RU2614258C2 - Method of producing l-amino acids - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology. Method of producing L-lysine using recombinant coryneform microorganism able to produce L-lysine is disclosed, where recombinant coryneform microorganism is transformed by means of insertion of acetate-inducible promoter below stop codon of target gene on chromosome for attenuation of target gene expression and enhancing of ability of recombinant coryneform microorganism to produce L-lysine, where target gene is gene located in nodal point of L-lysine biosynthesis path.

EFFECT: invention increases production of L-lysine in comparison with parent strain of microorganism.

4 cl, 2 dwg, 12 tbl, 13 ex

Description

FIELD OF TECHNOLOGY

This application claims priority for Korean Patent Application No. 10-2013-0121090, registered October 11, 2013, and Korean Patent Application No. 10-2014-0091307, registered July 18, 2014, in the Korean Intellectual Property Office, the description of which is included in this description as links in full.

One or more embodiments of the present invention relates to a method for producing L-amino acids using a method for inhibiting gene transcription.

BACKGROUND OF THE INVENTION

Pyruvate, obtained by glycolysis of various carbon sources in coryneform microorganisms, is converted to aspartate via oxaloacetate. Aspartate is converted into amino acids, such as threonine, methionine, isoleucine and lysine, using various biosynthesis pathways (Fig. 1). Thus, the expression of genes located at each nodal point of amino acid biosynthesis processes can be inhibited to reduce the formation of by-products and increase the production of target amino acids.

As described above, in order to obtain a strain of a microorganism capable of highly active production of target materials, using genetic engineering and metabolic engineering, it is necessary to selectively control the expression of genes associated with various metabolic processes of the microorganism. Recently, a technology for attenuating gene expression called “artificial convergent transcription” has been reported (Krylov et al., J Mol Microbiol Biotechnol, 18: 1-13, 2010). Artificial convergent transcription is a technology for attenuating expression of a target gene by inserting a promoter into the downstream region of the target gene transcription terminator in such a way that the opposite direction of the promoter causes a collision of RNA polymerase complexes originating from each promoter during transcription.

The inventors of the present invention developed a technology for selectively inhibiting expression of a target gene in the presence of acetate by inserting an acetate-inducible promoter in the opposite direction to transcribing the target gene, and effectively used technology to inhibit the expression of genes located at nodal points in a coryneform microorganism. Then, the inventors of the present invention confirmed the preparation of a coryneform microorganism producing an L-amino acid in high yield using technology and the present invention was carried out.

DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM

An object of the present invention is to provide a method for producing L-amino acids using an acetate-inducible promoter to inhibit transcription of a target gene.

One of the embodiments of the present invention relates to a method for producing L-amino acids, including

1) the cultivation of a recombinant coryneform microorganism capable of producing L-amino acids, where the recombinant coryneform microorganism is transformed by insertion of an acetate-inducible promoter below the stop codon of the target gene in the chromosome; and

2) the addition of acetate during cultivation to weaken the expression of the target gene and enhance the ability of the recombinant coryneform microorganism to produce L-amino acids.

SOLUTION

Embodiments will be described in detail below, examples of which are illustrated in the accompanying drawings, where throughout these like reference numbers refer to like elements. In this regard, these options for implementation may take various forms, and should not be construed as limiting the descriptions set forth in this application. Thus, embodiments are only described below with reference to the figures to explain aspects of the present description. Expressions such as “at least one” preceding the list of items modify the entire list of items and do not modify individual items in the list.

Further in the present description, the present invention is described in detail.

One of the embodiments of the present invention relates to a method for producing L-amino acids, including

1) the cultivation of a recombinant coryneform microorganism capable of producing L-amino acids, where the recombinant coryneform microorganism is transformed by insertion of an acetate-inducible promoter below the stop codon of the target gene in the chromosome; and

2) the addition of acetate during cultivation to weaken the expression of the target gene and enhance the ability of the recombinant coryneform microorganism to produce L-amino acids.

The term “acetate-inducible promoter”, as used herein, refers to a promoter having an activity inducing gene expression in the presence of acetate.

In the coryneform microorganism, acetate is converted by acetate acetate kinase (ackA, NCgl2656) and phosphotransacetylase (pta, NCgl2657) or succinyl-CoA: acetate-CoA-transferase (actA, NCgl2480) to acetyl-CoA, and then metabolized by isocytrate lyoxylate 24-cycloisylate glyoxylate acetylase glyoxylate . In Escherichia coli, acetate is converted to acetyl-CoA by acetyl-CoA synthase (acs, b4069) (Gerstmeir et al., J Biotechnol, 104: 99-122, 2003). Expression of these genes involved in the metabolism of acetate is induced in the presence of acetate. Thus, when using gene promoters, gene expression can be specifically induced in the presence of acetate.

Acetate-inducible promoters include the promoter of the gene encoding isocitratlyase (aceA, NCgl2248), or the promoter of the operon of the gene encoding acetate kinase (ackA, NCgl2656), and the gene encoding phosphotransacetylase (pta, NCgl2657), which is an upstream promoter. More specifically, among the acetate-inducible promoters described above, the aceA promoter is represented by the nucleotide sequence of SEQ ID NO: 1 and includes 486 base pairs above the aceA gene and 36 base pairs from the N-terminus of the open reading frame (ORF).

The upstream pta gene promoter, which is another acetate inducible promoter, is represented by the nucleotide sequence of SEQ ID NO: 2 and includes 340 base pairs above the pta gene.

In addition, it is obvious that any promoter capable of inducing expression of the target gene via acetate can be included in the scope of the present invention. For example, acetate-inducible promoters may include a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 or 2 or comprising the conservative nucleotide sequence of SEQ ID NO: 1 or 2, and one or more nucleotides (specifically, from 2 to 20, more specifically - from 2 to 10, more specifically, from 2 to 5 nucleotides, depending on the steric conformation of the amino acid residues of the protein) subjected to substitution, deletion, insertion, addition or inversion in one or more positions. Provided that the function of the inducible promoter is maintained or enhanced, it may include a nucleotide sequence having more than 80% homology with respect to the nucleotide sequence of SEQ ID NO: 1 or 2, specifically more than 90%, more specifically more than 95%, even more specifically, more than 97%. Provided that the function of the inducible promoter is maintained, replaced, deleted, inserted, added or inverted, the nucleotide sequence may include a spontaneous mutant sequence or even an artificial mutant sequence.

The term "homology", as used herein, refers to the identity between two different nucleotide sequences. Homology can be determined in a manner known in the art using BLAST 2.0 software, which calculates parameters such as score, identity and similarity. However, the method for determining homology is not limited to the above.

Unless otherwise indicated in the present description, the term “upstream” refers to the 5'-direction and the term “downstream” refers to the 3'-direction. Typically, the direction of transcription is 5'-3 'such that the position of the promoter is typically above (5') the target gene.

In the present description, the target gene on the chromosome can be a gene involved in the biosynthesis of amino acids, such as threonine, methionine, isoleucine and lysine, from various carbon sources, in particular, a gene located at the nodal point of the biosynthesis pathway.

For example, with regard to lysine, at the nodal points of the biosynthesis pathway are the E1 subunit gene of pyruvate dehydrogenase (aceE, NCgl2167), which is involved in the conversion of pyruvate to acetyl-CoA, the homoserine dehydrogenase gene (hom, NCgl1136), and the UDF-N gene acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase (murE, NCgl2083) using meso-2,6-diaminopimelate, which is a precursor of lysine in somatic synthesis.

In addition, with regard to threonine, at the nodal point is the dihydrodipicolinate synthase gene (dapA, NCgl1896), which is involved in the production of lysine from aspartate, and as for methionine, at the nodal point is the homoserine kinase gene (thrB, NCgl1137), which is involved in the production of threonine from homoserine . As for alanine and valine, which are amino acids formed from pyruvate, at the nodal point is the gene of the E1 subunit of pyruvate dehydrogenase (aceE, NCgl2167), which is involved in the conversion of pyruvate to acetyl-CoA.

Thus, the target gene, as non-limiting examples, can be selected from the group consisting of a gene encoding the E1 subunit of pyruvate dehydrogenase (aceE, NCgl2167), a gene encoding homoserine dehydrogenase (hom, NCgl1136), a gene encoding UDF-N-acetylmuramoilanilan D-glutamate-2,6-diaminopimelate lipase (murE, NCgl2083), and a gene encoding dihydrodipicolinate synthase (dapA, NCgl1896).

Specifically, the pyruvate dehydrogenase E1 subunit (aceE, NCgl2167) is one of the protein subunits of the pyruvate dehydrogenase complex (PDHC) involved in the delivery of pyruvate, which is the final metabolite of the glycolysis of the tricarboxylic acid cycle (TCA cycle). Thus, attenuation of aceE gene expression can decrease the supply of carbon sources to the TCA cycle and increase the supply of carbon sources to the lysine biosynthesis pathway to increase lysine production.

Homoserine dehydrogenase (hom, NCgl1136) is an enzyme that synthesizes homoserine from aspartate semi-aldehyde. Because aspartate semi-aldehyde is one of the intermediate precursors in the pathway of lysine biosynthesis; weakening the activity of the hom gene can decrease the supply of carbon sources to the pathway of homoserine biosynthesis and increase the supply of carbon sources to the pathway of lysine biosynthesis to increase lysine production.

UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase (murE, NCgl2083) uses meso-2,6-diaminopimelate for somatic synthesis. Because Meso-2,6-diaminopimelate is also used as a precursor for lysine biosynthesis; weakening of murE gene activity can reduce the supply of carbon sources to somatic synthesis and increase the supply of carbon sources to the lysine biosynthesis pathway to increase lysine production.

Dihydrodipicolinate synthase (dapA, NCgl1896) is an enzyme involved in the production of lysine using aspartate semi-aldehyde. Because aspartate semi-aldehyde is one of the intermediate precursors in the lysine biosynthesis pathway, the weakening of the dapA gene activity can reduce the supply of carbon sources to the lysine biosynthesis pathway and increase the supply of carbon sources to the threonine biosynthesis pathway to increase threonine production.

The term “stop codon,” as used herein, refers to codons that do not encode an amino acid in mRNA but act as a signal for terminating protein synthesis. It is generally accepted that three codons are used as stop codons, including UAA, UAG, and UGA.

The term "transcription terminator", as used herein, refers to a GC-rich inverted repeat sequence. The transcription terminator forms a hairpin for terminating gene transcription.

In the present invention, to attenuate expression of the target gene, as described above, an acetate-inducible promoter can be inserted below the stop codon of the target gene, specifically, between the stop codon and above the transcription terminator. An acetate-inducible promoter can be used to induce inversion of the transcription of the target gene in such a way that RNA polymerase complexes can interfere with each other with a weakening of the expression of the target gene.

The expression of the target gene can be attenuated with each cultivation. More specifically, expression of the target gene can be attenuated prior to or during cultivation.

The term "transformation" as used herein refers to the insertion of a vector comprising a polynucleotide encoding a target gene into a host cell such that a protein encoded by the polynucleotide can be expressed in the host cell. Provided that the inserted polynucleotide can be expressed in the host cell, the polynucleotide can be inserted into the chromosome of the host cell, or it can be located outside the chromosome. In addition, the polynucleotide includes DNA or RNA encoding a target protein. Provided that the polynucleotide can be inserted and expressed in a host cell, the polynucleotide can be inserted in any form.

In an embodiment of the invention, the coryneform microorganism may include microorganisms of the genus Corynebacterium, genus Brevibacterium, genus Arthrobacter sp. and the genus Microbacterium sp. Examples of the coryneform microorganism include Corynebacterium glutamicum, Corynebacterium thermoaminogenes, Brevibacterium flavum, Brevibacterium lactofermentum, and L-amino acid producing variants derived from them. Specifically, a coryneform microorganism may be, but not limited to, Corynebacterium glutamicum.

More specifically, coryneform microorganisms in the present invention may include Corynebacterium glutamicum KCCM11016P (formerly Registration No.: KFCC10881, Korean Patent Reference No. 10-0159812), Corynebacterium glutamicum KCCM10770P (Korean Patent Reference No. 10-0924065bum, Korea Cumneum Glutamicum), (former registration number: KFCC10750, reference to the Korean patent No.: 10-0073610).

In the present invention, coryneform microorganisms may also include Corynebacterium glutamicum CJ3P. According to an article by Binder et al. (Binder et al., Genome Biology, 13: R40, 2012), it is possible to obtain CJ3P having the ability to produce lysine by embedding mutations in three genes that contribute to the efficiency of lysine production (pyc (P458S), hom (V59A) and lysC (T311I)), in the parent wild-type Corynebacterium glutamicum strain (ATCC13032).

In addition, in the present invention, another coryneform microorganism may be Corynebacterium glutamicum KCCM11222P, which is an L-threonine-producing strain (reference to Korean Patent No.: 10-1335853).

According to one embodiment of the present invention, in all coryneform microorganisms in which the promoter represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 was inserted, the production of L-lysine or L-threonine was increased compared to that of the parent strain.

Regarding the method presented in the present invention, the cultivation of coryneform microorganism can be carried out using any cultivation conditions and cultivation method known in this field.

A culture medium that can be used in the cultivation of a strain of a coryneform microorganism can be, for example, the culture media described in Manual of Methods for General Bacteriology, American Society for Bacteriology (Washington D.C., USA, 1981).

Sources of carbon that can be used in the culture medium may include carbohydrate, such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, oil or lipid, such as soybean oil, sunflower oil, castor oil and coconut oil, fatty an acid such as palmitic acid, stearic acid and linoleic acid, an alcohol such as glycerin and ethanol, and an organic acid such as acetic acid. These substances may be present separately or as a mixture.

Sources of nitrogen that can be used in the culture medium may include peptone, yeast extract, beef extract, malt extract, corn extract, soy and urea, and an inorganic source of nitrogen such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and nitrate ammonium These nitrogen sources may also be present separately or as a mixture.

Sources of phosphorus that can be used in the culture medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their sodium salt. In addition, the culture medium may include a metal salt, such as magnesium sulfate and iron sulfate, necessary for growth. In addition to the substances described above, substances necessary for growth, such as amino acids and vitamins, can be used. In addition, suitable precursors may be used in the culture medium. Raw materials can be added to the culture solution in a batch manner or in a continuous manner during cultivation.

During the cultivation of the microorganism, the pH of the culture medium can be adjusted by adding a basic compound, such as ammonium hydroxide, potassium hydroxide and ammonia, or an acidic compound, such as phosphoric acid and sulfuric acid, to the culture medium in a suitable manner. In addition, bubble formation can be suppressed using an antifoam such as a fatty acid ester and polyglycol. To maintain aerobic conditions, oxygen or an oxygen-containing gas (e.g., air) can be introduced into the culture medium. The temperature of the culture medium, as a rule, can be from about 20 ° C to about 45 ° C, specifically from about 25 ° C to about 40 ° C. Cultivation may continue until the desired amount of L-amino acid is obtained, but a suitable culture time may be from about 10 to 160 hours.

Regarding the method presented in the present invention, the cultivation can be carried out in a continuous way or in a batch method, such as a batch method, a batch method with recharge and a batch method with recharge. These cultivation methods are known in the art, and any of the cultivation methods can be used.

The term "cultivation", as used in the present description, may include obtaining a medium for cultivation and time during the growth of microorganisms.

As for the method presented in the present invention, it may include an additional purification or isolation step. The desired L-amino acid can be purified or isolated from the culture solution using a suitable method known in the art, in accordance with a method such as batch cultivation, continuous cultivation and batch culture with water.

The L-amino acid produced by cultivation of the present invention may be an amino acid selected from the group consisting of threonine, methionine, isoleucine, lysine, valine and alanine, specifically lysine or threonine.

Brief Description of the Drawings

These and / or other aspects will become apparent and more apparent from the following description of embodiments presented in combination with the accompanying drawings, in which:

In FIG. 1 shows the nodal points of the process of biosynthesis of amino acids in a coryneform microorganism; and

FIG. 2 is a schematic diagram showing inhibition of aceE gene expression by insertion of the aceA gene promoter (a) between the stop codon and the transcription terminator above the aceE gene or (b) below the transcription terminator in the opposite direction of the aceE gene transcription.

The method according to the invention

Hereinafter, the present invention will be described in more detail with reference to examples. These examples are presented for illustrative purposes only and do not limit the scope of the present invention.

Examples

Example 1: Selection of an acetate-inducible promoter

Isocytratliase (aceA, NCgl2248) is a key enzyme in the glyoxylate cycle, and the gene encoding isocytratliase is expressed in the presence of acetate. In addition, acetate kinase (ackA, NCgl2656) and phosphotransacetylase (pta, NCgl2657), which are enzymes involved in the metabolism of acetate, form an operon, and their expression is enhanced in the presence of acetate. The promoter regions of the aceA gene and the pta-ackA operon are already known (Gerstmeir et al., J Biotechnol, 104, 99-122, 2003).

In Example 1, the aceA gene promoter and the pta-ack operon promoter, which is the upstream promoter region of the pta gene, were selected to inhibit the transcription of the target gene in the presence of acetate. Based on the aceA gene registered in the US NIH GenBank (Registration No. NCBI: NCgl2248), a nucleotide sequence (SEQ ID NO: 1) was obtained comprising 486 base pairs above the aceA gene and 36 base pairs from the N-terminus of the open reading frame (ORF) . In addition, based on the pta gene registered in the US NIH GenBank (Registration No. NCBI: NCgl2657), a nucleotide sequence (SEQ ID NO: 2) was obtained comprising 340 base pairs above the pta gene.

Example 2: Preparation of a vector for inhibiting aceE gene expression

The pyruvate dehydrogenase E1 subunit (aceE, NCgl2167) is one of the protein subunits of the pyruvate dehydrogenase complex (PDHC) involved in the delivery of pyruvate, which is the final glycolysis metabolite in the TCA cycle. Thus, attenuation of aceE gene expression can decrease the supply of carbon sources to the TCA cycle and increase the supply of carbon sources to the lysine biosynthesis pathway to increase lysine production (Blombach et al., Appl Microbiol Biotechnol, 76 (3): 615-23, 2007).

To selectively inhibit the expression of the aceE gene in the presence of acetate, the aceA gene promoter was inserted below the aceE gene so that transcription from this promoter can occur in a direction opposite to the original direction of transcription of the aceE gene (Fig. 2).

First, CLC main workbench software (CLC Bio, Denmark) was used to predict the aceE gene transcription terminator. The transcription terminator is a GC-rich inverted repeat sequence and forms a hairpin loop for terminating gene transcription. The result of predicting the aceE gene transcription terminator indicates that 36 base pairs, from the 21st base pair to the 56th base pair below the stop codon of the aceE gene, form a hairpin loop as a transcription terminator. Based on this result, two vectors were obtained for embedding the aceE promoter higher or lower, respectively, of the aceE gene transcription terminator so that transcription from this promoter can occur in the opposite direction to the original one.

<2-1> Preparation of pDZ-aceE1-PaceA vector for inhibiting aceE gene expression

The vector was obtained by insertion of the aceA gene promoter below the stop codon of the aceE gene located between the stop codon and the upstream transcription terminator.

To obtain the aceE gene fragment of Corynebacterium glutamicum, Corynebacterium glutamicum KCCM11016P chromosomal DNA was used as a template for the synthesis of primers (SEQ ID NOs: 3 and 4) designed to contain the XbaI restriction enzyme recognition site at the 5'-end of the fragment and the Spe restriction enzyme recognition site 3 for Spe 'is the end of the fragment. PCR was performed using synthesized primers to obtain a DNA fragment comprising 296 base pairs from the 2474th nucleotide from the aceE initiation codon to the 2769th nucleotide, aceE stop codon. In addition, primers were synthesized (SEQ ID NOs: 5 and 6) constructed with SpeI recognition at the 5'-end of the fragment and XbaI recognition at the 3'-end of the fragment, and PCR was performed using primers to produce a DNA fragment of 300 base pairs below the stop codon of the aceE gene. PfuUltra ™ High-Fidelity (Stratagene) DNA polymerase was used as polymerase and PCR was performed with 30 denaturation cycles at 95 ° C for 30 seconds; annealing at 55 ° C for 30 seconds and polymerizing at 72 ° C for 30 seconds, and then polymerizing at 72 ° C for 7 minutes.

Two PCR amplification products and a pDZ vector (reference to Korean patent No. 10-0924065) for incorporation into a chromosome already obtained by XbaI restriction enzyme digestion were cloned using the In-fusion Cloning Kit (TAKARA, JP) to obtain the pDZ vector -aceE1.

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 '

To obtain the aceA gene promoter fragment of Corynebacterium glutamicum, primers were synthesized designed containing the SpeI restriction enzyme recognition site at the 5 ′ end and 3 ′ end of the fragment (SEQ ID NOs: 7 and 8). PCR was performed using Corynebacterium glutamicum KCCM11016P chromosomal DNA as a template and synthesized primers to amplify the promoter region from approximately 500 base pairs represented by the nucleotide sequence of SEQ ID NO: 1. PCR amplification product and DNA fragment obtained by treatment with pDZ-aceE1 restriction enzyme SpeI, cloned using the In-fusion Cloning Kit (TAKARA, JP) to obtain the pDZ-aceE1-PaceA vector.

SEQ ID NO: 7:

PaceA-P3F 5'-gtcccttgagactagtagcactctgactacctctg-3 '

SEQ ID NO: 8:

PaceA-P3R 5'-ctgaggaataactagtttcctgtgcggtacgtggc-3 '

<2-2> Preparation of pDZ-aceE2-PaceA vector for inhibiting aceE gene expression

The vector was obtained by inserting the aceA gene promoter below the aceE gene transcription terminator.

To obtain the aceE gene fragment of Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as a template and primers designed containing the XbaI restriction enzyme recognition site at the 5'-end of the fragment and the SpeI restriction enzyme recognition site at the 3'-NO end of the fragment ( 9 and 10). PCR was performed using the synthesized primers to obtain a DNA fragment comprising 294 base pairs from the 2538th nucleotide from the initiating codon of the aceE gene to the 62nd nucleotide below the stop codon. In addition, primers designed to contain the SpeI restriction enzyme recognition site at the 5'-end of the fragment and the XbaI restriction enzyme recognition site at the 3'-end of the fragment (SEQ ID NO: 11 and 12) were used to perform PCR to obtain a DNA fragment comprising 294 base pairs from the 69th nucleotide below the stop codon of the aceE gene to the 362nd nucleotide. PfuUltra ™ High-Fidelity (Stratagene) DNA polymerase was used as polymerase and PCR was performed with 30 denaturation cycles at 95 ° C for 30 seconds; annealing at 55 ° C for 30 seconds and polymerizing at 72 ° C for 30 seconds, and then polymerizing at 72 ° C for 7 minutes.

Two PCR amplification products and a pDZ vector for insertion into the chromosome, already obtained by XbaI restriction enzyme digestion, were cloned using the Infusion Cloning Kit (TAKARA, JP) to obtain the pDZ-aceE2 vector.

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 '

To obtain the aceA gene promoter fragment of Corynebacterium glutamicum, primers were synthesized designed containing the SpeI restriction enzyme recognition site at the 5 ′ end and 3 ′ end of the fragment (SEQ ID NOs: 13 and 14). PCR was performed using Corynebacterium glutamicum KCCM11016P chromosomal DNA as a template and synthesized primers for amplification of the promoter region from approximately 500 base pairs represented by the nucleotide sequence of SEQ ID NO: 1. PCR amplification product and DNA fragment obtained by processing the vector pDZ-aceE2 enzyme restriction SpeI, cloned using the In-fusion Cloning Kit (TAKARA, JP) to obtain the vector pDZ-aceE2-PaceA.

SEQ ID NO: 13:

PaceA-P6F 5'-aaaaagagctactagtagcactctgactacctctg-3 '

SEQ ID NO: 14:

PaceA-P6R 5'-gattcgtcgtactagtttcctgtgcggtacgtggc-3 '

Example 3: Preparation of strains in which the aceA promoter was inserted below the aceE gene

The vectors pDZ-aceE1-PaceA and pDZ-aceE2-PaceA obtained in Example 2 were inserted by electroporation into Corynebacterium glutamicum KCCM11016P, which is an L-lysine-producing strain (the transformation method is described in Van der Rest et al., Appl Microbiol Biotechnol 52: 541-545, 1999). The appropriate strains were selected in which the aceA promoter was inserted below the stop codon of the aceE gene on the chromosome so that transcription from this promoter can occur in the direction opposite to the original direction, by PCR to obtain an L-lysine-producing strain. The selected strains were named Corynebacterium glutamicum KCCM11016P :: aceE1-PaceA and Corynebacterium glutamicum KCCM11016P :: aceE2-PaceA, respectively. Corynebacterium glutamicum KCCM11016P :: aceE1-PaceA was internationally deposited under the name Corynebacterium glutamicum CA01-2271 at the Korean Culture Center of Microorganism (KCCM) on June 12, 2013 with registration number KCCM11432P. The obtained strains were checked by analyzing the nucleotide sequence of the obtained target region by PCR using SEQ ID NO: 3 and SEQ ID NO: 6 as primers for KCCM11016P :: aceE1-PaceA and SEQ ID NO: 9 and SEQ ID NO: 12 as primers for KCCM11016P :: aceE2-PaceA.

Example 4: Comparison of lysine production by strains in which the aceA promoter was inserted below the aceE gene

The strain Corynebacterium glutamicum KCCM11016P, used as the parent strain, and Corynebacterium glutamicum KCCM11016P :: aceE1-PaceA and Corynebacterium glutamicum KCCM11016P :: aceE2-PaceA, which are the L-lysing examples described below.

Corynebacterium glutamicum KCCM11016P, KCCM11016P :: aceE1-PaceA and KCCM11016P :: aceE2-PaceA were inoculated respectively in 25 ml of the following medium for sowing in 250 ml baffles, followed by shaking at 200 ° C / min. within 20 hours. 1 ml of the seed culture solution was added to a 250 ml baffle flask containing 24 ml of the production medium described below, followed by cultivation with shaking at 200 rpm at 30 ° C for 72 hours. Appropriate composition of the medium for sowing and production environment are described below.

<Sowing 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 ⋅7 (H ₂ O) 0.5 g, biotin 100 μg , thiamine HCl 1000 μg, calcium pantothenate 2000 μg, nicotinamide 2000 μg (per 1 liter of distilled water).

<Production medium (pH 7.0)>

Glucose 100 g, (NH ₄ ) ₂ SO ₄ 40 g, soy protein 2.5 g, solid corn extract 5 g, urea 3 g, KH ₂ PO ₄ 1 g, MgSO ₄ ⋅ 7 (H ₂ O) 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium pantothenate 2000 μg, nicotinamide 3000 μg, CaCO ₃ 30 g (per 1 liter of distilled water).

After cultivation using HPLC, the concentration of L-lysine was measured. For cases where acetate was not added, the concentration of L-lysine in culture solutions of Corynebacterium glutamicum KCCM11016P, KCCM11016P :: aceE1-PaceA and KCCM11016P :: aceE2-PaceA is presented in Table 1.

Table 1
Differences in L-lysine production (no acetate added) Strain Lysine (g / l) Party 1 Party 2 Party 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, the strains were cultured in the same way, except that 5 g / L acetate was added to the production medium to compare the production of L-lysine. The concentration of L-lysine in the culture solutions is presented in table 2.

table 2
Differences in L-Lysine Production (5 g / L Acetate Added) Strain Lysine (g / l) Party 1 Party 2 Party 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, in the absence of acetate, the production of L-lysine by strains KCCM11016P :: aceE1-PaceA and KCCM11016P :: aceE2-PaceA did not differ from the parent strain KCCM11016P.

However, as shown in Table 2, in the presence of acetate, L-lysine production by strain KCCM11016P :: aceE1-PaceA was 3.6% higher than that of the parent strain KCCM11016P, and strain KCCM11016P :: aceE2-PaceA was 2.5% higher than the parent strain KCCM11016P.

In addition, a comparison of the KCCM11016P :: aceE1-PaceA strain and the KCCM11016P :: aceE2-PaceA strain showed that L-lysine production by the KCCM11016P :: aceE1-PaceA strain, in which the aceA promoter was inserted above the aceE gene transcription terminator between the stop codon and the overlying area of the transcription terminator, was more effective. This suggests that the region between the stop codon and the overlying region of the transcription terminator can be used to more effectively inhibit gene expression.

Example 5: Preparation of pDZ-aceE-Ppta vector for inhibiting aceE gene expression

Acetate kinase (ackA, NCgl2656) and phosphotransacetylase (pta, NCgl2657), which are enzymes involved in the metabolism of acetate, form an operon, and their expression is enhanced in the presence of acetate. Thus, when using gene promoters, gene expression can be specifically induced in the presence of acetate.

In Example 5, to inhibit aceE gene expression in the presence of acetate, a vector was prepared for using the pta-ack operon promoter, which was the upstream promoter region of the pta gene.

To inhibit the expression of the aceE gene, a vector was constructed that can contain the pta gene promoter below the stop codon of the aceE gene located between the stop codon and the overlying region of the transcription terminator, so that transcription from the pta gene promoter can occur in the direction opposite to the original direction of transcription aceE gene.

To obtain the aceE gene fragment of Corynebacterium glutamicum, the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as a template to obtain the pDZ-aceE1 vector in the same manner as in Example 2.

To obtain the pta Corynebacterium glutamicum pta gene promoter fragment, primers were synthesized designed containing the SpeI restriction enzyme recognition site at the 5 ′ end and 3 ′ end of the fragment (SEQ ID NO: 15 and SEQ ID NO: 16). To perform PCR to amplify the promoter region of approximately 340 base pairs represented by the nucleotide sequence of SEQ ID NO: 2, Corynebacterium glutamicum KCCM11016P chromosomal DNA was used as a template and synthesized primers. The PCR amplification product and DNA fragment obtained by treating the pDZ-aceE1 vector with SpeI restriction enzyme were cloned using the Infusion Cloning Kit (TAKARA, JP) to obtain the pDZ-aceE1-Ppta vector.

SEQ ID NO: 15:

Ppta-P7F 5'-gtcccttgagactagtctttgctggggtcagatttg-3 '

SEQ ID NO: 16:

Ppta-P7R 5'-ctgaggaataactagtacatcgcctttctaatttc-3 '

Example 6: Preparation of strains in which the pta gene promoter was inserted below the aceE gene, and comparison of their production of lysine

Corynebacterium glutamicum KCCM11016P was transformed using the pDZ-aceE1-Ppta vector obtained in Example 5 in the same manner as in Example 3. A strain was selected in which the pta gene promoter was inserted below the stop codon of the aceE gene on the chromosome so that transcription can occur in the opposite direction to the original direction of transcription of the aceE gene, by performing PCR to obtain an L-lysine-producing strain called KCCM11016P :: aceE1-Ppta. The resulting KCCM11016P :: aceE1-Ppta strain was checked by analyzing the obtained nucleotide sequence of the target region by PCR using SEQ ID NO: 3 and SEQ ID NO: 6 as primers.

The resulting strain was cultured in the same manner as in Example 4, and the concentration of L-lysine isolated from the culture solution was measured. For cases where acetate was not added, the concentration of L-lysine in the culture solution is presented in table 3.

Table 3
Differences in L-lysine production (no acetate added) Strain Lysine (g / l) Party 1 Party 2 Party 3 KCCM11016P 42.9 43.5 43,4 KCCM11016P :: aceE1-Ppta 43,2 43.3 43.6

In addition, the strain was cultured in the same manner, except that 5 g / L acetate was added to the production medium to compare the production of L-lysine. The concentration of L-lysine in the culture solution is presented in table 4.

Table 4
Differences in L-Lysine Production (5 g / L Acetate Added) Strain Lysine (g / l) Party 1 Party 2 Party 3 KCCM11016P 45.5 45.7 45.3 KCCM11016P :: aceE1-Ppta 46.7 46.5 46.6

As shown in table 3, in the absence of acetate, the production of L-lysine by strain KCCM11016P :: aceE1-Ppta did not differ from the production by the parent strain KCCM11016P.

However, as shown in Table 4, in the presence of acetate, L-lysine production by strain KCCM11016P :: aceE1-Ppta was 2.4% higher than that of the parent strain KCCM11016P.

In addition, since in the presence of acetate, L-lysine production by strain KCCM11016P :: aceE1-PaceA was higher than that of strain KCCM11016P :: aceE1-Ppta, expression of the target gene can be more effectively inhibited using the aceA gene promoter than using the pta gene promoter.

Example 7: Preparation of strains in which the aceA promoter was inserted below the aceE gene

Three L-lysine-producing strains, which were Corynebacterium glutamicum KFCC10750, KCCM10770P and CJ3P, were accordingly transformed using the pDZ-aceE1-PaceA vector obtained in example 2 in the same manner as in example 3. Strains in which the promoter was selected the aceA gene was inserted below the stop codon of the aceE gene on the chromosome so that transcription can occur in the direction opposite to the original direction of transcription of the aceE gene by PCR. The obtained three L-lysine-producing strains were KFCC10750 :: aceE1-PaceA, KCCM10770P :: aceE1-PaceA and CJ3P :: aceE1-PaceA. The obtained strains were checked by analyzing the obtained nucleotide sequence of the target region by PCR using SEQ ID NO: 3 and SEQ ID NO: 6 as primers.

The obtained strains were cultured in the same manner as in example 4, and the concentration of L-lysine isolated from the culture solutions was measured. For cases where acetate was not added, the concentration of L-lysine in the culture solutions is presented in table 5.

Table 5
Differences in L-lysine production (no acetate added) Strain Lysine (g / l) Party 1 Party 2 Party 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, the strains were cultured in the same manner, except that 5 g / L acetate was added to the production medium to compare the production of L-lysine. The concentration of L-lysine in the culture solutions is presented in table 6.

Table 6
Differences in L-Lysine Production (5 g / L Acetate Added) Strain Lysine (g / l) Party 1 Party 2 Party 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, in the absence of acetate, L-lysine production by the three strains KFCC10750 :: aceE1-PaceA, KCCM10770P :: aceE1-PaceA, CJ3P :: aceE1-PaceA did not differ from the production by the parent strain.

However, as shown in Table 6, in the presence of acetate, L-lysine production by strain KFCC10750 :: aceE1-PaceA was 5% higher than that of the parent strain, production by strain KCCM10770P :: aceE1-PaceA was 2.8% higher than in the parent strain, and production by strain CJ3P :: aceE1-PaceA was 15% higher than in the parent strain.

Example 8: Preparation of a vector for inhibiting hom gene expression

To increase the production of L-lysine, one can weaken the pathway of L-threonine biosynthesis using the same substrate as the pathway of L-lysine biosynthesis. An example of ways to weaken the pathway of L-threonine biosynthesis is to reduce the enzymatic activity of homoserine dehydrogenase (hom, NCgl1136) synthesizing homoserine from aspartate.

In Corynebacterium glutamicum, the hom gene forms the hom-thrB operon with the thrB gene, and the hom gene transcription terminator is located below the thrB gene. The promoter is reported to be located above the hom-thrB operon located above the hom gene. In addition, it is reported that the second promoter is located above the operon of the thrB gene (Mateos et al., J Bacteriol, 176: 7362-7371, 1994). Thus, to selectively inhibit the expression of the hom gene in the presence of acetate, the aceA promoter was inserted below the stop codon of the hom gene so that transcription from this promoter can occur in a direction opposite to the original direction of transcription of the hom gene. To preserve thrB gene expression, a second promoter sequence was added above the thrB gene ORF.

In Example 8, a recombinant vector was obtained by inserting the aceA gene promoter between the downstream stop codon of the hom gene and the upstream thrB gene.

To obtain a fragment of the Corynebacterium glutamicum hom gene, Corynebacterium glutamicum KCCM11016P chromosomal DNA was used as a template and the primers were synthesized that contain the XbaI restriction enzyme recognition site at the 5'-end of the fragment and SpeI restriction enzyme recognition site at the 3'-end of the NO fragment (SE 17 and 18). PCR was performed using synthesized primers to obtain a DNA fragment comprising 300 base pairs between the 1039th nucleotide from the initiating codon of the hom gene and the 1338th nucleotide, which is the stop codon. Even when aceA is inserted between hom-thrB operons, thrB gene expression must be maintained. Thus, when a DNA fragment having 300 base pairs below the stop codon of the hom gene was obtained, primers were synthesized designed to further include the thrB promoter sequence of 32 base pairs from the 5 ′ side (SEQ ID NOs: 19 and 20). PCR was performed using these primers (SEQ ID NO: 19 and 20) to obtain a DNA fragment of 334 base pairs containing a SpeI restriction enzyme recognition site at the 5'-end of the fragment and an XbaI restriction enzyme recognition site at the 3'-end of the fragment. PCR was carried out under the same conditions as in example 2.

Two PCR amplification products and a pDZ vector for insertion into the chromosome, already obtained by XbaI restriction enzyme digestion, were cloned using the Infusion Cloning Kit (TAKARA, JP) to obtain the pDZ-hom vector.

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 '

To obtain a promoter fragment, the aceA gene of Corynebacterium glutamicum synthesized primers designed with the SpeI restriction enzyme recognition site at the 5'-end and 3'-end of the fragment (SEQ ID NOs: 21 and 22). PCR was performed using Corynebacterium glutamicum KCCM11016P chromosomal DNA as a template and synthesized primers for amplification of the promoter region from approximately 500 base pairs represented by the nucleotide sequence of SEQ ID NO: 1. PCR amplification product and DNA fragment obtained by processing the vector with pDZ-hom enzyme restriction SpeI, cloned using the In-fusion Cloning Kit (TAKARA, JP) to obtain the vector pDZ-hom-PaceA.

SEQ ID NO: 21:

PaceA-h3F 5'-gaggcggatcactagtagcactctgactacctctg-3 '

SEQ ID NO: 22:

PaceA-h3R 5'-aagggactaaactagtttcctgtgcggtacgtggc-3 '

Example 9: Preparation of strains in which the aceA promoter was inserted below the hom gene and comparison of their production of lysine

Corynebacterium glutamicum KCCM11016P, which is an L-lysine-producing strain, was transformed using the pDZ-hom-PaceA vector obtained in Example 8 in the same manner as in Example 3. A strain was selected in which the aceA promoter was inserted below the stop codon the hom gene on the chromosome in such a way that transcription can occur in the opposite direction to the original direction of transcription of the hom gene by PCR to obtain an L-lysine-producing strain called KCCM11016P :: hom-PaceA. The resulting strain KCCM11016P :: hom-PaceA was checked by analyzing the nucleotide sequence of the obtained target region by PCR using SEQ ID NO: 17 and SEQ ID NO: 20 as primers.

The strain Corynebacterium glutamicum KCCM11016P used as the parent strain and the obtained strain KCCM11016P :: hom-PaceA were cultured in the same manner as in example 4, and the concentration of L-lysine isolated from the culture solutions was measured. For cases where acetate was not added, the concentration of L-lysine in the culture solutions of the strain Corynebacterium glutamicum KCCM11016P and KCCM11016P :: hom-PaceA are presented in table 7.

Table 7
Differences in L-lysine production (no acetate added) Strain Lysine (g / l) Party 1 Party 2 Party 3 KCCM11016P 43,2 43.3 43.6 KCCM11016P :: hom-PaceA 43.3 43.6 43,4

In addition, the strains were cultured in the same manner, except that 5 g / L acetate was added to the production medium to compare the production of L-lysine. The concentration of L-lysine in the culture solutions is presented in table 8.

Table 8
Differences in L-Lysine Production (5 g / L Acetate Added) Strain Lysine (g / l) Party 1 Party 2 Party 3 KCCM11016P 44.9 45.6 45,2 KCCM11016P :: hom-PaceA 46.3 46.6 46,4

As shown in table 7, in the absence of acetate, the production of L-lysine by strain KCCM11016P :: hom-PaceA did not differ from the production by the parent strain KCCM11016P.

However, as shown in Table 8, in the presence of acetate, L-lysine production by strain KCCM11016P :: hom-PaceA was 2.6% higher than that of the parent strain KCCM11016P.

Example 10: Preparation of a vector for inhibiting murE gene expression

UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase (murE, NCgl2083) uses meso-2,6-diaminopimelate, a precursor for lysine biosynthesis, in somatic synthesis. The weakening of murE gene activity can reduce the supply of carbon sources to the somatic synthesis pathway and increase the supply of carbon sources to the lysine biosynthesis pathway to increase lysine production.

In Corynebacterium glutamicum, the murE gene (NCgl2083) forms an operon with seven genes from NCgl2076 to NCgl2082. The operon transcription begins with NCgl2083 murE in the direction of the NCgl2076 gene. Thus, the transcription terminator is located below the NCgl2076 gene. Thus, to selectively inhibit murE gene expression in the presence of acetate, the aceA promoter was inserted below the stop codon of the murE gene so that transcription can occur in a direction opposite to the original direction of transcription of the murE gene. To maintain the expression of the other seven genes, with the exception of the murE gene located in the first region of the operon, the murE operon promoter above the ORF of the NCgl2082 gene was further included.

In Example 10, a recombinant vector was obtained by inserting the aceA gene promoter below the stop codon of the murE gene.

To obtain a fragment of the murE Corynebacterium glutamicum murE gene, Corynebacterium glutamicum KCCM11016P chromosomal DNA was used as a template and synthesized primers designed containing the XbaI restriction enzyme recognition site at the 5'-end of the fragment and the XhoI restriction enzyme recognition site at the 3'-end of the SEQ ID NO: 23 and 24). PCR was performed using synthesized primers to obtain a DNA fragment comprising 300 base pairs between the 1267th nucleotide from the initiator codon of the murE gene and the 1566th nucleotide, which was the stop codon. In addition, primers were synthesized designed containing the XhoI restriction enzyme recognition site at the 5'-end of the fragment and the XbaI restriction enzyme recognition site at the 3'-end of the fragment (SEQ ID NOs: 25 and 26). PCR was performed using synthesized primers to obtain a DNA fragment comprising 292 base pairs from the 10th nucleotide below the stop codon of the murE gene. PCR was carried out under the same conditions as in Example 2. Two PCR amplification products and a pDZ vector for incorporation into the chromosome, already obtained by XbaI restriction enzyme digestion, were cloned using the Infusion Cloning Kit (TAKARA, JP) to obtain pDZ-murE vectors.

SEQ ID NO: 23:

mur-m1F 5'-ccggggatcctctagaaaccctcgttcagaggtgc-3 '

SEQ ID NO: 24:

mur-m1R 5'-ttgtgatcatctctcgagctatccttcttccgtagtaag-3 '

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

SEQ ID NO: 26:

mur-m2R 5'-gcaggtcgactctagacatgagcataaatgtcagc-3 '

To obtain the aceA gene fragment of Corynebacterium glutamicum, primers were synthesized designed containing the XhoI restriction enzyme recognition site at the 5 ′ end of the fragment (SEQ ID NOs: 27 and 28). To perform PCR to amplify a promoter region of approximately 500 base pairs represented by the nucleotide sequence of SEQ ID NO: 1, Corynebacterium glutamicum KCCM11016P chromosomal DNA was used as a template and synthesized primers. In addition, to obtain the promoter region of the murE Corynebacterium glutamicum operon, Corynebacterium glutamicum KCCM11016P chromosomal DNA was used as a template and the primers were designed that contained the XhoI restriction enzyme recognition site at the 3'-end of the fragment (SEQ ID NO: 29 and 30). PCR was carried out using synthesized primers to obtain a DNA fragment comprising 300 base pairs above the murE ORF gene.

Two PCR amplification products and a DNA fragment obtained by treating the pDZ-murE vector with the XhoI restriction enzyme were cloned using the Infusion Cloning Kit (TAKARA, JP) to obtain the 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'-ttgtgatcatctcggggttttctctcctccacagg-3 '

Example 11: Preparation of strains in which the aceA promoter was inserted below the murE gene, and comparison of their production of lysine

Corynebacterium glutamicum KCCM11016P was transformed using the pDZ-murE-PaceA-PmurE vector obtained in Example 10 in the same manner as in Example 3.

A strain was selected in which the aceA promoter was inserted below the stop codon of the murE gene on the chromosome so that transcription can occur in the direction opposite to the original direction of transcription of the murE gene by PCR to obtain an L-lysine-producing strain called KCCM11016P :: murE -PaceA-PmurE. The resulting strain KCCM11016P :: murE-PaceA-PmurE was checked by analyzing the nucleotide sequences of the obtained target region by PCR using SEQ ID NO: 23 and SEQ ID NO: 26 as primers.

The strain Corynebacterium glutamicum KCCM11016P used as the parent strain and the resulting strain KCCM11016P :: murE-PaceA-PmurE were cultured in the same manner as in example 4, and the concentration of L-lysine isolated from the culture solutions was measured. For cases where acetate was not added, the concentration of L-lysine in the culture solutions of the strain Corynebacterium glutamicum KCCM11016P and strain KCCM11016P :: murE-PaceA-PmurE are presented in table 9.

Table 9
Differences in L-lysine production (no acetate added) Strain Lysine (g / l) Party 1 Party 2 Party 3 KCCM11016P 43.5 43.9 44.0 KCCM11016P :: murE-PaceA-PmurE 43.7 44.1 43.8

In addition, the strains were cultured in the same manner, except that 5 g / L acetate was added to the production medium to compare the production of L-lysine. The concentration of L-lysine in the culture solutions is presented in table 10.

Table 10
Differences in L-Lysine Production (5 g / L Acetate Added) Strain Lysine (g / l) Party 1 Party 2 Party 3 KCCM11016P 45,2 45.6 45.3 KCCM11016P :: murE-PaceA-PmurE 46.6 46.9 46.5

As shown in table 9, in the absence of acetate, the production of L-lysine by strain KCCM11016P :: murE-PaceA-PmurE did not differ from the production by the parent strain KCCM11016P.

However, as shown in Table 10, in the presence of acetate, L-lysine production by strain KCCM11016P :: murE-PaceA-PmurE was 2.8% higher than that of the parent strain KCCM11016P.

Example 12: Preparation of a vector for inhibiting dapA gene expression

To increase the production of L-threonine, one can weaken the pathway of L-lysine biosynthesis using the same substrate as the pathway of L-threonine biosynthesis. An example of ways to weaken the pathway of L-lysine biosynthesis is to reduce the enzymatic activity of dihydrodipicolinate synthase (dapA, NCgl1896), which is involved in the production of lysine from aspartate.

In Corynebacterium glutamicum, the dapA gene forms the dapA-ORF4 operon with the ORF4 gene (NCgl1895), and thus the dapA gene transcription terminator is located below the ORF4 gene. In addition, it is reported that the promoter is located above the dapA-ORF4 operon located above the dapA gene, and the second promoter is located above the ORF4 gene (Patek et al., Biotechnology letters, 19: 1113-1117, 1997). Thus, to selectively inhibit the expression of the dapA gene in the presence of acetate, the aceA promoter was inserted below the stop codon of the dapA gene so that transcription can occur in a direction opposite to the original direction of transcription of the dapA gene. To preserve ORF4 gene expression, a promoter region sequence of approximately 100 base pairs in the upstream ORF4 gene was added to the ORF of the upstream ORF4 gene.

In Example 12, a recombinant vector was obtained by inserting the aceA gene promoter below the stop codon of the dapA gene.

To obtain the Corynebacterium glutamicum dapA gene fragment, the Corynebacterium glutamicum KCCM11016P chromosomal DNA was used as a template and the primers were synthesized designed containing the XbaI restriction enzyme recognition site at the 5'-end of the fragment and the SpeI restriction enzyme recognition site at the 3'-end SEI ID fragment: 31 and 32). PCR was performed using synthesized primers to obtain a DNA fragment comprising 301 base pairs between the 606th nucleotide from the initiating codon of the dapA gene and the 906th nucleotide, which was the stop codon. In addition, primers were synthesized designed containing the SpeI restriction enzyme recognition site at the 5'-end of the fragment and the XbaI restriction enzyme recognition site at the 3'-end of the fragment (SEQ ID NO: 33 and 34). Using PCR, a DNA fragment was obtained that further included a sequence of the promoter region of approximately 100 base pairs between the 809th nucleotide from the initiating codon of the dapA gene and the 2nd nucleotide in the underlying stop codon to preserve ORF4 gene expression and 213 base pairs below the stop codon dapA gene. PCR was carried out under the same conditions as in Example 2. Two PCR amplification products and a pDZ vector for incorporation into the chromosome, already obtained by XbaI restriction enzyme digestion, were cloned using the Infusion Cloning Kit (TAKARA, JP) to obtain pDZ-dapA vectors.

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 '

To obtain the aceA gene promoter fragment of Corynebacterium glutamicum, primers were synthesized designed containing the SpeI restriction enzyme recognition site at the 5 ′ end and 3 ′ end of the fragment (SEQ ID NOs: 35 and 36). PCR was performed using Corynebacterium glutamicum KCCM11016P chromosomal DNA as a template and synthesized primers for amplification of the promoter region of approximately 500 base pairs represented by the nucleotide sequence of SEQ ID NO: 1. PCR amplification product and DNA fragment obtained by processing the vector pDZ-dapA enzyme restriction SpeI, cloned using the In-fusion Cloning Kit (TAKARA, JP) to obtain the vector pDZ-dapA-PaceA.

SEQ ID NO: 35:

PaceA-d3F 5'-acgttgatgcactagtagcactctgactacctctg-3 '

SEQ ID NO: 36:

PaceA-d3R 5'-agttctataaactagtttcctgtgcggtacgtggc-3 '

Example 13: Obtaining strains in which the aceA promoter was inserted below the dapA gene, and comparison of their production of threonine

To test the effect of inhibiting the expression of the dapA gene in the L-threonine-producing strain, the Corynebacterium glutamicum strain KCCM11222P (Korean Patent No .: 2013-0061570), which is the L-threonine-producing strain, was transformed using the pDZ-dapA-PaceA vector obtained in Example 12 in the same manner as in Example 3. By performing PCR, a strain was selected in which the aceA promoter was inserted below the stop codon of the dapA gene on the chromosome so that transcription can occur in the direction opposite to the original direction of transcription of the dapA gene, called KCCM11222P : : dapA-PaceA. The resulting strain KCCM11222P :: dapA-PaceA was checked by analyzing the nucleotide sequence of the obtained target region by PCR using SEQ ID NO: 31 and SEQ ID NO: 34 as primers.

The strain Corynebacterium glutamicum KCCM11222P used as the parent strain and the resulting strain KCCM11222P :: dapA-PaceA were cultured as described below.

Each of the strains was appropriately inoculated in 25 ml of medium for inoculation into flasks with 250 ml baffles, followed by cultivation with shaking at 200 rpm at 30 ° C for 20 hours. Then, 1 ml of the seed culture solution was added to a 250 ml flask with baffles containing 24 ml of production medium, followed by cultivation with shaking at 200 rpm at 30 ° C for 48 hours. The corresponding compositions of the medium for sowing and production environment are described below.

<Sowing 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 ⋅7 (H ₂ O) 0.5 g, biotin 100 μg , thiamine HCl 1000 μg, calcium pantothenate 2000 μg, nicotinamide 2000 μg (per 1 liter of distilled water).

<Production medium (pH 7.0)>

Glucose 100 g, (NH ₄ ) ₂ SO ₄ 20 g, soy protein 2.5 g, solid corn extract 5 g, urea 3 g, KH ₂ PO ₄ 1 g, MgSO ₄ ⋅ 7 (H ₂ O) 0.5 g, biotin 100 μg, thiamine HCl 1000 μg, calcium pantothenate 2000 μg, nicotinamide 3000 μg, CaCO ₃ 30 g (per 1 liter of distilled water).

After cultivation, the concentration of L-threonine in the culture solution was measured by HPLC. For cases where acetate was not added, the concentration of L-threonine in culture solutions of Corynebacterium glutamicum KCCM11222P and KCCM11222P :: dapA-PaceA is presented in table 11.

Table 11
Differences in L-threonine production (no acetate added) Strain Threonine (g / l) Party 1 Party 2 Party 3 KCCM11222P 7.0 6.9 7.2 KCCM11222P :: dapA-PaceA 7.1 7.3 7.0

In addition, the strains were cultured in the same way, except that 5 g / L acetate was added to the production medium to compare the production of L-threonine. The concentration of L-threonine in the culture solutions is presented in table 12.

Table 12
Differences in L-threonine production (5 g / L acetate was added) Strain Threonine (g / l) Party 1 Party 2 Party 3 KCCM11222P 7.6 7.4 7.7 KCCM11222P :: dapA-PaceA 11.2 11.5 11,4

As shown in table 11, in the absence of acetate, the production of L-threonine by strain KCCM11222P :: dapA-PaceA did not differ from the production by the parent strain KCCM11222P.

However, as shown in Table 12, in the presence of acetate, L-threonine production by strain KCCM11222P :: dapA-PaceA was 50% higher than that of the parent strain KCCM11222P.

[Registration No.]

Depository institution: Korean Culture Center of Microorganisms (International Depositary Authority)

Registration No: KCCM11432P

Registration Date: June 12, 2013

As described above, according to one or more of the above embodiments of the present invention, an acetate-inducible promoter can be used to efficiently produce L-amino acids, as the target L-amino acid can be obtained in high yield, weakening the expression of the target gene by adding acetate at the appropriate time.

It should be understood that the examples of embodiments presented in the present description, should be interpreted solely in a descriptive sense, and not for purposes of limitation. A description of the features or aspects in each embodiment should generally be considered suitable for other similar features and aspects in other embodiments.

Although one or more embodiments of the present invention are described with reference to the figures, those skilled in the art will understand that various changes in form and detail can be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims (8)

1. The method of obtaining L-lysine, including 1) cultivation of a recombinant coryneform microorganism capable of producing L-lysine, where the recombinant coryneform microorganism is transformed by insertion of an acetate-inducible promoter below the stop codon of the target gene on the chromosome; and 2) the addition of acetate during cultivation to weaken the expression of the target gene and enhance the ability of the recombinant coryneform microorganism to produce L-lysine, where the target gene is a gene located at the nodal point of the biosynthesis pathway of L-lysine, where the region below the stop codon is a region capable of attenuating the expression of the target gene by activating the acetate-induced promoter in the direction opposite to the direction of transcription of the target gene. 2. The method for producing L-lysine according to claim 1, wherein the region below the stop codon is between the stop codon and the region above the transcriptional terminator of the target gene. 3. The method for producing L-lysine according to claim 1, wherein the acetate-inducible promoter is represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 4. The method of producing L-lysine according to claim 1, wherein the target gene is at least one gene selected from the group consisting of a gene encoding the E1 subunit of pyruvate dehydrogenase, a gene encoding a homoserine dehydrogenase, and a gene encoding UDF-N-acetylmuramoylalanyl -D-glutamate-2,6-diaminopimelatligase.

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