RU2817768C2 - Microorganism having enhanced ability to produce l-amino acid with branched chain, and method of producing l-amino acid with branched chain using same - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a polynucleotide having promoter activity and containing a nucleotide substitution with another nucleotide in at least two or more corresponding positions selected from positions 34, 36, 37, 41 and 43 of the nucleotide sequence presented in SEQ ID NO: 1. Said polynucleotide comprises 34^th nucleotide substitution with T; replacement of 36^th nucleotide with T; replacing 37^th nucleotide with G; substitution of 41^st nucleotide with T; replacement of 43^rd nucleotide with A or a combination thereof in the nucleotide sequence presented in SEQ ID NO: 1. Also disclosed are a microorganism producing a branched L-amino acid, containing said polynucleotide, and a method of producing a branched L-amino acid using said microorganism.

EFFECT: invention enables to obtain a branched-chain L-amino acid with increased output.

8 cl, 10 tbl, 5 ex

Description

Technical field of the invention

The present invention relates to a branched chain L-amino acid producing microorganism having enhanced acetate metabolism regulator A activity, and a method for producing branched chain L-amino acid using it.

Prior Art

L-Amino acids, as the basic building blocks of proteins, are used as the main ingredients of pharmaceuticals, food additives, animal feed, nutritional supplements, pesticides, disinfectants, etc. In particular, branched chain amino acids (BCAAs), as an umbrella term for the essential amino acids L-valine, L-leucine and L-isoleucine, are known to have antioxidant actions and actions that directly promote protein synthesis in muscle cells.

However, the production of branched chain amino acids using microorganisms is mainly carried out using microorganisms of the genus Escherichia or microorganisms of the genus Corynebacterium, and it is known that branched chain amino acids are produced by biosynthesis using 2-ketoisocaproate as a precursor from pyruvic acid through various stages (US 10316297 B2, US 10526586 B2, and US 10072278 B2). However, the production of branched chain L-amino acids using microorganisms has the problem that mass production on an industrial scale is difficult.

Description of the invention

Technical problem

In this prior art, as a result of intensive attempts to enhance the ability to produce branched chain L-amino acids using microorganisms, the present inventors have discovered that the ability to produce branched chain L-amino acids is significantly increased by increasing the microorganisms' expression of acetate metabolism regulator A (hereinafter referred to as RamA), thus completing the present invention.

Technical solution

It is an object of the present invention to provide a branched chain L-amino acid producing microorganism having enhanced acetate metabolism regulator A activity.

Another object of the present invention is to provide a microorganism producing a branched chain L-amino acid comprising a polynucleotide having promoter activity and containing a substitution of a nucleotide for another nucleotide at one or more of the corresponding positions selected from the 34th positions , 36th, 37th, 41st and 43rd nucleotides of the nucleotide sequence shown in SEQ ID NO: 1.

Another object of the present invention is to provide a method for producing branched chain L-amino acid, where the method includes growing a microorganism in a culture medium.

Another object of the present invention is to provide a polynucleotide having promoter activity and containing a substitution of a nucleotide for another nucleotide at one or more of the corresponding positions selected from positions 34, 36, 37, 41 and 43 of the nucleotide sequence presented in SEQ ID NO: 1.

Beneficial Effects

Branched chain L-amino acids can be produced in high yields by growing a branched chain L-amino acid producing microorganism containing the polynucleotide of the present invention.

In addition, the amino acids obtained by the present invention can be used in various products, such as food, nutritional supplements or pharmaceutical drugs, as well as animal feed or animal feed additives.

Best Mode for Carrying Out the Invention

The present invention will be described in more detail. However, each description and embodiment disclosed in the present invention may be used in different descriptions and embodiments herein. Moreover, all combinations of the various components disclosed in the specification of the present invention are included within the scope of the specification of the present invention. Moreover, the scope of the present invention should not be limited by the descriptions given herein.

One skilled in the art will understand or be able to determine, using no more than experimentation, many equivalents to specific embodiments of the disclosure of the present invention. Such equivalents are intended to be included within the scope of the claims.

In one aspect of the present invention, there is provided a branched chain L-amino acid producing microorganism having enhanced acetate metabolism regulator A activity.

As used herein, the term "acetate metabolism regulator A" refers to a regulatory protein associated with acetic acid metabolism as a target protein as described herein, and may be encoded by the ramA gene.

In the present invention, the expression of acetate metabolism regulator A can be enhanced, and the enhanced expression can result in an increase in the ability to produce branched chain L-amino acid.

As used herein, the term "enhancing" the activity of acetate metabolism regulator A means that the activity of acetate metabolism regulator A is increased compared to the baseline activity. Enhancement can be used interchangeably with activation, up-regulation, overexpression, increase, etc. In this regard, activation, enhancement, upregulation, overexpression and enhancement can include all activities that exhibit activity that was not initially detected, or exhibit improved activity compared to the original activity or activity before modification. "Initial activity" refers to the activity of a particular polypeptide originally present in the parent strain or unmodified microorganism before transformation, when the microorganism is transformed by genetic modification caused by a natural or artificial factor. This term may be used interchangeably with the term "activity before modification". "Amplification", "up-regulation", "overexpression" or "increase" in the activity of a polypeptide compared to the original activity means that the activity and/or concentration (expression level) of a particular polypeptide is improved compared to the activity and/or concentration (expression level). ), originally present in the parent strain or unmodified microorganism before transformation.

Enhancement can be achieved by introducing a foreign polypeptide or by enhancing the activity and/or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of acetate metabolism regulator A is enhanced or not can be identified based on the improvement/increase in activity or expression level of the polypeptide or the amount of product released from the polypeptide.

Enhancement of the activity of acetate metabolism regulator A can be achieved by using various methods well known in the art, and these methods are not limited as long as the activity of the target polypeptide is enhanced relative to the activity of the microorganism before modification. In particular, any genetic engineering and/or protein engineering methods well known in the art can be used (for example, Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012).

In particular, enhancing the activity of acetate metabolism regulator A can be achieved by:

1) increasing the number of copies of the polynucleotide encoding the polypeptide in cells;

2) replacing the regulatory sequence of gene expression on the chromosome encoding the polypeptide with a sequence that improves the expression of the polypeptide, or introducing a modification into it;

3) modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

4) modification of the amino acid sequence of the polypeptide to enhance the activity of the polypeptide;

5) modifying the sequence of a polynucleotide encoding a polypeptide to enhance the activity of the polypeptide (for example, by modifying the nucleotide sequence of a polypeptide gene to encode a polypeptide modified such that it has enhanced activity);

6) introducing a foreign polypeptide having the activity of a polypeptide that encodes a foreign polynucleotide;

7) optimization of the codon of the polynucleotide encoding the polypeptide;

8) modification or chemical modification of the exposed region, selected by analysis of the three-dimensional structure of the polypeptide; or

9) any combination of two or more than two selected from, but not limited to, 1)-8) above.

Specifically, increasing the copy number of a polynucleotide encoding a polypeptide as described in 1) above can be achieved by introducing a vector that replicates and functions independently of the host cell and is operably linked to the polynucleotide encoding the polypeptide in the host cell. Alternatively, increasing the copy number can be achieved by introducing one copy or two or more than two copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. Introduction into a chromosome can be accomplished by introducing, but not limited to, a vector capable of inserting a polynucleotide into the chromosome of a host cell into the host cell. The vector is as described below.

Replacing a gene expression regulatory sequence (or expression regulatory region) on a chromosome encoding a polypeptide with a sequence that improves expression of the polypeptide, or introducing a modification thereto as described in 2) above, can be achieved, for example, by inducing a mutation in the sequence by deletion , insertion, non-conservative or conservative substitution, or any combination thereof, or by replacing a gene expression regulatory sequence with a sequence capable of improving expression of the polypeptide in order to further enhance the activity of the expression regulatory region. The expression regulating region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating transcription or translation termination, an enhancer, etc. The replacement can be carried out, in particular, but is not limited to, using a method of replacing an endogenous promoter with a strong heterologous promoter.

Examples of a strong promoter known in the art may include the CJ1-CJ7 promoters (US Patent No. US 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US Patent No. US 10584338 B2), 02 promoter (US Patent No. US 10273491 B2), tkt promoter and yccA promoter, but are not limited to.

Modification of the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide described in 3) above can be accomplished, for example, by replacing the nucleotide sequence with a nucleotide sequence encoding a different start codon having a higher level of expression of the polypeptide than the endogenous start codon, but is not limited to this.

Modification of the amino acid sequence or nucleotide sequence described in 4) and 5) above can be achieved by inducing a mutation in the amino acid sequence of the polypeptide of the nucleotide sequence encoding the polypeptide, by deletion, insertion, non-conservative or conservative substitution, or any combination thereof, or by replacing an amino acid sequence or nucleotide sequence with an amino acid sequence or nucleotide sequence modified to have greater activity, or an amino acid sequence or nucleotide sequence modified to increase activity, to further enhance the activity of the polypeptide, but not limited to. The replacement may be carried out, in particular, but is not limited to, by inserting the polynucleotide into the chromosome by homologous recombination. In this regard, the vector used for this purpose may additionally include a selection marker to confirm its integration into the chromosome. The selection marker will be described below.

Introduction of a foreign polypeptide having the activity of the polypeptide described in 6) above can be achieved by introducing into a cell a foreign polynucleotide encoding a polypeptide exhibiting activity identical/similar to that of the polypeptide. The origin or sequence of the foreign polynucleotide is not particularly limited as long as the foreign polynucleotide exhibits activity identical/similar to that of the polypeptide. The method used for administration can be suitably selected by one skilled in the art. Since the introduced polynucleotide is expressed in the host cell, a polypeptide is produced and its activity can be increased.

Codon optimization of a polynucleotide encoding a polypeptide described in 7) above can be achieved by codon optimization to enhance transcription or translation of an endogenous polynucleotide in a host cell, or by codon optimization to allow optimized transcription or translation of a foreign polynucleotide in a cell -the owner.

Modification or chemical modification of the exposed region selected by analyzing the three-dimensional structure of the polypeptide described in 8) above can be achieved, for example, by determining a candidate protein template according to the similarity between the sequences based on a comparison of the sequence information of the polypeptide being analyzed and a database, in which stores sequence information about existing proteins based on identification of their structure, selection of the exposed region to be modified or chemically modified, and modification or chemical modification of the exposed region.

An increase in the activity of acetate metabolism regulator A, as described above, may be an increase in the activity or concentration (level of expression) of the polypeptide compared to the activity or concentration of acetate metabolism regulator A expressed in wild-type or unmodified strains of microorganisms, or an increase in the amount of product derived from, but not limited to, a polypeptide.

More specifically, as used herein, the term “gene expression regulatory sequence,” used interchangeably with “gene expression regulatory region,” refers to a sequence operably linked to a target gene so as to express the target gene, and may include a modified polynucleotide according to with a description of the present invention. As described above, a gene expression regulatory sequence as described in the present invention may refer to a promoter, an enhancer, etc. to effect transcription of a gene, and may be a concept further comprising an operator sequence to control transcription, a sequence encoding a suitable mRNA ribosome binding site, and DNA regulating transcription and translation termination.

In one embodiment of the disclosure of the present invention, the gene expression control sequence may be a promoter, but is not limited to this.

In one embodiment of the disclosure of the present invention, expression of acetate metabolism regulator A may be enhanced by, but is not limited to, introducing a modification to the promoter or replacing the promoter with a promoter having stronger activity.

As used herein, the term "promoter" refers to an untranslated nucleotide sequence comprising a polymerase binding site located upstream of the coding region and having the activity of initiating transcription of a target gene into mRNA, i.e. a region of DNA that initiates gene transcription when a polymerase binds to it. The promoter may be located in the 5' region of the mRNA transcription initiation site.

As used herein, the term “operably linked” refers to functional linkage to a target gene sequence such that a polynucleotide having promoter activity in accordance with the present invention initiates and mediates transcription of the target gene. Functional linking can be carried out using a genetic recombination method known in the art, and site-specific DNA cleavage and ligation can be carried out using, but not limited to, a restriction enzyme, ligation, etc.

In the present invention, a target gene refers to a gene encoding a target protein whose expression is to be controlled in a microorganism, in particular, the gene may be, but is not limited to, a gene encoding acetate metabolism regulator A. More specifically, the gene may be, but is not limited to, a ramA gene.

In addition, the ramA gene may be an endogenous gene or a foreign gene, and may include a mutation to correct activity. The ramA gene sequence can be readily obtained from a known database such as the National Institutes of Health (U.S.A.) GenBank by those skilled in the art.

In yet another aspect of the present invention, there is provided a branched chain L-amino acid producing microorganism comprising a polynucleotide having promoter activity and comprising replacing the nucleotide with a different nucleotide at one or more of the corresponding positions selected from positions 34, 36, 37, 41 and 43 nucleotide sequence presented in SEQ ID NO: 1.

As used herein, the term "polynucleotide" refers to a DNA strand having a certain minimum length in the form of a polymer of nucleotides in which the nucleotide monomers are linked to each other in the form of a long chain through covalent bonds.

When considering the description of a “promoter,” the term “polynucleotide having promoter activity” may be used in the description of the present invention interchangeably with “modified polynucleotide,” “modified promoter,” or “modified ramA promoter,” and all of the above terms may be used herein.

In this regard, the term "modification" refers to a genetic or non-genetic stable change in phenotype, and may be used interchangeably herein with the term "mutation".

In particular, a modified polynucleotide as described herein may have modified (increased) promoter activity compared to an unmodified polynucleotide. Thus, the expression of the ramA gene, which is a target gene operably linked to the modified polynucleotide according to the description of the present invention, and the activity of the protein encoded by the ramA gene can be adjusted (increased), and the expression of other genes can also be adjusted, as well as the target gene.

In light of the objectives of the present invention, a polynucleotide having promoter activity refers to a polynucleotide capable of expressing a protein involved in increasing the production of amino acids, in particular branched chain amino acids, more specifically amino acids including, but not limited to, leucine, valine and isoleucine this.

As used herein, the term “polynucleotide sequence disclosed in SEQ ID NO: 1” may refer to the promoter sequence of the gene encoding acetate metabolism regulator A (RamA).

A polynucleotide having promoter activity as described herein is a modified polynucleotide having no natural sequence but having promoter activity and the expression of a target protein operably linked thereto can be increased compared to the polynucleotide sequence set forth in SEQ ID NO: 1.

In particular, the modified polynucleotide as described in the present invention may be a polynucleotide in which the nucleotide sequence shown in SEQ ID NO: 1, i.e. the promoter sequence of the ramA gene is modified, and at least one nucleotide selected from the 34th, 36th, 37th, 41st and 43rd nucleotides of the sequence may be replaced by a different nucleotide.

More specifically, the modified polynucleotide as described herein may include replacing the 34th nucleotide with T; replacement of the 36th nucleotide with T; replacement of the 37th nucleotide with G; replacement of the 41st nucleotide with T; replacement of the 43rd nucleotide with A; or any combination thereof in the nucleotide sequence set forth in, but not limited to, SEQ ID NO: 1. When modified, the polynucleotide as described herein may consist of a single nucleotide sequence selected from SEQ ID NO: 3-5.

In one embodiment, a modified polynucleotide in accordance with the description of the present invention may include replacing the 34th nucleotide with T; replacement of the 36th nucleotide with T; substitution of the 37th nucleotide with G in the nucleotide sequence presented in SEQ ID NO: 1. In this case, the modified polynucleotide in accordance with the description of the present invention may consist of SEQ ID NO: 5.

In yet another embodiment, a modified polynucleotide in accordance with the description of the present invention may include replacing the 41st nucleotide with T; and replacing the 43rd nucleotide with A in the nucleotide sequence shown in SEQ ID NO: 1. In this case, the modified polynucleotide in accordance with the description of the present invention may consist of SEQ ID NO: 4.

In yet another embodiment, a modified polynucleotide in accordance with the description of the present invention may include replacing the 34th nucleotide with T; replacement of the 36th nucleotide with T; replacement of the 37th nucleotide with G; replacement of the 41st nucleotide with T; and replacing the 43rd nucleotide with A in the nucleotide sequence shown in SEQ ID NO: 1. In this case, the modified polynucleotide in accordance with the description of the present invention may consist of SEQ ID NO: 3.

The microorganism producing the branched chain L-amino acid according to the present invention may be a microorganism comprising a polynucleotide that has promoter activity and includes the replacement of the 34th nucleotide with T; replacement of the 36th nucleotide with T; replacement of the 37th nucleotide with G; replacement of the 41st nucleotide with T; replacement of the 43rd nucleotide with A; or any combination thereof in the nucleotide sequence set forth in SEQ ID NO: 1.

In particular, the branched chain L-amino acid producing microorganism according to the present invention may be a microorganism comprising a polynucleotide having promoter activity and consisting of a single nucleotide sequence selected from SEQ ID NO: 3-5, or a nucleotide sequence having at least 80% or more and less than 100% sequence homology with it. The modified polynucleotide as described in the present invention will be described in more detail below.

As used herein, the term "branched chain amino acid" refers to an amino acid having a branched alkyl group on its side chain and includes valine, leucine and isoleucine. Specifically, in the present invention, the branched chain amino acid may be a branched chain L-amino acid, and the branched chain L-amino acid may be, but is not limited to, L-valine, L-leucine and L-isoleucine.

As used herein, the term "branched-chain amino acid-producing microorganism" includes all wild-type microorganisms and microorganisms in which natural or artificial genetic modification has been carried out, and may be a microorganism that includes genetic modification to produce a target branched-chain amino acid or having an improved activity, in which a particular mechanism is weakened or enhanced by introducing a foreign gene or enhancing or inactivating the activity of an endogenous gene. In the description of the present invention, "microorganism capable of producing branched chain L-amino acid" may be used interchangeably with "microorganism producing branched chain amino acid" and "microorganism having ability to produce branched chain amino acid".

In light of the objectives of the present invention, the microorganism may be any microorganism capable of producing a branched chain amino acid and comprising a modified polynucleotide as described in the present invention. In particular, the branched chain amino acid producing microorganism may be a microorganism characterized in that the ability to produce the target branched chain amino acid is increased by including a modified polynucleotide. Specifically, in the present invention, the branched-chain amino acid-producing microorganism or the microorganism having the ability to produce the branched-chain amino acid may be a microorganism in which some of the genes in the branched-chain amino acid biosynthetic pathway are enhanced or weakened, or a microorganism in which some of the genes in the branched chain amino acid degradation pathway are upregulated or downregulated, but are not limited to.

In an embodiment in the description of the present invention, a microorganism of the genus Corynebacterium having the ability to produce a branched chain amino acid may be a microorganism of the genus Corynebacterium including a modified polynucleotide in accordance with the description of the present invention, or transformed with a vector including a gene encoding a polynucleotide in accordance with the description of the present invention , having an improved ability to produce branched chain amino acids. “A microorganism of the genus Corynebacterium having an enhanced ability to produce a branched-chain amino acid” refers to a microorganism having an enhanced ability to produce a branched-chain amino acid compared to the amino acid-producing ability of a parent strain before transformation or an unmodified microorganism. "Unmodified microorganism" does not exclude strains having a mutation that may occur naturally in microorganisms and refers to a microorganism not comprising a polynucleotide as described herein or a microorganism not transfected with a vector comprising a polynucleotide as described herein. .

The microorganism of the genus Corynebacterium may specifically include, but is not limited to, Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes, Corynebacterium efficiens, Corynebacterium stationis, etc.

As used herein, the term "vector" refers to an artificial DNA molecule comprising genetic material expressing a target polypeptide in a suitable host cell, particularly a DNA construct comprising the nucleotide sequence of a polynucleotide encoding the target polypeptide and operably linked to a suitable regulatory region expression capable of expressing the target gene. Once a suitable host cell is transformed with a vector, the vector can replicate or function independently of the host genome or can be integrated into the genome.

The vector used in the present invention is not particularly limited, and any vector known in the art can be used. Examples of common vectors may include natural or recombinant plasmid, cosmid, virus and bacteriophage. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or cosmid vector. .d. Vectors based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. can be used as a plasmid vector. In particular, the vectors pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118 and pCC1BAC can be used.

For example, the target polynucleotide can be inserted into a chromosome using a chromosome insertion vector. Incorporation of a polynucleotide into a chromosome can be accomplished using any of the methods known in the art, such as, but not limited to, homologous recombination. The polynucleotide may further include a selection marker to confirm chromosomal integration. The selection marker is used to select cells transformed with the vector, i.e. to identify whether a target nucleic acid molecule is integrated or not, and markers conferring selective phenotypes such as drug resistance, nutrient requirements, resistance to cytotoxic agents or surface polypeptide expression of proteins can be used. In a medium treated with a selection agent, transformed cells can be selected because only cells expressing the selection marker are able to survive or exhibit different phenotypes.

As used herein, the term “transformation” refers to the introduction of a vector comprising a target polynucleotide into a host cell or microorganism such that the polynucleotide is expressed in the host cell. The transformed polynucleotide may be present in a form integrated into the chromosome of the host cell, or in a form located outside the chromosome, provided that the polypeptide is expressed in the host cell. In addition, the polynucleotide may include DNA and RNA encoding the target polypeptide. The polynucleotide can be introduced into a host cell in any form, provided that the polynucleotide can be introduced into and expressed in the host cell. For example, the polynucleotide can be introduced into a host cell in the form of an expression cassette, which is a genetic construct that includes all the elements necessary for self-replication. Typically, the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into a host cell in its original form and is operably linked to, but is not limited to, a sequence required for expression in the host cell.

In yet another aspect of the present invention, there is provided a method for producing a branched chain L-amino acid, comprising culturing a microorganism in a culture medium.

In addition, this method of producing a branched chain L-amino acid may further include isolating or separating the target substance from the culture medium or microorganism.

“Polynucleotide”, “microorganism of the genus Corynebacterium”, “vector” and “branched chain L-amino acid” are described above.

As used herein, the term "cultivation" refers to growing a microorganism as described herein in an appropriately selected medium. The culture process in accordance with the description of the present invention can be carried out using suitable culture media and culture conditions well known in the art. The cultivation process can be suitably adjusted by one skilled in the art according to the selected strain. In particular, the cultivation can be carried out using a batch culture method, a continuous culture method and a fed culture method, but is not limited to it.

As used herein, the term "culture medium" refers to a substance in which the essential elements are mixed with the nutrients required for the growth of a microorganism as described herein and the amount of nutrients and growth factors, as well as water, that are necessary for life and growth. . In particular, although the culture media and other conditions for growing a microorganism of the genus Corynebacterium in accordance with the description of the present invention are not particularly limited, provided that these media are generally used in the cultivation of microorganisms, the microorganism in accordance with the present invention can be grown in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and/or vitamins, under aerobic conditions when temperature, pH, etc. are adjusted.

In particular, the culture medium for the microorganism of the genus Corynebacterium is disclosed in the document ("Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., U.S.A., 1981)).

In the present invention, carbohydrates such as glucose, sucrose, lactose, fructose and maltose can be used as carbon sources; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid and citric acid and amino acids such as glutamic acid, methionine and lysine. In addition, natural organic nutrients such as starch hydrolysates, molasses, raw molasses, rice bran, cassava, sugar cane pomace and liquid corn extract may be used, and in particular carbohydrates such as glucose and sterile pre-processed molasses may be used. (ie, molasses converted to reduced sugars), and suitable amounts of any other carbon sources, may also be used without limitation. These carbon sources can be used alone or in combination, but are not limited to, at least two of them.

As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used; and organic nitrogen sources such as amino acids, e.g. glutamic acid, methionine and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, liquid corn extract, casein hydrolysate, fish or its degradation products, and defatted soybean meal or its degradation products. These nitrogen sources may be used alone or in combination with, but not limited to, at least two of them.

As sources of phosphorus, potassium dihydrogen phosphate, potassium hydrogen phosphate or corresponding sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, calcium carbonate, etc. can be used. In addition, amino acids, vitamins and/or corresponding precursors may be further included. These components or precursors may be added to the culture medium in batches or through a continuous process, but are not limited to this.

Moreover, during the microorganism cultivation process of the present invention, the pH value of the culture medium can be adjusted by suitably adding a compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid. Moreover, during the culture process, foam formation can be prevented by using an antifoaming agent such as a polyglycol fatty acid ester. In addition, oxygen or an oxygen-containing gas may be introduced into the culture medium to maintain the culture medium under aerobic conditions, or nitrogen, hydrogen, or carbon dioxide may be introduced into the culture medium to maintain the culture medium under anaerobic and microaerobic conditions without introducing any other gases, but not limited to this.

In the present invention, the culture temperature can be maintained at 20°C to 45°C, in particular 25°C to 40°C, and the culture can be carried out for, but not limited to, about 10 to 160 hours.

The branched chain L-amino acid produced by culture according to the present invention can be released into the culture medium or remain in the cells.

The method for producing a branched chain L-amino acid according to the present invention may further include obtaining a microorganism according to the present invention, obtaining a culture medium for growing the strain, or any combination thereof (regardless of the sequence, in any sequence), for example, before culturing.

The method for producing branched chain L-amino acid in accordance with the present invention may further include isolating the branched chain L-amino acid from a culture medium (in which the growth was carried out) or a microorganism in accordance with the description of the present invention. The isolation step may further be carried out after the cultivation process.

The isolation step can be carried out by collecting the target branched chain L-amino acid using a suitable method known in the art in accordance with the microorganism cultivation methods of the present invention, such as a batch culture method, a continuous culture method, or a fed-batch culture method. For example, centrifugation, filtration, treatment with a protein precipitating agent (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographic methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography can be used. chromatography, HPLC (high performance liquid chromatography), SMB (pseudo moving bed chromatography) and any combination thereof. The desired branched chain L-amino acid target can be isolated from the culture medium or microorganism using any suitable method known in the art.

In addition, the method for producing branched chain L-amino acid according to the present invention may further include a purification step. Purification can be accomplished using a suitable method known in the art. As an example, when the method for producing branched chain L-amino acid according to the present invention includes an isolation step and a purification step, then the isolation step and the purification step can be carried out continuously or intermittently regardless of the sequence, or can be carried out simultaneously or as one combined stage, but not limited to this.

In another aspect of the present invention, there is provided a polynucleotide having promoter activity and comprising a substitution of at least one nucleotide selected from the 34th, 36th, 37th, 41st and 43rd nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, to another nucleotide.

"Polynucleotide" and "promoter" are as described above.

In addition, the sequence of a modified polynucleotide as described herein may be modified by mutagenesis well known in the art, such as directed evolution and site-directed mutagenesis.

Thus, the modified polynucleotide in accordance with the present invention may include a polynucleotide having a nucleotide sequence in which the 34th base is fixed as T; The 36th base is fixed as T; The 37th base is fixed as G; The 41st base is fixed as T; and the 43rd base is fixed as A, and another portion of the nucleotide sequence has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the nucleotide sequence SEQ ID NO: 3.

In addition, the modified polynucleotide in accordance with the present invention may include a polynucleotide having a nucleotide sequence in which the 41st base is fixed as T; and the 43rd base is fixed as A, and another portion of the nucleotide sequence has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with the nucleotide sequence SEQ ID NO: 4.

In addition, the modified polynucleotide as described in the present invention may include a polynucleotide having a nucleotide sequence in which the 34th base is fixed as T; The 36th base is fixed as T; and the 37th base is fixed as G and the other portion of the sequence has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to the nucleotide sequence of SEQ ID NO: 5.

In this case, the nucleotide sequence having homology or identity may exclude a sequence having 100% identity, or may be a sequence having less than 100% identity.

It is clear that any polynucleotide having a nucleotide sequence including the deletion, modification, substitution or insertion of one or more nucleotides other than the 34th, 36th, 37th, 41st or 43rd position is included in the scope of the present invention, provided that the nucleotide sequence retains homology and biological activity identical or equivalent to the homology and biological activity of at least one nucleotide sequence selected from SEQ ID NO: 3-5.

As used herein, the term “homology” or “identity” refers to the degree of similarity between two given amino acid sequences or nucleotide sequences, and may be expressed as a percentage. Often the terms homology and identity can be used interchangeably.

The homology or sequence identity of conserved polynucleotides or polypeptides can be determined by a standard alignment algorithm and can be coupled with a sequence gap penalty set by default in the program being used. Essentially homologous or identical sequences can generally hybridize to each other in whole or in part under moderately stringent or very stringent conditions. It is apparent that hybridization involves the hybridization of a polynucleotide with a polynucleotide comprising a common codon or a degenerate codon.

Homology, similarity or identity between two polynucleotide or polypeptide sequences can be determined using any computer algorithm known in the art, for example, the "FASTA" program, using the default parameters introduced by Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, homology, similarity or identity can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) built into the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (including the GCG software package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984) )), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073). For example, homology, similarity or identity can be determined using the National Center for Biotechnology Information BLAST or ClustalW database.

Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the computer program GAP, as introduced by Needleman et al., (1970), J Mol Biol. 48:443, as disclosed by Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program determines similarity by the number of aligned identical characters (i.e., nucleotides or amino acids) divided by the total number of characters in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (containing a value of 1 if identical and 0 if not identical) and a weighted comparison matrix according to Gribskov, et al. (1986), Nucl. Acids Res. 14:6745, as described in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence and Structure, National Biomedical Research Foundation, pp.353-358 (1979) (or EDNAFULL (EMBOSS NCBI NUC4.4 version); (2) penalty 3.0 for each gap and an additional penalty of 0.10 for each character in each gap (or alternatively, penalty for entering a gap of 10, penalty for extending a gap of 0.5) and (3) no penalty for ending a gap.

In addition, a modified polynucleotide as described herein may include various modifications in the coding region provided not to change the nucleotide sequence due to codon degeneracy or to take into account codon preferences of the living organism in which the polynucleotide is expressed. In addition, the polynucleotide may include any nucleotide sequence having promoter activity and hybridized to a probe designed using a known genetic sequence, for example, a nucleotide sequence that is fully or partially complementary to the nucleotide sequence under stringent conditions, including, but not limited to, substitution of at least one nucleotide in nucleotide sequence SEQ ID NO: 1.

The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are disclosed in detail in known documents (eg, J. Sambrook et al.). For example, the conditions may include hybridizing between genes having high homology, for example, 40% homology or greater, in particular 70% or greater, 80% or greater, 85% or greater, 90% or greater, more particularly 95% or more, more specifically, 97% or more, and most specifically, 99% or more, without hybridizing between genes having homology or identity less than that described above, or performing hybridization once, in particular two or three times, under normal washing conditions for Southern hybridization at salt concentration and temperature 60°C, lxSSC, and 0.1% SDS (sodium dodecyl sulfate), in particular 60°C, 0.1xSSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, and 0.1% SDS.

Hybridization requires that two nucleic acids have complementary sequences, even though there is a mismatch between the bases according to the degree of hybridization stringency. The term "complementary" is used to describe the interaction between nucleotide bases that are capable of hybridizing with each other. For example, in relation to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the description of the present invention may include not only substantially similar nucleic acid sequences, but also an isolated nucleic acid fragment that is complementary to the full-length sequence.

In particular, polynucleotides having homology or identity can be detected using the above-described hybridization conditions, including a hybridization process at a Tm value of 55°C. In addition, the Tm value may be 60°C, 63°C, or 65°C, but not limited to, and can be adjusted accordingly by one skilled in the art according to the intended application. The appropriate degree of stringency of polynucleotide hybridization may depend on the length of the polynucleotides and the degree of complementarity and parameters well known in the art (Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

In particular, the expression "the modified polynucleotide consists of one nucleotide sequence selected from SEQ ID NO: 3-5, or a nucleotide sequence having at least 80% or greater and less than 100% sequence homology" does not exclude insertion and/or deletion , and/or mutation of the nucleotide, which may occur during ligation with the target gene, for example, using a restriction enzyme, in the case where the polynucleotide is used in a state ligated with the target gene as a promoter.

For example, a polynucleotide consisting of one nucleotide sequence selected from SEQ ID NO: 3-5 and having promoter activity may also include a polynucleotide hybridized in full or partially complementary manner to one nucleotide sequence selected from SEQ ID NO: 3-5 in under strict conditions, having promoter activity in accordance with the description of the present invention.

A microorganism comprising a modified polynucleotide as described herein is characterized in that the production of a branched chain amino acid including valine, leucine or isoleucine is increased. Although wild-type strains belonging to the genus Corynebacterium cannot produce branched-chain amino acids or produce them in trace amounts, a polynucleotide having promoter activity as described in the present invention is significant because its use increases the production of branched-chain amino acids. chain.

Method according to the invention

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are provided merely to provide examples of the description of the present invention, and the scope of the description of the present invention is not limited to them.

Example 1. Selection of mutant strains with enhanced ability to produce valine by artificial mutagenesis

Example 1-1. Induction of artificial mutation using UV radiation

In order to select a mutant strain having an enhanced ability to produce valine, which is a typical branched-chain amino acid, Corynebacterium glutamicum KCCM11201P (Korean Patent No. 10-1117022) as a valine-producing strain was spread on an agar-containing nutrient medium and grown at 30 °C for 36 hours. Several hundred colonies were selected from it and exposed to UV radiation at room temperature in order to cause a random mutation in the genomes of the strains.

Example 1-2. Assessment of fermentation titer and selection of a strain with an induced mutation

In order to select a mutant strain having an enhanced ability to produce L-valine compared to Corynebacterium glutamicum KCCM11201P used as the parent strain, fermentation titer testing was carried out on strains in which a random mutation was induced. After subculturing each of the colonies in a nutrient medium, each strain was inoculated into a 250 ml flask with corner partitions containing 25 ml of production medium and grown with stirring at 30°C for 72 hours at 200 rpm. L-valine concentrations were then analyzed using HPLC, and the L-valine concentrations analyzed are presented in Table 1 below.

Nutrient medium (pH 7.2)

10 g glucose, 5 g meat broth, 10 g polypeptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (based on 1 liter of distilled water).

Production medium (pH 7.0)

100 g glucose, 40 g ammonium sulfate, 2.5 g soy protein, 5 g corn extract, 3 g urea, 1 g dibasic potassium phosphate, 0.5 g magnesium sulfate heptahydrate, 100 mcg biotin, 1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide and 30 g calcium carbonate (based on 1 liter of distilled water).

Based on comparison with strain KCCM11201P, used as a control, strain A7 was selected as having the greatest increase in valine production (see Table 1).

Example 2: Confirmation of mutation by gene sequencing

The main genes of strain A7, which has an enhanced ability to produce valine, were sequenced and compared with the genes of strain KCCM11201P and Corynebacterium glutamicum strain ATCC14067. As a result, it was confirmed that strain A7 contains a mutation in the promoter position of the regulator of acetate metabolism A.

In particular, it was confirmed that strain A7 has the nucleotide sequence SEQ ID NO: 2, including a mutation in the promoter region (SEQ ID NO: 1) of the ramA gene.

In the following examples, the effects of modification introduced at a specific position in the promoter region of the ramA gene and the effects of enhancing RamA expression by improving or replacing the ramA gene promoter to produce valine, isoleucine and leucine, which are branched chain amino acids, in a microorganism of the genus Corynebacterium were examined.

Example 3. Construction of a modified strain and confirmation of the ability to produce valine

Example 3-1. Introduction of a modified promoter into the Corynebacterium glutamicum strain KCCM11201P and assessment of the ability to produce L-valine

In order to introduce the modified promoter polynucleotide shown in SEQ ID NO: 2 into the ramA gene in Corynebacterium glutamicum KCCM11201P, a vector including the desired modification was prepared. Specifically, genomic DNA of strain A7 was extracted using the G-spin Total DNA Extraction Mini Kit (Intron, catalog no. 17045) according to the kit protocols, and PCR (polymerase chain reaction) was performed using the genomic DNA as a template. Under the following conditions: denaturation at 94°C for 5 minutes; 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 150 seconds; and then polymerization at 72°C for 7 minutes, and a PCR product (hereinafter referred to as “modified fragment 1”) of 1114 bp in length. prepared using SEQ ID NOs: 9 and 10.

After treating the resulting modified fragment 1 with the restriction enzyme XbaI (New England Biolabs, Beverly, MA), the modified fragment 1 was ligated with the pDZ vector (Korean Patent No. 10-0924065 and International Patent Application Publication No. 2008-033001) treated with the same restriction enzyme using T4 ligase (New England Biolabs, Beverly, MA). After transformation of E. coli DH5α with the constructed gene, the transformed strains were selected in LB (Luria-Bertani) medium containing kanamycin, and DNA from them was obtained using the DNA-spin plasmid DNA purification kit (iNtRON) to obtain the vector pDZ-Pm-ramA, including fragment 1 with the introduced modification.

Corynebacterium glutamicum KCCM1120IP was transformed with the pDZ-Pm-ramA vector by chromosomal homologous recombination (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector was introduced into the chromosome by homologous sequence recombination were selected in a culture medium containing kanamycin (25 mg/l). PCR was then performed on Corynebacterium glutamicum transformants in which secondary recombination had been completed using SEQ ID NOs: 9 and 10, and strains in which a modification had been introduced into the promoter in the region upstream of ramA were confirmed (SEQ ID NO: 1) on the chromosome. The recombinant strain was named Corynebacterium glutamicum KCCM11201P-Pm-ramA.

To compare the ability to produce valine between valine-producing Corynebacterium glutamicum KCCM11201P and KCCM11201P-Pm-ramA, flask evaluation was performed. After subculturing each of the strains in a nutrient medium, each of the strains was inoculated into a 250 ml corner flask containing 25 ml of production medium and grown with stirring at 30°C for 72 hours at 200 rpm. L-valine concentrations were then analyzed using HPLC, and the L-valine concentrations analyzed are presented in Table 3 below.

Nutrient medium (pH 7.2)

10 g glucose, 5 g meat broth, 10 g polypeptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (based on 1 liter of distilled water).

Production medium (pH 7.0)

100 g glucose, 40 g ammonium sulfate, 2.5 g soy protein, 5 g corn extract, 3 g urea, 1 g dibasic potassium phosphate, 0.5 g magnesium sulfate heptahydrate, 100 mcg biotin, 1 mg thiamine-HCl, 2 mg calcium pantothenate, 3 mg nicotinamide, 30 g calcium carbonate (based on 1 liter of distilled water).

As a result, it was confirmed that the L-valine production ability of strain KCCM11201P-Pm-ramA was enhanced by approximately 23% compared with KCCM11201P.

Example 3-2. Construction of a mutant strain of Corynebacterium Glutamicum KCCM11201P, in which the promoter is improved and replaced, and evaluation of the ability of the constructed strain to produce L-valine

According to the results of Example 3-1 above, it was confirmed that the ability to produce valine was enhanced by modifying the promoter of the ramA gene, and thus vectors for improving or replacing the ramA promoter were constructed based on the modified promoter in accordance with SEQ ID NO: 2 to further increase ramA expression.

In order to construct vectors including the modification, primer 3 (SEQ ID NO: 11) - primer 10 (SEQ ID NO: 18) in Table 4 was synthesized such that they contain a region for the xbaI restriction enzyme at the 5' end and 3' end.

The improved ramA promoters were named Pm1, Pm2 and Pm3-ramA, and a pair of primers according to SEQ ID NOs: 11 and 13 and a pair of primers according to SEQ ID NOs: 12 and 14 were used to construct Pm1-ramA, and a pair of primers in according to SEQ ID NO: 11 and 15 and a pair of primers according to SEQ ID NO: 12 and 14 were used to construct Pm2-ramA. In addition, a pair of primers in accordance with SEQ ID NO: 11 and 17; and a pair of primers according to SEQ ID NO: 12 and 18 were used to construct Pm3-ramA.

PCR was performed using each of the primers and wild-type Corynebacterium glutamicum chromosomal DNA as a template [Sambrook et al., Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories].

In this case, PCR was carried out under the following conditions: denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, and polymerization at 72°C for 1 minute; and then polymerization at 72°C for 7 minutes.

Then, the PCR product obtained by the above process and the previously obtained vector pDZ-Pm-ramA were treated with the restriction enzyme xbaI, and then subjected to seamless cloning. Seamless cloning was performed using the In-Fusion® HD Cloning Kit (Clontech). E. coli DH5α was transformed with it and applied to solid LB medium containing kanamycin (25 mg/L). Colonies transformed with plasmids into which the target gene was introduced were selected by PCR, and the plasmids were obtained by extraction and named pDZ-Pm1-ramA, pDZ-Pm2-ramA and pDZ-Pm3-ramA, respectively.

In addition, separately, in order to replace the ramA promoter with Pcj7, which is a stronger promoter, primer 11 (SEQ ID NO: 19) - primer 16 (SEQ ID NO: 24) in Table 4, having a region for a restriction enzyme, was synthesized xbaI at the 5' end and 3' end.

The pDZ-Pcj7-ramA vector was constructed in the same manner as the vector construction method in Example 3-1 described above, using a pair of primers according to SEQ ID NO: 19 and 20; primer pairs according to SEQ ID NO: 21 and 22; and primer pairs according to SEQ ID NO: 23 and 24.

Corynebacterium glutamicum KCCM11201P was transformed with the vectors pDZ-Pm1-ramA, pDZ-Pm2-ramA, pDZ-Pm3-ramA and pDZ-Pcj7-ramA by chromosomal homologous recombination (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999 ). Strains in which the vector was introduced into the chromosome by homologous sequence recombination were selected in a culture medium containing kanamycin (25 mg/l). PCR was then performed using Corynebacterium glutamicum transformants in which secondary recombination was completed using SEQ ID NOs: 9 and 10, and strains in which the ramA promoter had been improved and replaced with the Pcj7 promoter were confirmed.

Among the recombinant Corynebacterium glutamicum strains, KCCM11201P-Pm1-ramA, KCCM11201P-Pm2-ramA and KCCM11201P-Pm3-ramA were named CA08-1518, CA08-1519 and CA08-1520, respectively, and deposited in the Korean Center for Culture of Microorganisms (KCCM), recognized as an international depositary in accordance with the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure on April 27, 2020 under the numbers KCCM12704P, KCCM12705P and KCCM12706P respectively.

In addition, the Pcj7 promoter replacement strain was named KCCM11201P-Pcj7-ramA. The ability to produce valine was then assessed in the same manner as in Example 3-1 above, and the results are presented in Table 5 below.

Based on the results in Table 5, it was confirmed that strains KCCM11201P-Pm1-ramA (CA08-1518), KCCM11201P-Pm2-ramA (CA08-1519) and KCCM11201P-Pm3-ramA (CA08-1520), including an improved promoter compared to the strain KCCM11201P exhibited an increase in L-valine production of approximately 27%, 23%, and 19%, respectively, which was similar to or greater than the ability of strain KCCM11201P-Pcj7-ramA to produce L-valine in which the promoter was replaced with a stronger one.

Example 3-3: Construction of a mutant strain of Corynebacterium glutamicum CJ7V in which the promoter of the ramA gene was improved and replaced, and evaluation of the ability of the constructed strain to produce L-valine

In order to identify whether the effect of enhancing the ability to produce L-valine is achieved in other L-valine-producing strains related to Corynebacterium glutamicum, one type of modification [ilvN(A42V); Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp 456-467] to obtain a strain with enhanced ability to produce L-valine.

Specifically, genomic DNA of the wild-type Corynebacterium glutamicum strain ATCC14067 was extracted using the G-spin Total DNA Extraction Mini Kit (Intron, catalog #17045) according to the protocols in the kit. PCR was performed using genomic DNA as a template. In order to construct a vector for introducing the A42V modification into the ilvN gene, fragments A and B of the gene were obtained using, respectively, a pair of primers in accordance with SEQ ID NO: 25 and 26; and primer pairs according to SEQ ID NO: 27 and 28. PCR was carried out under the following conditions: denaturation at 94°C for 5 minutes; 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds and polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 7 minutes.

The result was fragments A and B of the polynucleotide, both of which included 537 bp. PCR product 1044 bp long. (hereinafter referred to as “modified fragment 2”) was obtained by performing PCR with overlapping primers using the two fragments as templates according to SEQ ID NOs: 25 and 26.

After treating the resulting fragment 2 with the introduced modification with the restriction enzyme XbaI (New England Biolabs, Beverly, MA), fragment 2 with the introduced modification was ligated with the pDZ vector treated with the same restriction enzyme using T4 ligase (New England Biolabs, Beverly, MA) . After transformation of E. coli DH5α with the engineered gene, transformed strains were selected in LB medium containing kanamycin, and DNA was obtained from it using the DNA-spin plasmid DNA purification kit (iNtRON). The vector used to introduce the A42V modification into the ilvN gene was named pDZ-ilvN(A42V).

Wild-type Corynebacterium glutamicum ATCC14067 was then transformed with the vector pDZ-ilvN(A42V) by chromosomal homologous recombination (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector was introduced into the chromosome by homologous recombination, the sequences were selected in a culture medium containing kanamycin (25 mg/l). The gene fragments were then amplified by PCR performed on Corynebacterium glutamicum transformants in which secondary recombination had been completed using SEQ ID NOs: 25 and 26, and the strains into which the modification had been introduced were confirmed by gene sequencing. The recombinant strain was named Corynebacterium glutamicum CJ7V.

Finally, Corynebacterium glutamicum CJ7V was transformed with vectors in the same manner as in Examples 3-1 and 3-2, and the strains were named Corynebacterium glutamicum CJ7V-Pm1-ramA, CJ7V-Pm2-ramA, CJ7V-Pm3-ramA, and CJ7V, respectively. -Pcj7-ramA. To compare the L-valine production ability of the engineered strains, they were grown in the same manner as in Example 3-1 above, the concentrations of L-valine were analyzed, and the analyzed concentrations of L-valine are presented in Table 7 below.

According to the data presented in Table 7, it was confirmed that strains CJ7V-Pm1-ramA, CJ7V-Pm2-ramA and CJ7V-Pm3-ramA, including an improved promoter, showed an increase in L-valine production by approximately 23%, 18%, respectively. and 14%, which was similar to or greater than the ability of strain CJ7V-Pcj7-ramA to produce L-valine, in which the promoter was replaced with a stronger one.

Example 3-4: Construction of a mutant strain of Corynebacterium glutamicum CJ8V in which the promoter of the ramA gene is improved and replaced, and evaluation of the constructed strain to produce L-valine

In order to identify whether the effect of improving the ability to produce L-valine was achieved by other L-valine-producing strains belonging to Corynebacterium glutamicum, one type of modification [ilvN(A42V)] was introduced into wild-type Corynebacterium glutamicum ATCC13869 in the same manner, as in the method according to Example 3-3 for obtaining a strain having the ability to produce L-valine, and the recombinant strain was named Corynebacterium glutamicum CJ8V.

Finally, Corynebacterium glutamicum CJ8V was transformed with vectors in the same way as in the method in Examples 3-1 and 3-2, and the strains were named Corynebacterium glutamicum CJ8V-Pm1-ramA, CJ8V-Pm2-ramA, CJ8V-Pm3-ramA, respectively and CJ8V-Pcj7-ramA. To compare the ability to produce L-valine between the engineered strains, they were grown in the same manner as in Example 3-1 above, the concentrations of L-valine were analyzed, and the analyzed concentrations of L-valine are presented in Table 8 below.

According to the data presented in Table 8, it was confirmed that strains CJ8V-Pm1-ramA, CJ8V-Pm2-ramA and CJ8V-Pm3-ramA, including an improved promoter compared to strain CJ8V, showed an increase in L-valine production, respectively, approximately by 21%, 16% and 10%, which was similar to or greater than the ability of the CJ8V-Pcj7-ramA strain to produce L-valine, in which the promoter was replaced with a stronger one.

Example 4. Construction of mutant strains of Corynebacterium glutamicum KCCM11661P and KCCM11662P producing L-leucine, into which a modification of the promoter was introduced, and evaluation of the ability to produce L-leucine

Corynebacterium glutamicum KCCM11661P and KCCM11662P were transformed with the vectors pDZ-Pm1-ramA, pDZ-Pm2-ramA, pDZ-Pm3-ramA and pDZ-Pcj7-ramA by chromosomal homologous recombination (van der Rest et al., Appl Microbiol Biotechnol 52:541 545, 1999). Strains in which the vector was introduced into the chromosome by homologous sequence recombination were selected in a culture medium containing kanamycin (25 mg/l). PCR was then performed on Corynebacterium glutamicum transformants in which secondary recombination had been completed using SEQ ID NOs: 9 and 10, and strains in which the ramA promoter had been improved and replaced with Pcj7 were confirmed. The recombinant strains were named, respectively, Corynebacterium glutamicum KCCM1166lP-Pm1-ramA, KCCM1166IP Pm2-ramA, K KCCM11661P-Pm3-ramA, KCCM1166lP-Pcj7-ramA and KCCM11662P-Pm1-ramA, KCCM11662P Pm2-ramA, KCC M11662P-Pm3-ramA and KCCM11662P-Pcj7-ramA.

The constructed strains were grown according to the following method and the ability to produce leucine was compared.

After subculturing each strain in a nutrient medium, each strain was inoculated into a 250 ml corner flask containing 25 ml of production medium and grown with stirring at 30°C for 72 hours at 200 rpm. Next, L-leucine concentrations were analyzed using HPLC, and the analyzed L-leucine concentrations are presented in Table 9 below.

<Nutritional medium (pH 7.2)>

10 g glucose, 5 g meat broth, 10 g polypeptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, 2 g urea (based on 1 liter of distilled water)

<Producing medium (pH 7.0)>

50 g glucose, 20 g ammonium sulfate, 20 g corn extract, 1 g dibasic potassium phosphate, 0.5 g magnesium sulfate heptahydrate, 100 mcg biotin, 1 mg thiamine-HCl and 15 g calcium carbonate (based on 1 L distilled water)

The results confirmed that strains KCCM1166lP-Pm1-ramA, KCCM11661P Pm2-ramA and KCCM11661P-Pm3-ramA, including an improved promoter, showed an increase in L-leucine production, respectively, by 11%, 7% and 11% compared to strain KCCM11661P , which was similar to or greater than the ability of strain KCCM1166lP-Pcj7-ramA to produce L-leucine, in which the promoter was replaced with a stronger one.

In addition, it was confirmed that strains KCCM11662P-Pm1-ramA, KCCM11662P Pm2-ramA and KCCM11662P-Pm3-ramA, including the improved strain, showed an increase in L-leucine production, respectively, by 10%, 6% and 10% compared to the strain KCCM11662P, which was similar to or greater than the ability of strain KCCM11662P-Pcj7-ramA to produce L-leucine, in which the promoter was replaced with a stronger one.

Example 5: Construction of the L-isoleucine-producing mutant strain Corynebacterium glutamicum KCCM11248P, in which the ramA gene promoter is improved and replaced, and evaluation of the ability to produce L-isoleucine

In order to identify whether the L-isoleucine-producing ability-improving effect is achieved in other L-isoleucine-producing strains related to Corynebacterium glutamicum, the L-isoleucine-producing Corynebacterium glutamicum strain KCCM11248P was transformed with vectors in the same manner as in Examples 3-1 and 3-2 above, and the transformed strains were named Corynebacterium glutamicum KCCM11248P-Pm-ramA, KCCM11248P-Pm1-ramA, KCCM11248P-Pm2-ramA, KCCM11248P-Pm3-ramA and KCCM11248P-Pcj7-ramA, respectively. Strains KCCM11248P-Pm-ramA, KCCM11248P-Pm1-ramA, KCCM11248P-Pm2-ramA, KCCM11248P-Pm3-ramA and KCCM11248P-Pcj7-ramA were grown according to the following method, and the ability to produce isoleucine was assessed.

Each strain was inoculated into a 250 ml corner flask containing 25 ml of inoculation medium and grown with stirring at 30°C for 20 hours at 200 rpm. Then, 1 ml of inoculation medium was inoculated into a 250 ml corner flask containing 24 ml of production medium and grown with stirring at 30°C for 48 hours at 200 rpm. The compositions of the inoculation medium and production medium are as follows.

<Inoculation medium (pH 7.0)>

20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH2PO4, 8 g K2HPO4, 0.5 g MgSO4⋅7H2O, 100 µg biotin, 1000 µg thiamine HCl, 2000 µg calcium pantothenate and 2000 mcg nicotinamide (based on 1 liter of distilled water)

<Producing medium (pH 7.0)>

50 g glucose, 12.5 g (NH4)2SO4, 2.5 g soy protein, 5 g corn extract, 3 g urea, 1 g KH2PO4, 0.5 g MgSO4⋅7H2O, 100 µg biotin, 1000 µg thiamine HCl, 2000 mcg calcium pantothenate, 3000 mcg nicotinamide, 30 g CaCO3 (based on 1 liter of distilled water)

After completion of cultivation, L-isoleucine concentrations were measured using HPLC, the measured L-isoleucine concentrations are presented in Table 10 below.

The results confirmed that strains KCCM11248P-Pm1-ramA, KCCM11248P-Pm2-ramA and KCCM11248P-Pm3-ramA, including an improved promoter, showed an increase in L-isoleucine production, respectively, by 39%, 32% and 18%, compared with strain KCCM11248P, which was similar to or greater than the ability of strain KCCM11248P-Pcj7-ramA to produce L-isoleucine, in which the promoter was replaced with a stronger one.

The above description of the present invention is offered for illustration purposes, and it will be understood by those skilled in the art that various changes and modifications can be made without changing the technical concept and essential features in accordance with the description of the present invention. Thus, it is clear that the above-described embodiments are illustrative in all aspects and are not limiting of the description of the present invention. Thus, the scope of the specification is determined not by the detailed description, but by the claims and their equivalents, and all changes to the scope of the claims and their equivalents are to be considered as included in this specification.

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LIST OF SEQUENCES

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<223> RamA Pm2 gene promoter variant

<400> 4

gcgatccgcc tcgactatgt tcacccccaa aggggaagta taatggaccc ttgtc

55

<210> 5

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> RamA Pm3 gene promoter variant

<400> 5

gcgatccgcc tcgactatgt tcacccccaa aggtgtggta cactggaccc ttgtc

55

<210> 6

<211> 512

<212> DNA

<213> Artificial sequence

<220>

<223> RamA Pcj7 gene promoter variant

<400> 6

gcgatccgcc tcgactatgt tcacccccaa aggggaagta cactgtaccc ttgtcgaatg

60

attgttactc gtgacgcgcc ctatgggtgt accagcacgg gtgtaaagca gggaggaaatc

120

tgaaggtacc gccggcatag cctaccgatg tagattccac cccatctgtc tcccagtaca

180

ttttttcatg accccagaaa catcccagcg ctactaatag ggagcgttga ccttccttcc

240

acggaccggt aatcggagtg cctaaaaccg catgcggctt aggctccaag ataggttctg

300

cgcggccggg taatgcatct tctttagcaa caagttgagg ggtaggtgca aataagaacg

360

acatagaaat cgtctccttt ctgtttttaa tcaacataca ccaccaccta aaaattcccc

420

gaccagcaag ttcacagtat tcgggcacaa tatcgttgcc aaaatattgt ttcggaatat

480

catgggatac gtacccaacg aaaggaaaca ct

512

<210> 7

<211> 846

<212> DNA

<213> Artificial sequence

<220>

<223> RamA gene ATCC14067

<400> 7

gtggataccc agcggattaa agatgacgaa gatgctattc gttcggcgct gacatcgctg

60

aaaaccgcaa caggcatccc agtcaccatg ttcgccactg tgttgcagga caatcgcctg

120

caaattactc agtgggttgg gttgcgtacc ccggctctgc agaatctggt cattgaacca

180

ggtgtgggcg ttggtggacg cgtcgtcgca acccgtcgtc cggttggtgt gagtgattac

240

accagggcaa atgtcatttc acatgagaag gattccgcga ttcaggatga gggccttcat

300

tccattgtcg cagttcccgt gatcgtgcac cgcgaaatcc gtggcgtttt gtatgttggc

360

gttcactctg cggtgcgtct cggcgacact gttattgaag aagtcaccat gactgcgcgc

420

acgttggaac aaaacctggc gatcaactcc gcgcttcgcc gcaatggcgt tcctgatggt

480

cgcggttccc tcaaagctag ccgcgtgatg aatggggcgg agtgggagca ggttcgttcc

540

actcattcca agctgcgcat gctggcaaat cgtgtgaccg atgaggatct gcgccgcgat

600

ttggaagagc tttgcgatca gatggtcacc ccagtccgca tcaagcagac caccaagctg

660

tccgcgcgtg agttggacgt gctggcttgt gtcgcgctcg gtcacaccaa cgtcgaagct

720

gctgaagaga tgggcatcgg cgcggaaacc gtcaagagct acctgcgctc ggtcatgcgc

780

aagctcggcg cccacacgcg ctacgaggca gtcaacgcag cacgccggat cggcgcactg

840

ccttaa

846

<210> 8

<211> 281

<212>PRT

<213> Artificial sequence

<220>

<223> RamA gene ATCC14067 a.a.

<400> 8

Met Asp Thr Gln Arg Ile Lys Asp Asp Glu Asp Ala Ile Arg Ser Ala

1 5 10 15

Leu Thr Ser Leu Lys Thr Ala Thr Gly Ile Pro Val Thr Met Phe Ala

20 25 30

Thr Val Leu Gln Asp Asn Arg Leu Gln Ile Thr Gln Trp Val Gly Leu

35 40 45

Arg Thr Pro Ala Leu Gln Asn Leu Val Ile Glu Pro Gly Val Gly Val

50 55 60

Gly Gly Arg Val Val Ala Thr Arg Arg Pro Val Gly Val Ser Asp Tyr

65 70 75 80

Thr Arg Ala Asn Val Ile Ser His Glu Lys Asp Ser Ala Ile Gln Asp

85 90 95

Glu Gly Leu His Ser Ile Val Ala Val Pro Val Ile Val His Arg Glu

100 105 110

Ile Arg Gly Val Leu Tyr Val Gly Val His Ser Ala Val Arg Leu Gly

115 120 125

Asp Thr Val Ile Glu Glu Val Thr Met Thr Ala Arg Thr Leu Glu Gln

130 135 140

Asn Leu Ala Ile Asn Ser Ala Leu Arg Arg Asn Gly Val Pro Asp Gly

145 150 155 160

Arg Gly Ser Leu Lys Ala Ser Arg Val Met Asn Gly Ala Glu Trp Glu

165 170 175

Gln Val Arg Ser Thr His Ser Lys Leu Arg Met Leu Ala Asn Arg Val

180 185 190

Thr Asp Glu Asp Leu Arg Arg Asp Leu Glu Glu Leu Cys Asp Gln Met

195 200 205

Val Thr Pro Val Arg Ile Lys Gln Thr Thr Lys Leu Ser Ala Arg Glu

210 215 220

Leu Asp Val Leu Ala Cys Val Ala Leu Gly His Thr Asn Val Glu Ala

225 230 235 240

Ala Glu Glu Met Gly Ile Gly Ala Glu Thr Val Lys Ser Tyr Leu Arg

245 250 255

Ser Val Met Arg Lys Leu Gly Ala His Thr Arg Tyr Glu Ala Val Asn

260 265 270

Ala Ala Arg Arg Ile Gly Ala Leu Pro

275 280

<210> 9

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 9

gctctagata ggccggttcg gactcgccct gcc

33

<210> 10

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 10

gctctagaaa cgtgcgcgca gtcatggtga ctt

33

<210> 11

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 11

gctcggtacc cggggatcct ctagataggc cggttcggac tcgccctgcc

50

<210> 12

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 12

ttacgccaag cttgcatgct ctagaaacgt gcgcgcagtc atggtgactt

50

<210> 13

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 13

cgacaagggt ccattatacc acacctttgg gggt

34

<210> 14

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 14

acccccaaag gtgtggtata atggaccctt gtcg

34

<210> 15

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 15

tcgacaaggg tcatattatac ttccccttt

29

<210> 16

<211> 29

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 16

aaaggggaag tataatgtac ccttgtcga

29

<210> 17

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 17

aagggtacag tgtaccacac ctttgggggt

thirty

<210> 18

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 18

acccccaaag gtgtggtaca ctgtaccctt

thirty

<210> 19

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 19

attcgagctc ggtacccggt ctagatcaag aaactgcagg tgtgtaccga

50

<210> 20

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 20

catcggtagg ctatgccggc ggtaccttca gatttcctcc tgctttacac

50

<210> 21

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 21

gtaccgccgg catagcctac cgatg

25

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 22

agtgtttcct ttcgttgggt acgta

25

<210> 23

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 23

tacgtaccca acgaaaggaa acactgtgga tacccagcgg attaaagatg

50

<210> 24

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 24

tgcatgcctg caggtcgact ctagaatcgc ggcgcagatc ctcatcggtc

50

<210> 25

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 25

aatttctaga ggcagaccct attctatgaa gg

32

<210> 26

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 26

agtgtttcgg tctttacaga cacgagggac

thirty

<210> 27

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 27

gtccctcgtg tctgtaaaga ccgaaacact

thirty

<210> 28

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<223> primer

<400> 28

aatttctaga cgtgggagtg tcactcgctt gg

32

<---

Claims (8)

1. A polynucleotide having promoter activity and containing a substitution of a nucleotide for another nucleotide at at least two or more corresponding positions selected from positions 34, 36, 37, 41 and 43 of the nucleotide sequence presented in SEQ ID NO: 1, where the specified polynucleotide contains a replacement of the 34th nucleotide with T; replacement of the 36th nucleotide with T; replacement of the 37th nucleotide with G; replacement of the 41st nucleotide with T; replacement of the 43rd nucleotide with A or a combination thereof in the nucleotide sequence presented in SEQ ID NO: 1. 2. The polynucleotide according to claim 1, containing one nucleotide sequence selected from SEQ ID NO: 3-5. 3. A microorganism producing a branched-chain L-amino acid containing a polynucleotide according to claim 1 or 2. 4. The microorganism according to claim 3, where the specified polynucleotide contains one nucleotide sequence selected from SEQ ID NO: 3-5. 5. The microorganism according to claim 3, which is a microorganism of the genus Corynebacterium . 6. The microorganism according to claim 5, where the microorganism of the genus Corynebacterium includes Corynebacterium glutamicum . 7. A method for producing branched-chain L-amino acids, comprising cultivating a microorganism according to any one of claims. 3-5 in culture medium. 8. The method of claim 7, further comprising isolating or separating the branched chain L-amino acid from the culture medium or microorganism.