Novel polynucleotide and method for producing l-alanine using same

Abstract

The present invention relates to a novel polynucleotide and a method for producing L-alanine using the same. A microorganism into which a novel polynucleotide of the present invention, in which a specific position in a promoter region of a gene encoding AvtA is mutated, is introduced, has a significantly increased ability to produce L-alanine, and thus the novel polynucleotide can be effectively used for efficiently producing L-alanine.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0190976, filed on December 30, 2022, and all contents disclosed in the corresponding Korean patent application file are incorporated as a part of this specification.

[0003] The present invention relates to a novel polynucleotide and a method for producing L-alanine using the same, and more particularly, to a novel polynucleotide, a recombinant vector comprising the polynucleotide, a host cell transformed with the vector, and a method for producing L-alanine using the host cell. Background Art

[0004] L-alanine is a colorless or white amino acid crystal with no odor but a unique sweetness. It is widely used in fields such as chemistry, food, and medicine. In the food industry, it enhances umami flavor and has various physiological functions, such as promoting alcohol metabolism, protecting liver function, and promoting insulin secretion. Compared to other amino acids, L-alanine has the function of inhibiting browning reactions, so it is used as an acidity regulator and is used in various ways, such as in medications for benign prostatic hyperplasia and sports products. As of 2020, the annual production of alanine is approximately 500 tons, and the market size is estimated to be US$250 million or more.

[0005] Most food additives or color additives are obtained by extraction from natural resources, chemical synthesis or biological production, and L-alanine is mainly produced by chemical synthesis or enzymatic conversion. Recently, with the change of consumers' awareness of healthy living, although the price is high due to low production, natural substances are still more popular than artificial products. Therefore, in addition to the method of producing petroleum-derived L-alanine, various studies have been carried out, for example, efforts have been made to develop microorganisms or fermentation process technologies that produce high-concentration amino acids.

[0006] Common methods for producing L-alanine include fermentation methods using microorganisms such as Corynebacterium or Escherichia coli, Brevibacterium, Lactobacillus sp., etc. ( US 5559016 A ).

[0007] However, as the demand for L-alanine increases, research into more efficient production of high-concentration L-alanine has become increasingly important.

[0008] Therefore, as a result of developing a microorganism that produces high-concentration L-alanine, the present inventors have developed a novel polynucleotide in which a specific position of the promoter region of the gene encoding AvtA is mutated; and have confirmed that the use of this polynucleotide can improve L-alanine production, thereby completing the present invention. Summary of the Invention

[0009] [Technical Issues]

[0010] One embodiment of the present invention provides a polynucleotide, wherein based on the nucleotide sequence represented by SEQ ID NO: 2, the nucleotide corresponding to position 47 is substituted with T, and the nucleotides corresponding to positions 48 and 49 are substituted with A, respectively.

[0011] Another embodiment of the present invention provides a vector comprising the polynucleotide; and a vector comprising a gene encoding a target protein operably linked to the polynucleotide.

[0012] Other embodiments of the present invention provide a host cell comprising the polynucleotide; and a vector comprising a gene encoding a target protein operably linked to the polynucleotide.

[0013] Other embodiments of the present invention provide a method for producing an amino acid, comprising culturing the host cell in a culture medium.

[0014] Another embodiment of the present invention provides a composition comprising a host cell for producing amino acids.

[0015] Other embodiments of the present invention provide a polynucleotide, a vector comprising the polynucleotide, and a host cell comprising the vector for producing amino acids, wherein the polynucleotide, based on the nucleotide sequence represented by SEQ ID NO: 2, the nucleotide corresponding to position 47 is substituted with T, and the nucleotides corresponding to positions 48 and 49 are respectively substituted with A.

[0016] [Technical solution]

[0017] Detailed explanation is as follows. On the other hand, each description and embodiment disclosed in the present invention can be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of this application cannot be considered to be limited to the specific description described below.

[0018] The present invention provides a polynucleotide, wherein based on the nucleotide sequence represented by SEQ ID NO: 2, the nucleotide corresponding to position 47 is substituted with T, and the nucleotides corresponding to positions 48 and 49 are substituted with A, respectively.

[0019] In the present specification, the term "nucleotide sequence represented by SEQ ID NO: 2" may refer to a part of the promoter sequence of a gene encoding an aminotransferase.

[0020] In this specification, the term "aminotransferase" is used interchangeably with "transaminase" and refers to an enzyme that can reversibly produce L-alanine from pyruvate. The use of cofactor L-valine at that time can affect the reduction of by-products.

[0021] In the present specification, the term "L-alanine" is one of the essential amino acids and refers to an L-amino acid having a chemical formula of HO2CCH(NH2)CH3.

[0022] The polynucleotide may have promoter activity.

[0023] As used herein, the term "promoter" refers to an untranslated nucleotide sequence upstream of a coding region that contains a polymerase binding site and has transcription initiation activity for the mRNA of the promoter's target gene. In other words, it refers to a DNA region that binds to the polymerase to initiate gene transcription. A promoter can be located 5' of the mRNA transcription start site. The target gene of a promoter can be, but is not limited to, a gene encoding an aminotransferase.

[0024] The polynucleotide of the present invention is a portion of the nucleotide sequence represented by SEQ ID NO: 2, that is, the promoter sequence of the gene encoding aminotransferase, which is mutated. Specifically, the mutation may be that the nucleotide corresponding to position 47 is substituted with T, and the nucleotides corresponding to positions 48 and 49 are respectively substituted with A. Therefore, the polynucleotide may consist of the nucleotide sequence of SEQ ID NO: 1.

[0025] The term "mutation" refers to a genetic or non-genetic stable phenotypic change, and in this specification, it may be designated by being used interchangeably with "mutant".

[0026] Specifically, the polynucleotide may have increased promoter activity compared to a polynucleotide that does not contain a mutation (wild-type polynucleotide). Thus, the polynucleotide can control (increase) the expression of a target gene operably linked to the polynucleotide and the activity of a protein encoded by the target gene. In addition, it can control the expression of genes other than the target gene.

[0027] These polynucleotides can be used to improve the amino acid production capacity (output) of the host cell by importing into a suitable host cell, for example the activity of L-Alanine production capacity (output). Therefore, these polynucleotides can be used to improve amino acid production capacity (output), and particularly, they can be used to improve L-Alanine production capacity (output).

[0028] Specifically, the polynucleotide may consist of the nucleotide sequence of SEQ ID NO: 1.

[0029] In addition, the nucleotide sequence of the present invention can be modified by conventionally known mutagenesis, such as directed evolution and site-directed mutagenesis.

[0030] Therefore, the polynucleotide may include a polynucleotide comprising a nucleotide sequence having at least 60% or more, specifically 70% or more, more specifically 80% or more, more specifically 83% or more, 84% or more, 88% or more, 90% or more, 93% or more, 95% or more, or 97% or more homology to the nucleotide sequence of SEQ ID NO: 1. As long as it is a sequence having homology to the sequence and is a polynucleotide sequence having substantially the same or corresponding biological activity (promoter activity) and / or desired activity (e.g., activity of increasing the production of L-alanine in a host cell) as the nucleotide sequence of SEQ ID NO: 1, even if some sequences are deleted, modified, substituted, or added in the polynucleotide sequence, it is also included in the scope of the present application.

[0031] In this specification, the term "identity (or homology)" refers to the degree of correspondence to a specified nucleic acid sequence or amino acid sequence and can be expressed as a percentage (%). In the case of nucleic acid homology, it can be determined using the documented algorithm BLAST (see: Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873, 1993) or Pearson's FASTA (see: Methods Enzymol., 183, 63, 1990). Based on this algorithm BLAST, programs named BLASTN or BLASTX have been developed (see: http: / / www.ncbi.nlm.nih.gov).

[0032] In one embodiment, the polynucleotides provided herein comprising a specific nucleic acid sequence can be interpreted as comprising a polynucleotide fragment comprising not only the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, but also a nucleic acid sequence complementary to the specific nucleic acid sequence. Specifically, complementary polynucleotides can hybridize at a Tm value that can be appropriately adjusted by those skilled in the art according to the purpose, for example, a Tm value of 55°C, 60°C, 63°C, or 65°C; and complementary polynucleotides can be analyzed under the conditions described below: these conditions are specifically described in known literature. For example, the following conditions can be listed: conditions that allow genes with high complementarity of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99.5% or more, or 99.9% or more to hybridize, and conditions that do not hybridize genes with complementarity lower than the above, or conditions of washing once, specifically twice or three times, at salt concentrations and temperatures corresponding to: 60° C., 1× SSC (salt-sodium citrate buffer) and 0.1% (w / v) SDS (sodium dodecyl sulfate); 60° C., 0.1× SSC and 0.1% SDS; or 68° C., 0.1× SSC and 0.1% SDS, which are washing conditions for general southern hybridization, and similar conditions, but are not limited thereto. Hybridization requires that two nucleotides have complementary sequences, but depending on the stringency of hybridization, mismatches between bases may be allowed. The term "complementary" can be used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in the case of DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. The appropriate stringency for polynucleotide hybridization depends on the length and degree of complementarity of the polynucleotide, and this is well known in the relevant art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0033] In particular, when the corresponding polynucleotide is used as a promoter linked to a target gene, the expression "consisting of the nucleotide sequence of SEQ ID NO: 1" does not exclude the case of addition and / or deletion and / or mutation of nucleotides, which may occur during the process of linking to the target gene using restriction enzymes.

[0034] For example, polynucleotides having biological activity (promoter activity) and / or target activity (e.g., activity of increasing L-alanine production in a host cell) consisting of the nucleotide sequence represented by SEQ ID NO: 1 may be included but are not limited thereto, as long as it is a nucleotide sequence that has the biological activity (promoter activity) and / or target activity (e.g., activity of increasing L-alanine production) of the present application by hybridizing with a complementary sequence of all or part of the nucleotide sequence of SEQ ID NO: 1 under stringent conditions.

[0035] Furthermore, the polynucleotide of the present invention can be operably linked to a gene encoding a target protein.

[0036] In the present specification, the term "gene expression regulatory sequence" refers to a sequence that comprises the polynucleotide of the present invention and can express a target gene operably linked thereto.

[0037] As used herein, the term "operably linked" refers to functionally linking a polynucleotide having promoter activity of the present invention to a gene sequence to initiate and mediate transcription of a target gene. Operable linkages can be prepared using genetic recombination techniques known in the art, and site-specific DNA cleavage and ligation can be constructed using cleavage and ligation enzymes known in the art, but are not limited thereto.

[0038] Furthermore, the gene expression regulatory sequence of the present invention may further include, in addition to a promoter for gene transcription, any operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, and DNA for regulating transcription and translation termination.

[0039] For example, in addition to the promoter, the regulatory sequence suitable for prokaryotes may further include a ribosome binding site, but is not limited thereto. If desired by those skilled in the art, the polynucleotide having promoter activity of the present invention may constitute the sequence described above for regulating gene expression.

[0040] In the present invention, the target gene refers to a gene encoding a target protein to regulate expression in a microorganism.

[0041] For example, it can be a gene related to amino acid production, but is not limited thereto. Specifically, the gene can be a gene encoding an enzyme related to amino acid biosynthesis, but is not limited thereto. More specifically, the gene can be a gene encoding an aminotransferase, but is not limited thereto.

[0042] In addition, the present invention provides a vector comprising a polynucleotide, in which, based on the nucleotide sequence represented by SEQ ID NO: 2, the nucleotide corresponding to the mutation may be that the nucleotide corresponding to position 47 is substituted with T, and the nucleotides corresponding to positions 48 and 49 are substituted with A, respectively.

[0043] The polynucleotide is as described above.

[0044] The vector may further comprise a gene encoding a target protein operably linked to the polynucleotide.

[0045] In this specification, the term "vector" refers to a DNA product containing a base sequence of a polynucleotide encoding a target protein, which is operably linked to an appropriate regulatory sequence to express the target protein in an appropriate host. The regulatory sequence may include a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, and / or a sequence regulating transcription and / or translation termination. The vector can be transformed into a suitable host cell and then expressed independently of the host cell's genome, or integrated into the host cell's genome.

[0046] In the present invention, the vector is not particularly limited as long as it is replicable in the host, and it can be selected from all commonly used vectors. Examples of commonly used vectors may include plasmids, cosmids, viruses, phages, etc. in a natural state or a recombinant state. For example, as vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ vectors, pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based, and pET-based vectors can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be exemplified, but are not limited thereto.

[0047] The available vectors in this specification sheets can be known expression vectors and / or vectors for inserting polynucleotides into host cell chromosomes.Polynucleotides are inserted into host cell chromosomes and can be carried out by any method known in the art, such as homologous recombination or CRISPR system, but are not limited thereto.The vector can further include a selection marker for confirming whether to insert it into chromosome.Selection marker is used to select cells transformed using a vector, that is, to confirm whether to insert polynucleotides, and it can be selected from the gene providing selective phenotype (such as drug resistance, auxotrophy, expression of tolerance or surface protein to cytotoxic agents) and used.Under the environment of processing with selection agent, only the cells expressing selection marker survive or show other phenotypes, so the cells transformed can be selected.

[0048] In addition, the present invention provides a host cell comprising a polynucleotide and a gene encoding a target protein operably linked to the polynucleotide.

[0049] The polynucleotide and the gene encoding the target protein operably linked to the polynucleotide are as described above.

[0050] The host cell may be a microorganism, but is not limited thereto.

[0051] In this specification, the term "microorganism" is a concept that includes all wild-type microorganisms or microorganisms in which natural or artificial genetic modification has occurred, and includes all microorganisms in which a specific mechanism is weakened or strengthened due to reasons such as insertion of foreign genes or strengthening or weakening of endogenous gene activity.

[0052] In the present invention, the microorganism may comprise a polynucleotide, and specifically, may comprise a polynucleotide and / or a gene encoding a target protein operably linked to the polynucleotide. In addition, the microorganism may comprise a vector comprising a polynucleotide or a gene encoding a gene expression regulatory sequence and a target protein, but is not limited thereto. In addition, the polynucleotide, the gene encoding the target protein, and the vector may be introduced into the microorganism by transformation, but is not limited thereto. In addition, as long as the microorganism is capable of expressing the gene, it does not matter whether the polynucleotide and the gene encoding the target protein are located on the chromosome or outside the chromosome.

[0053] A microorganism comprising the polynucleotide and the gene encoding the target protein may have improved amino acid production ability, and specifically, it may have improved L-alanine production ability.

[0054] For example, the microorganism may have enhanced aminotransferase activity.

[0055] In the present invention, any microorganism is included without limitation as long as it can function as a promoter when the polynucleotide having promoter activity of the present invention is introduced.

[0056] Specifically, the microorganism can be a Corynebacterium microorganism, and more specifically, can include Corynebacterium stationis, Corynebacterium thermoaminogenes, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum and bacterial strains prepared therefrom, but is not limited thereto. Specifically, it can be Corynebacterium stationis or Corynebacterium glutamicum bacterial strain.

[0057] In addition, the present invention provides a method for producing an amino acid, comprising culturing a host cell containing a polynucleotide in a culture medium, in which, based on the nucleotide sequence represented by SEQ ID NO: 2, the nucleotide corresponding to position 47 is substituted with T, and the nucleotides corresponding to positions 48 and 49 are respectively substituted with A.

[0058] The polynucleotide and host cell are as described above.

[0059] The method may further include recovering the amino acid from the cultured microorganism, the culture product, or both after the culturing.

[0060] The amino acid may be L-alanine, but is not limited thereto.

[0061] As used herein, the term "cultivation" refers to growing a microorganism under appropriately artificially adjusted environmental conditions. The methods for culturing the microorganisms of the present invention can be performed using methods for culturing Corynebacterium glutamicum that are widely known in the art. Specifically, examples of culturing methods include, but are not limited to, batch culture, continuous culture, and fed-batch culture. These various methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp. 138-176, 1991).

[0062] In this specification, the term "culture product" refers to a substance comprising a culture medium in which microorganisms grow or have grown under appropriately artificially adjusted environmental conditions. In a narrow sense, grown microorganisms are not included in the culture product, but they can be included in the culture product in a broad sense. "Culture products" include various substances secreted by microorganisms during their growth together with the culture medium components used to cultivate the microorganisms, and specifically include the target substance L-alanine.

[0063] The culture medium used for cultivation should meet the requirements of the specific strain in an appropriate manner. The culture medium for Corynebacterium strains is known. For example, the microorganism of the present application can be cultured in a universal culture medium containing a suitable carbon source, nitrogen source, vitamins, etc. under an aerobic state, while adjusting the temperature, pH, etc. At this time, the carbon source includes carbohydrates such as glucose, fructose and sucrose; amino acids such as glutamic acid, cysteine, etc. Specifically, natural organic nutrients such as starch hydrolysates and molasses can be used, and preferably, the natural organic nutrients are carbohydrates such as glucose, fructose, sterilized pretreated molasses (i.e., molasses converted into reducing sugars), etc., and appropriate other carbon sources can be used in various ways without restriction, but are not limited thereto. As a nitrogen source, inorganic nitrogen sources such as ammonia can be used; and organic nitrogen sources such as amino acids such as glutamic acid and cysteine; and peptone, meat extract, yeast extract, etc. These nitrogen sources can be used alone or in combination, but are not limited thereto. In the culture medium, as a phosphorus source, phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium salts can be used, but are not limited thereto. As inorganic compounds, magnesium sulfate, iron sulfate, manganese sulfate, calcium chloride, etc. can be used, and in addition, amino acids, vitamins, and appropriate precursors can be contained. These media or precursors can be added to the culture product in a batch or continuous method, but are not limited thereto.

[0064] During the culture period, the pH of the culture product can be adjusted by adding compounds such as potassium hydroxide, ammonia and phosphoric acid to the culture product in an appropriate manner. In addition, during the culture period, a defoaming agent such as fatty acid polyethylene glycol ester can be used to suppress foam generation. In addition, in order to maintain the aerobic state of the culture product, oxygen or oxygen-containing gas can be injected into the culture product. The temperature of the culture product can be 27°C to 37°C, specifically, 30°C to 33°C. The culture period can be continued until the yield of the desired useful substance is obtained, and specifically, it is 20 to 120 hours.

[0065] Recovering the amino acid can be performed by collecting the target amino acid from the culture medium, culture solution, or microorganism using appropriate methods known in the art according to the culture method. For example, recovery can be performed by at least one method selected from the group consisting of, but not limited to, centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC. The method for producing the amino acid can further include a purification step before, simultaneously with, or after recovery.

[0066] [Beneficial Effects]

[0067] The present invention relates to a novel polynucleotide and a method for producing L-alanine using the same. A microorganism into which the novel polynucleotide is introduced at a specific position of the promoter region of the gene encoding AvtA of the present invention has significantly increased L-alanine-producing ability. Therefore, the novel polynucleotide can be effectively used for producing L-alanine. DETAILED DESCRIPTION

[0068] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only intended to illustrate the present invention, but the scope of the present invention is not limited by these examples.

[0069] Example 1. Construction of a strain with a mutation introduced into the promoter region of the avtA gene

[0070] A mutant strain was constructed in which mutations were introduced into the promoter sequence of the avtA gene, wherein nucleotide G at position 47 was substituted with T, nucleotide G at position 48 was substituted with A, and nucleotide C at position 49 was substituted with A.

[0071] Specifically, in order to introduce a mutation into wild-type Corynebacterium glutamicum (ATCC13869) (in the promoter region represented by SEQ ID NO: 2, the 47th nucleotide G is substituted with T, the 48th nucleotide G is substituted with A, and the 49th nucleotide C is substituted with A), a gene fragment containing the corresponding mutation was obtained by PCR using primer pairs of SEQ ID NO: 3 and SEQ ID NO: 4 and SEQ ID NO: 5 and SEQ ID NO: 6. PCR conditions were repeated 30 times: denaturation at 95°C for 5 minutes, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, a 501bp polynucleotide amplified using primers of SEQ ID NO: 3 and SEQ ID NO: 4 and a 501bp polynucleotide amplified using primers of SEQ ID NO: 5 and SEQ ID NO: 6 were obtained. The two gene fragments obtained were ligated to pDCM2 cut with restriction enzymes BamHI and SalI (Korean Patent Publication No. 10-2020-0136813) using infusion enzyme to construct a gene replacement vector containing avtA promoter mutation, and this was named pDCM2-Pm_avtA. The information of the primer sequences used to construct the vector is shown in Table 1 below.

[0072] [Table 1]

[0073]

[0074] By electric pulse method (Appl.Microbiol.Biotechnol., 1999), carrier is transformed into Corynebacterium glutamicum ATCC13869, and is subsequently applied on the composite plate culture medium containing 25mg / L kanamycin to fix bacterium colony.After this, by conventional secondary hybridization process, obtain and comprise corresponding mutation and remove the final bacterial strain of the carrier imported.Use the primer pair of SEQID NO:7 (5'-CGTACACCACCATCAAGGACA-3') and SEQ ID NO:8 (5'-CAAAGGACCCGAGCGAAGAGC-3'), finally confirmed the bacterial strain of importing corresponding mutation by gene sequence analysis.By importing the bacterial strain named as CJ0011 of target mutation.

[0075] Example 2. Confirmation of L-alanine production in strains into which new promoter mutant genes were introduced

[0076] In order to compare the L-alanine production capabilities of the mutant strain CJ0011 constructed in Example 1 and the wild-type Corynebacterium glutamicum ATCC13869 strain, they were cultured by the following method.

[0077] Specifically, after the parent strain Corynebacterium glutamicum ATCC 13869 strain and the mutant strain CJ0011 strain were inoculated into 250 mm baffled flasks containing 25 ml of production medium, they were cultured at 200 rpm at 30° C. for 46 hours.

[0078] After the cultivation was completed, the L-alanine concentration in each culture medium was measured using high performance liquid chromatography, and the L-alanine concentration in the culture solution for each test strain is shown in Table 2 below.

[0079] [Table 2]

[0080] Comparison of L-alanine production capacity between Corynebacterium glutamicum ATCC13869 and Corynebacterium glutamicum CJ0011

[0081]

[0082] As a result, as shown in Table 2, it was confirmed that the Corynebacterium glutamicum CJ0011 strain into which the mutation was introduced produced L-alanine at a concentration of 7.5 g / L and had an L-alanine productivity of about 132% compared to that of the parent strain.

[0083] The above results indicate that the mutations in the sequence of the promoter region represented by SEQ ID NO: 2, i.e., substitution of nucleotide G at position 47 with T, substitution of nucleotide G at position 48 with A, and substitution of nucleotide C at position 49 with A, significantly improve the L-alanine-producing ability of the microorganism, and that the promoter in which nucleotide G at position 47 with T, nucleotide G at position 48 with A, and nucleotide C at position 49 with A in the sequence of the promoter region represented by SEQ ID NO: 2 can be effectively used in the method for producing L-alanine.

[0084] The composition of the culture medium used in Example 2 is as follows.

[0085] <Activation Medium>

[0086] Beef extract 5g / L, polypeptone 10g / L, yeast extract 5g / L, urea 2g / L, sodium chloride (NaCl) 2.5g / L, agar 20g / L, glucose 10g / L, 10N sodium hydroxide (NaOH)

[0087] <Seed culture medium>

[0088] Glucose (anhydrous glucose) 20g / L, polypeptone 10g / L, yeast extract 10g / L, ammonium sulfate [(NH4)2SO4] 10g / L, urea 1.5g / L, potassium dihydrogen phosphate (KH2PO4) 5.2g / L, dipotassium hydrogen phosphate (K2HPO4) 10.7g / L, d-biotin 1.8mg / L, thiamine hydrochloride 9mg / L, CAPA 9mg / L, NCA 60mg / L, magnesium sulfate (MgSO4) 0.5g / L

[0089] <Production Medium>

[0090] Calcium carbonate (CaCO3) 30g / L, sucrose 57g / L, BM 6g / L, magnesium sulfate (MgSO4) 0.5g / L, (NH4)2SO4 50g / L, KH2PO4 1g / L, yeast extract 2g / L, ammonium acetate 6.28g / L, d-biotin 0.05mg / L, thiamine hydrochloride 0.1mg / L, MnSO4 6.7mg / L, FeSO4 10mg / L.

Claims

1. A polynucleotide, wherein based on the nucleotide sequence represented by SEQ ID NO: 2, the nucleotide corresponding to position 47 is substituted with T, and the nucleotides corresponding to positions 48 and 49 are respectively substituted with A. 2 . The polynucleotide according to claim 1 , wherein the polynucleotide consists of the nucleotide sequence of SEQ ID NO:

1.

3. The polynucleotide according to claim 1 or 2, wherein the polynucleotide has promoter activity. 4 . A recombinant vector comprising the polynucleotide according to claim 3 ; and a gene encoding a target protein operably linked to the polynucleotide. The recombinant vector according to claim 4 , wherein the target protein is an aminotransferase. A Corynebacterium microorganism comprising the polynucleotide according to claim 3; and a gene encoding a target protein operably linked to the polynucleotide. 7 . The Corynebacterium microorganism according to claim 6 , wherein the polynucleotide consists of the nucleotide sequence of SEQ ID NO:

1. The Corynebacterium microorganism according to claim 6 , wherein the target protein is an aminotransferase. 9 . The Corynebacterium microorganism according to claim 6 , wherein the Corynebacterium microorganism is Corynebacterium glutamicum. 10 . A method for producing an amino acid, comprising culturing the Corynebacterium microorganism according to claim 6 in a culture medium. The method for producing an amino acid according to claim 10 , wherein the amino acid is L-alanine. 12 . A composition for producing amino acids, comprising the Corynebacterium microorganism according to claim 6 .

13. The composition for producing an amino acid according to claim 12, wherein the amino acid is L-alanine.

Images