted the aceA gene promoter in the reverse direction of the hom gene transcription direction at the bottom of the hom gene end codon to selectively inhibit the hom gene expression in the presence of acetate. In order to maintain the expression of thrB gene, Was added to the second promoter sequence.

In this Example, a recombination vector for inserting the aceA gene promoter between the lower end of the hom gene end codon and the upper end of the thrB gene was constructed.

In order to obtain a hom gene fragment derived from Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as a template. A restriction enzyme XbaI recognition site and a restriction enzyme SpeI Primers (SEQ ID NOS: 17 and 18) designed to have recognition sites were synthesized. A DNA fragment containing 300 bp from the 1039th base to the 1338th base, which is the termination codon, was obtained from the homogenous initiation codon through PCR using the synthesized primer. The expression of the thrB gene should be maintained even when the aceA promoter is inserted between hom-thrB operons for the inhibition of hom gene expression. Thus, primers (SEQ. ID. NO. 19 and 20) designed to add a thrB promoter sequence of 32 bp to the 5 'side of the DNA fragment were synthesized in the production of a DNA fragment containing 300 bp at the lower end of the hom gene end codon. PCR was performed with these primers (SEQ ID NOS: 19 and 20) to obtain a 344 bp DNA fragment having a restriction enzyme SpeI recognition site at the 5 'end of the fragment and a restriction enzyme XbaI recognition site at the 3' end. PCR was carried out using the same conditions as in Example 2.

The pDZ-hom vector was prepared by cloning the two PCR amplification products with the In-fusion Cloning Kit (TAKARA, JP) for chromosome introduction pDZ vector prepared by digesting with restriction enzyme XbaI in advance.

SEQ ID NO: 17: hom-h1F 5'- ccggggatcctctagaccaggtgagtccacctacg -3 '

SEQ ID NO: 18: hom-h1R 5'- gaggcggatcactagtttagtccctttcgaggcgg -3 '

SEQ ID NO: 19: hom-h2F 5'-actagtgatccgcctcgaaagggac -3 '

SEQ ID NO: 20: hom-h2R 5'- gcaggtcgactctagagactgcggaatgttgttgtg -3 '

Primers (SEQ ID NOS: 21 and 22) were designed to have a restriction enzyme SpeI recognition site at the 5 'and 3' ends of the fragment, respectively, in order to secure a promoter fragment of the aceA gene derived from Corynebacterium glutamicum. Using the chromosomal DNA of Corynebacterium glutamicum KCCM11016P as a template, a promoter region of about 500 bp having the nucleotide sequence of SEQ ID NO: 1 was amplified by PCR using the synthesized primer. The DNA fragment obtained by treating the above PCR amplification product and the pDZ-hom vector with the restriction enzyme SpeI was cloned using an In-fusion Cloning Kit (TAKARA, JP) to prepare a pDZ-hom-PaceA vector.

SEQ ID NO: 21: PaceA-h3F 5'-gaggcggatcactagtagcactctgactacctctg -3 '

SEQ ID NO: 22: PaceA-h3R 5'- aagggactaaactagtttcctgtgcggtacgtggc -3 '

Example  9: hom  Gene bottom aceA  Generation of gene promoter insertion and comparison of lysine production ability

The vector pDZ-hom-PaceA prepared in Example 8 was transformed into L-lysine producing strain Corynebacterium glutamicum KCCM11016P in the same manner as in Example 3, and the aceA gene L-lysine producing KCCM11016P :: hom-PaceA was obtained by isolating the inserted strain so that the promoter would operate in the reverse direction of the hom gene transfer direction. The construction strain KCCM11016P :: hom-PaceA was confirmed by sequencing the target site by carrying out PCR using SEQ ID NO: 17 and SEQ ID NO: 20 as primers.

The strain Corynebacterium glutamicum KCCM11016P used as parent strain and the KCCM11016P :: hom-PaceA strain prepared were cultured in the same manner as in Example 4 for production of L-lysine, and the concentration of recovered L-lysine Respectively. When acetate was not added, the concentrations of L-lysine in the culture medium for Corynebacterium glutamicum KCCM11016P and KCCM11016P :: hom-PaceA were as shown in Table 7 below.

Changes in L-lysine production (no acetate added) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3   KCCM11016P 43.2 43.3 43.6 KCCM11016P ::
hom-PaceA 43.3 43.6 43.4

In addition, L-lysine producing ability was compared by culturing the strain in the same manner, except that 5 g / L of acetate was added to the production medium. L-lysine concentration in the culture was as shown in Table 8 below.

Changes in L-lysine production (added with 5 g / L acetate) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3   KCCM11016P 44.9 45.6 45.2 KCCM11016P ::
hom-PaceA 46.3 46.6 46.4

As shown in Table 7, it was confirmed that the KCCM 11016P :: hom-PaceA strain had no change in the productivity of the parent strain KCCM 11016P and lysine under the condition that no acetate was present.

However, as shown in the results of Table 8, it was confirmed that the KCCM 11016P :: hom-PaceA strain increased the lysine production capacity by 2.6% or more as compared with the parent strain KCCM 11016P under the condition of acetate.

Example  10: murE  Generation of vectors for gene expression inhibition

(UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase, murE, NCgl2083) was synthesized by lysine biosynthesis (UDP-N-acetylmuramoylalanyl- Meso-2,6-Diaminopimelate, which is used as a precursor of the lysine biosynthesis, is used for the synthesis of mycelial cells. The activity of the murE gene is weakened to weaken the carbon source introduction into the cell synthesis, By increasing the carbon source input into the pathway, lysine production can be increased.

The murE gene (NCgl2083) in Corynebacterium glutamicum is an operon with seven genes from NCgl2076 to NCgl2082. The transcription of the operon starts from the NCgl2083 murE gene and proceeds toward the NCgl2076 gene. Thus, the transcriptional terminator is located at the bottom of the NCgl2076 gene. The murE gene clone The aceA gene promoter was inserted at the bottom of the codon to operate in the reverse direction of the murE gene transcription direction to selectively inhibit the expression of murE gene in the presence of acetate. The other seven genes other than the murE gene located at the beginning of operon The promoter of the murE operon was further inserted at the top of the NCgl2082 gene ORF.

In this Example, a recombinant vector for inserting the aceA gene promoter at the bottom of the murE gene termination codon was constructed.

In order to obtain a murE gene fragment derived from Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as a template. A restriction enzyme XbaI recognition site and a restriction enzyme XhoI Primers (SEQ ID NOS: 23 and 24) designed to have recognition sites were synthesized. A DNA fragment containing 300 bp from the 1267th base to the 1566th nucleotide, which is the termination codon, was obtained from the murE gene initiation codon through PCR using the synthesized primer. The primers (SEQ ID NOS: 25 and 26) designed to have the restriction enzyme XhoI recognition site and the restriction enzyme XbaI recognition site at the 3 'end were synthesized at the 5' end of the fragment, A DNA fragment containing 292 bp was obtained. PCR was carried out using the same conditions as in Example 2. PDZ-murE vector was prepared by cloning the above two PCR amplification products and a pDZ vector for introduction of chromosome prepared by digesting with restriction enzyme XbaI in advance using an In-fusion Cloning Kit (TAKARA, JP).

SEQ ID NO: 23: mur-m1F 5'- ccggggatcctctagaaaccctcgttcagaggtgc -3 '

SEQ ID NO: 24: mur-m1R 5'- ttgtgatcatctcgagctatccttcttccgtagtaag -3 '

SEQ ID NO: 25: mur-m2F 5'-agctcgagatgatcacaatgacccttgg -3 '

SEQ ID NO: 26: mur-m2R 5'- gcaggtcgactctagacatgagcataaatgtcagc -3 '

In order to obtain a promoter fragment of the aceA gene derived from Corynebacterium glutamicum, a primer (SEQ ID NOS: 27 and 28) designed to have a restriction enzyme XhoI recognition site at the 5 'end of the fragment was synthesized. Using the chromosomal DNA of Corynebacterium glutamicum KCCM11016P as a template, a promoter region of about 500 bp having the nucleotide sequence of SEQ ID NO: 1 was amplified by PCR using the synthesized primer. In order to secure the promoter region of the murE operon derived from Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as a template and a restriction enzyme XhoI recognition site was designed at the 3 'end of the fragment Primers (SEQ ID NOS: 29 and 30) were synthesized. A DNA fragment containing 300 bp upstream of the murE gene ORF was obtained by PCR using the synthesized primer. The DNA fragment obtained by treating the two PCR amplification products and pDZ-murE vector with restriction enzyme XhoI was cloned using an In-fusion Cloning Kit (TAKARA, JP) to construct pDZ-murE-PaceA-PmurE vector.

SEQ ID NO: 27: mur-m3F 5'- tcatcagcagcactctgactacctctg -3 '

SEQ ID NO: 28: mur-m3R 5'-agaaggatagctcgagttcctgtgcggtacgtggc -3 '

SEQ ID NO: 29: mur-m4F 5'-agagtgctgctgatgatcctcgatttg -3 '

SEQ ID NO: 30: mur-m4R 5'- ttgtgatcatctcgagggttttctctcctccacagg -3 '

Example  11: murE  Gene bottom aceA  Insertion of gene promoter Caution and lysine Productive  compare

The vector pDZ-murE-PaceA-PmurE prepared in Example 10 was transformed into L-lysine producing strain Corynebacterium glutamicum KCCM11016P in the same manner as in Example 3, The KCCM11016P :: murE-PaceA-PmurE producing L-lysine was obtained by isolating the inserted strains of the aceA gene promoter so as to operate in the reverse direction of the murE gene transcription direction through PCR. The construction strain KCCM11016P :: murE-PaceA-PmurE was confirmed by sequencing the target site by carrying out PCR using SEQ ID NO: 23 and SEQ ID NO: 26 as primers.

The KCCM11016P :: murE-PaceA-PmurE prepared from the strain Corynebacterium glutamicum KCCM11016P used as a parent strain was cultured in the same manner as in Example 4 for the production of L-lysine, and the concentration of recovered L-lysine Were measured. When acetate was not added, the concentration of L-lysine in the culture medium for Corynebacterium glutamicum KCCM11016P and KCCM11016P :: murE-PaceA-PmurE was as shown in Table 9 below.

Changes in L-lysine production (no acetate added) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3   KCCM11016P 43.5 43.9 44.0 KCCM11016P ::
murE-PaceA-PmurE 43.7 44.1 43.8

In addition, L-lysine producing ability was compared by culturing the strain in the same manner, except that 5 g / L of acetate was added to the production medium. The concentration of L-lysine in the culture was as shown in Table 10 below.

Changes in L-lysine production (added with 5 g / L acetate) Strain Lysine (g / L) Batch 1 Batch 2 Batch 3   KCCM11016P 45.2 45.6 45.3 KCCM11016P ::
murE-PaceA-PmurE 46.6 46.9 46.5

As shown in Table 9, it was confirmed that KCCM11016P :: murE-PaceA-PmurE strain had no change in the production ability of the parent strain KCCM11016P and lysine under the condition that no acetate was present.

However, as shown in the results of Table 10, it was confirmed that the KCCM11016P :: murE-PaceA-PmurE strain increased the lysine production capacity by 2.8% or more as compared with the parent strain KCCM11016P under the condition of the presence of acetate.

Example  12: dapA  Generation of vectors for gene expression inhibition

Lysine biosynthetic pathway using a substrate such as the L-threonine biosynthetic pathway, thereby increasing L-threonine production capacity. A method of reducing the enzyme activity of a dihydrodipicolinate synthase (dapA, NCgl1896) gene involved in lysine production from aspartate in order to weaken the L-lysine biosynthesis pathway.

In the Corynebacterium glutamicum, the dapA gene forms the ORF4 (NCgl1895) gene and the dapA-ORF4 operon, so that the transcription terminator is located at the bottom of the ORF4 gene. It has been reported that a promoter exists at the upper end of the dapA-ORF4 operon, that is, at the top of the dapA gene, and furthermore, a promoter sequence exists also at the upper end of the ORF4 gene (Patek et al ., Biotechnology letters , Vol 19, No 11, 1997). Therefore, the insertion of the aceA gene promoter at the lower end of the dapA gene codon to act in the reverse direction of the transcription of the dapA gene selectively inhibited the expression of the dapA gene in the presence of acetate. To maintain the expression of the ORF4 gene, Of the promoter site was added to the top of the ORF4 gene ORF.

In this Example, a recombination vector for inserting the aceA gene promoter at the bottom of the dapA gene termination codon was constructed.

In order to obtain the dapA gene fragment derived from Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as a template, and a restriction enzyme XbaI recognition site at the 5 'end of the fragment and a restriction enzyme SpeI Primers (SEQ ID NOS: 31 and 32) designed to have recognition sites were synthesized. A DNA fragment containing 301 bp was obtained from the 606th base to the 906th base, which is the termination codon, at the dapA gene initiation codon through PCR using the synthesized primers. (SEQ ID NOS: 33 and 34) designed to have a restriction enzyme SpeI recognition site at the 5 'end of the fragment and a restriction enzyme XbaI recognition site at the 3' end were synthesized and PCR was performed to maintain the expression of the ORF4 gene a DNA fragment containing a promoter sequence of 100 bp from the 809th base to the second base of the termination codon at the dapA gene start codon and containing 213 bp at the lower end of the dapA gene termination codon was obtained. PCR was carried out using the same conditions as in Example 2. The pDZ-dapA vector was prepared by cloning the two PCR amplification products with the In-fusion Cloning Kit (TAKARA, JP) for chromosome introduction pDZ vector prepared by cutting with restriction enzyme XbaI in advance.

SEQ ID NO: 31: dapA-d1F 5'- ccggggatcctctagatgtttggcttgctttgggc -3 '

SEQ ID NO: 32: dapA-d1R 5'-gttgatgcactagtttatagaactccagcttt -3 '

SEQ ID NO: 33: dapA-d2F 5'- ttctataaactagtgcatcaacgtaggagatcc -3 '

SEQ ID NO: 34: dapA-d2R 5'- gcaggtcgactctagacgttctgggaaccctgag -3 '

Primers (SEQ ID NOs: 35 and 36) were designed to have a restriction enzyme SpeI recognition site at the 5 'end and the 3' end of the fragment, respectively, in order to secure a promoter fragment of the aceA gene derived from Corynebacterium glutamicum. Using the chromosomal DNA of Corynebacterium glutamicum KCCM11016P as a template, a promoter region of about 500 bp having the nucleotide sequence of SEQ ID NO: 1 was amplified by PCR using the synthesized primer. The DNA fragment obtained by treating the above PCR amplification product and the pDZ-dapA vector with restriction enzyme SpeI was cloned using an In-fusion Cloning Kit (TAKARA, JP) to prepare a pDZ-dapA-PaceA vector.

SEQ ID NO: 35: PaceA-d3F 5'-acgttgatgcactagtagcactctgactacctctg -3 '

SEQ ID NO: 36: PaceA-d3R 5'-agttctataaactagtttcctgtgcggtacgtggc -3 '

Example  13: dapA  Gene bottom aceA  Insertion of gene promoter Threonine Productive  compare

In order to confirm the inhibitory effect of dapA gene expression in the L-threonine producing strain, the vector pDZ-dapA-PaceA prepared in Example 12 was applied to L-threonine producing host KCCM11222P (Korean Patent Publication No. 2013-0061570) The strain transformed in the same manner as in Example 3, and the strain in which the aceA gene promoter was inserted at the lower end of the dapA gene on the chromosome so as to operate in the reverse direction of the dapA gene transcription, was isolated by PCR to obtain L-threonine-producing KCCM11222P :: dapA -PaceA. The construction strain KCCM11222P :: dapA-PaceA was finally confirmed by sequencing the target site by performing PCR using SEQ ID NO: 31 and SEQ ID NO: 34 as primers.

Corynebacterium glutamicum KCCM11222P strain used as a parent strain and KCCM11222P :: dapA-PaceA strain prepared were cultured in the following manner to produce L-threonine.

Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed culture and incubated at 30 ° C for 20 hours with shaking at 200 rpm. A 250 ml corner-baffle flask containing 24 ml of production medium was then inoculated with 1 ml of the seed culture and incubated at 30 DEG C for 48 hours with shaking at 200 rpm. The composition of the seed medium and the production medium are as follows.

&Lt; Batch medium (pH 7.0) >

Glucose 20 g, peptone 10 g, yeast extract 5 g, urea _{1.5 g, KH 2 PO 4 4} g, K 2 HPO 4 8 g, MgSO 4 · 7H 2 O 0.5 g, biotin 100 ㎍, thiamine HCl 1000 ㎍, calcium - 2000 ㎍ of pantothenic acid, 2000 ㎍ of nicotinamide (1 liter of distilled water)

&Lt; Production medium (pH 7.0) >

Glucose _{100 g, (NH 4) 2} SO 4 20 g, soy protein 2.5 g, corn steep solids (Corn Steep Solids) 5 g, urea _{3 g, KH 2 PO 4 1} g, MgSO 4 · 7H 2 O 0.5 g, 100 ㎍ of biotin, 1000 티 of thiamine hydrochloride, 2000 칼 of calcium-pantothenic acid, 3000 니 of nicotinamide, 30 g of CaCO ₃ (based on 1 liter of distilled water).

After completion of the culture, the concentration of L-threonine in the culture solution was analyzed by HPLC. When acetate was not added, the concentrations of L-threonine in the culture medium for Corynebacterium glutamicum KCCM11222P and KCCM11222P :: dapA-PaceA were as shown in Table 11 below.

Change in L-threonine production (no acetate added) Strain Threonine (g / L) Batch 1 Batch 2 Batch 3   KCCM11222P 7.0 6.9 7.2 KCCM11222P ::
dapA-PaceA 7.1 7.3 7.0

In addition, L-threonine production was compared by culturing the strain in the same manner except that 5 g / L of acetate was added to the production medium. The concentration of L-threonine in the culture was as shown in Table 12 below.

Changes in L-threonine production (added with 5 g / L acetate) Strain Threonine (g / L) Batch 1 Batch 2 Batch 3   KCCM11222P 7.6 7.4 7.7 KCCM11222P ::
dapA-PaceA 11.2 11.5 11.4

As shown in Table 11, it was confirmed that the KCCM11222P :: dapA-PaceA strain had no change in the parent strain KCCM11222P and threonine production ability under the condition that no acetate was present.

However, as shown in the above Table 12, it was confirmed that in the presence of acetate, the KCCM11222P :: dapA-PaceA strain increased the threonine production ability by more than 50% as compared with the parent strain KCCM11222P.

Korea Microorganism Conservation Center (overseas) KCCM11432P 20130612

<110> CJ CheilJedang Corporation <120> Method for producing L-amino acid <130> PN106397 <150> KR 2013/0121090 <151> 2013-10-11 <160> 36 <170> Kopatentin 2.0 <210> 1 <211> 522 <212> DNA <213> Corynebacterium glutamicum <220> <221> gene <222> (1). (486) <223> aceA <400> 1 agcactctga ctacctctgg aatctaggtg ccactcttct ttcgatttca acccttatcg 60 tgtttggcga tgtgatcaga ctaagtgatc accgtcacca gcaaaagggg tttgcgaact 120 ttactaagtc attacccccg cctaaccccg acttttatct aggtcacacc ttcgaaacct 180 acggaacgtt gcggtgcctg cattttccca tttcagagca tttgcccagt acatccgtac 240 tagcaactcc cccgcccact ttttctgcga agccagaact ttgcaaactt cacaacaggg 300 gtgaccaccc ccgcacaaaa cttaaaaacc caaaccgatt gacgcaccaa tgcccgatgg 360 agcaatgtgt gaaccacgcc accacgcaaa ccgatgcaca tcacgtcgaa acagtgacaga 420 tgcattagct catactttgt ggtcggcacc gcccattgcg aatcagcact taaggaagtg 480 actttgatgt caaacgttgg aaagccacgt accgcacagg aa 522 <210> 2 <211> 340 <212> DNA <213> Corynebacterium glutamicum <220> <221> gene &Lt; 222 > (1) .. (340) <223> pta <400> 2 ctttgctggg gtcagatttg tcacgctgcg cgctttcata gaccccatta atggggggtg 60 aagagctgta aagtaccgct aaaaactttg caaagggtgc ttcgcaactt gtaaccgctc 120 cgtattgttt tctacggcaa taagcatttg tgctgctcaa agcgtggaat tgagatcggt 180 ttgaaaatta caaaataaaa ctttgcaaac cgggctgtac gcaaggcgga cgaacgctaa 240 actatgtaag aaatcacaac ctcccctcat tagtgccagg aggcacaagc ctgaagtgtc 300 atcaatgaga aggttcaggc tgaaattaga aaggcgatgt 340 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> aceE P1F <400> 3 ccggggatcc tctagacctc cggcccatac gttgc 35 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> aceE P1R <400> 4 ttgagactag ttattcctca ggagcgtttg 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> aceE P2F <400> 5 gaataactag tctcaaggga cagataaatc 30 <210> 6 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> aceE P2R <400> 6 gcaggtcgac tctagagacc gaaaagatcg tggcag 36 <210> 7 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pace A P3F <400> 7 gtcccttgag actagtagca ctctgactac ctctg 35 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PaceA P3R <400> 8 ctgaggaata actagtttcc tgtgcggtac gtggc 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> aceE P4F <400> 9 ccggggatcc tctagaggtc ccaggcgact acacc 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> aceE P4R <400> 10 gagctactag tacgacgaat cccgccgcca gacta 35 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> aceE P5F <400> 11 gtcgtactag tagctctttt tagccgagga acgcc 35 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> aceE P5R <400> 12 gcaggtcgac tctagacatg ctgttggatg agcac 35 <210> 13 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Pace A P6F <400> 13 aaaaagagct actagtagca ctctgactac ctctg 35 <210> 14 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PaceA P6R <400> 14 gattcgtcgt actagtttcc tgtgcggtac gtggc 35 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Ppta P7F <400> 15 gtcccttgag actagtcttt gctggggtca gatttg 36 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Ppta P7R <400> 16 ctgaggaata actagtacat cgcctttcta atttc 35 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> hom h1F <400> 17 ccggggatcc tctagaccag gtgagtccac ctacg 35 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> hom h1R <400> 18 gaggcggatc actagtttag tccctttcga ggcgg 35 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hom h2F <400> 19 actagtgatc cgcctcgaaa gggac 25 <210> 20 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> hom h2R <400> 20 gcaggtcgac tctagagact gcggaatgtt gttgtg 36 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PaceA h3F <400> 21 gaggcggatc actagtagca ctctgactac ctctg 35 <210> 22 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PaceA h3R <400> 22 aagggactaa actagtttcc tgtgcggtac gtggc 35 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> mur m1F <400> 23 ccggggatcc tctagaaacc ctcgttcaga ggtgc 35 <210> 24 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> mur m1R <400> 24 ttgtgatcat ctcgagctat ccttcttccg tagtaag 37 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> mur m2F <400> 25 agctcgagat gatcacaatg acccttgg 28 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> mur m2R <400> 26 gcaggtcgac tctagacatg agcataaatg tcagc 35 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> mur m3F <400> 27 tcatcagcag cactctgact acctctg 27 <210> 28 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> mur m3R <400> 28 agaaggatag ctcgagttcc tgtgcggtac gtggc 35 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> mur m4F <400> 29 agagtgctgc tgatgatcct cgatttg 27 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> mur m4R <400> 30 ttgtgatcat ctcgagggtt ttctctcctc cacagg 36 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> dapA d1F <400> 31 ccggggatcc tctagatgtt tggcttgctt tgggc 35 <210> 32 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> dapA d1R <400> 32 gttgatgcac tagtttatag aactccagct tt 32 <210> 33 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> dapA d2F <400> 33 ttctataaac tagtgcatca acgtaggaga tcc 33 <210> 34 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> dapA d2R <400> 34 gcaggtcgac tctagacgtt ctgggaaccc tgag 34 <210> 35 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PaceA d3F <400> 35 acgttgatgc actagtagca ctctgactac ctctg 35 <210> 36 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PaceA d3R <400> 36 agttctataa actagtttcc tgtgcggtac gtggc 35

Claims (7)

1) culturing a recombinant coryneform microorganism having an ability to produce L-amino acid, which is transformed by introducing an acetate inducible promoter into a lower end codon of a target gene in a chromosome; And
2) enhancing L-amino acid producing ability of the recombinant coryneform microorganism by weakening expression of the target gene during culturing by adding acetate in the culturing step;
Amino acid. &Lt; / RTI &gt; The method according to claim 1,
Wherein the lower end of the termination codon is between the termination codon of the target gene and the upper end of the transcription terminator. The method according to claim 1,
Wherein the acetate inducible promoter has the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The method according to claim 1,
The target gene includes a gene coding for a pyruvate dehydrogenase subunit E1 (NCgl12167), a gene encoding homoserine dehydrogenase (NCgl1136) and a gene encoding a yupipie-ene-acetyl muramoylenyl-di-glutamate- Wherein at least one gene is selected from the group consisting of a gene coding for 2,6-diamino pyrimidate ligase (NCgl2083). 5. The method of claim 4,
Wherein the L-amino acid is L-lysine. The method according to claim 1,
Wherein the target gene is a gene encoding dihydrodipicolinate synthase (-NCgl1896). The method according to claim 6,
Wherein said L-amino acid is L-threonine.

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