Novel polynucleotide and method for producing L-alanine using the same

Abstract

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 of the present invention, in which a specific site in the promoter region of the gene encoding AvtA has been mutated, has significantly increased L-alanine-producing ability, and therefore the novel polynucleotide can be usefully used for efficient L-alanine production.

Description

[Technical Field]

[0001] Cross-Citation of Related Applications This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0190976 dated December 30, 2022, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] 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 containing the polynucleotide, a host cell transformed with the vector, and a method for producing L-alanine using the host cell. [Background technology]

[0003] L-alanine is a colorless or white crystalline amino acid with no odor but a distinctive sweet taste, and is widely used in fields such as chemistry, food, and medicine. In the food industry, it is used as a flavor enhancer or nutritional fortifier to enhance flavor and has various physiological effects, such as promoting alcohol metabolism, protecting liver function, and promoting insulin secretion. Compared to other amino acids, it has the ability to inhibit browning reactions and is also used as a sourness corrector. It is used in a wide range of applications, including medicines for benign prostatic hyperplasia and sports products. As of 2020, the annual production of alanine was approximately 500 tons, and the market size was estimated to be more than USD 250 million.

[0004] Most food additives or color additives are obtained through natural resource extraction, chemical synthesis, or biological production, and L-alanine is mainly produced by chemical synthesis or enzymatic conversion. Recently, as consumers' awareness of a healthy lifestyle has changed, natural substances have become more popular than artificial products, despite their higher prices due to their low production volume. Therefore, various research efforts are being conducted to deviate from petroleum-derived L-alanine production methods, such as developing microorganisms and fermentation process technologies that can produce high concentrations of amino acids.

[0005] Common methods for producing L-alanine include fermentation using microorganisms such as Corynebacterium, Escherichia coli, Brevibacterium, and Lactobacillus (US Pat. No. 5,559,016A).

[0006] However, as the demand for L-alanine increases, there is an increasing need for research into more efficient production of high-concentration L-alanine.

[0007] Therefore, the present inventors have made efforts to develop a microorganism capable of producing high concentrations of L-alanine, and as a result, have developed a novel polynucleotide in which a specific site in the promoter region of the AvtA-encoding gene has been mutated. They have confirmed that the amount of L-alanine produced can be improved using this polynucleotide, and have thus completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] One example of the present invention provides a polynucleotide in which, based on the nucleotide sequence set forth in SEQ ID NO: 2, the nucleotide at position 47 is substituted with T, and the nucleotides at positions 48 and 49 are each substituted with A.

[0009] Another embodiment of the present invention provides a vector comprising the polynucleotide; and a gene encoding a protein of interest operably linked to the polynucleotide.

[0010] Another embodiment of the invention provides a host cell comprising the polynucleotide; and a gene encoding a protein of interest operably linked to the polynucleotide.

[0011] Another embodiment of the invention provides a method for producing an amino acid comprising culturing the host cell described above in a medium.

[0012] Another embodiment of the present invention provides a composition for producing an amino acid, comprising the host cell.

[0013] Another example of the present invention provides a polynucleotide in which the nucleotide at position 47 of the nucleotide sequence set forth in SEQ ID NO: 2 is substituted with T and the nucleotides at positions 48 and 49 of the nucleotide sequence are substituted with A, a vector containing the polynucleotide, and a host cell containing the vector for use in amino acid production. [Means for solving the problem]

[0014] This will be explained in detail as follows. Meanwhile, 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 belong to the scope of the present invention. Furthermore, it cannot be said that the scope of the present application is limited by the specific descriptions described below.

[0015] The present invention provides a polynucleotide in which the nucleotide at position 47 of the nucleotide sequence represented by SEQ ID NO: 2 is substituted with T, and the nucleotides at positions 48 and 49 of the nucleotide sequence are substituted with A.

[0016] As used herein, the term "nucleotide sequence represented by SEQ ID NO: 2" may refer to a portion of the promoter sequence of a gene encoding an aminotransferase.

[0017] As used herein, the term "aminotransferase" can be used interchangeably with "aminotransferase" and refers to an enzyme that can reversibly produce L-alanine from pyruvate. The cofactor L-valine used in this process can affect the reduction of by-products.

[0018] As used herein, the term "L-alanine" refers to an L-amino acid that is one of the essential amino acids and has the chemical formula HO2CCH(NH2)CH3.

[0019] The polynucleotide may have promoter activity.

[0020] As used herein, the term "promoter" refers to a non-coding nucleotide sequence upstream of a coding region that contains a polymerase binding site and has the activity of initiating transcription of a target gene into mRNA, i.e., a DNA region that allows polymerase to bind and initiate transcription of the gene. The promoter is located 5' to the mRNA transcription initiation site. In this case, the target gene of the promoter can be, but is not limited to, a gene encoding an aminotransferase.

[0021] The polynucleotide of the present invention is a nucleotide sequence represented by SEQ ID NO: 2, i.e., a partial mutation in the promoter sequence of a gene encoding aminotransferase, and specifically, the mutation may be a substitution of the 47th nucleotide of the sequence with T and the 48th and 49th nucleotides with A, respectively. Thus, the polynucleotide may consist of the nucleotide sequence of SEQ ID NO: 1.

[0022] The term "mutation" refers to a stable phenotypic change, either genetic or non-genetic, and may be interchangeably referred to herein as "mutation."

[0023] Specifically, the polynucleotide may have increased promoter activity compared to a polynucleotide that does not contain a mutation (wild-type polynucleotide), and thus may regulate (increase) the expression of a gene of interest operably linked to the polynucleotide and the activity of a protein encoded by the gene of interest, thereby regulating the expression of genes other than the gene of interest.

[0024] The polynucleotide may have an activity of increasing the amino acid production (production amount) of the host cell, for example, L-alanine production (production amount), when introduced into a suitable host cell. Thus, the polynucleotide may be used to increase amino acid production (production amount), specifically, L-alanine production (production amount).

[0025] Specifically, the polynucleotide may consist of the nucleotide sequence of SEQ ID NO:1.

[0026] The nucleotide sequences of the present invention can also be modified by conventional mutagenesis techniques, such as directed evolution and site-directed mutagenesis.

[0027] 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, and even 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. The scope of the present application also includes polynucleotide sequences with partial deletions, modifications, substitutions, or additions of sequences that are homologous to the above sequence and have substantially the same or corresponding biological activity (promoter activity) and / or a desired activity (e.g., activity of increasing L-alanine production in a host cell).

[0028] As used herein, the term "identity" or "homology" refers to the degree of identity between a given nucleic acid sequence or amino acid sequence, and can be expressed as a percentage (%). Homology between nucleic acid sequences can be determined, for example, using the literature-based algorithm BLAST (see Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (see Methods Enzymol., 183, 63, 1990). Based on the BLAST algorithm, a program called BLASTN or BLASTX has been developed (see http: / / www.ncbi.nlm.nih.gov).

[0029] For example, a polynucleotide comprising a specific nucleic acid sequence provided herein can be interpreted as including not only the specific nucleic acid sequence or a nucleic acid sequence substantially equivalent thereto, but also a polynucleotide fragment comprising a nucleic acid sequence complementary to the specific nucleic acid sequence. Specifically, the complementary polynucleotide can be hybridized at a Tm value that can be appropriately adjusted by those skilled in the art depending on the purpose, for example, a Tm value of 55°C, 60°C, 63°C, or 65°C, and analyzed under the conditions described below; such conditions are specifically described in known literature. Examples of conditions that can be used include, but are not limited to, conditions under which 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, 99% or more, 99.5% or more, or 99.9% or more are hybridized, while genes with lower complementarity are not hybridized, or conditions for washing once, specifically two to three times, at salt concentrations and temperatures equivalent to those used in standard Southern hybridization: 60°C, 1x SSC (saline-sodium citrate buffer), and 0.1% (w / v) SDS (sodium dodecyl sulfate); 60°C, 0.1x SSC, and 0.1% SDS; or 68°C, 0.1x SSC, and 0.1% SDS. Hybridization requires that two nucleotides have complementary sequences, or mismatches between bases may be tolerated depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. The appropriate stringency for hybridizing polynucleotides depends on the length and degree of complementarity of the polynucleotides, and is well known in the art (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0030] In particular, the expression "consist of the nucleotide sequence of SEQ ID NO: 1" does not exclude cases where nucleotide additions, deletions, and / or mutations may occur during the process of linking the polynucleotide to a target gene, such as using a restriction enzyme, when the polynucleotide is linked to the target gene as a promoter.

[0031] For example, the polynucleotide having a biological activity (promoter activity) and / or a desired activity (e.g., an activity to increase L-alanine production in a host cell) consisting of a nucleotide sequence set forth in SEQ ID NO: 1 may include, without limitation, any nucleotide sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the nucleotide sequence of SEQ ID NO: 1 and has the biological activity (promoter activity) and / or a desired activity (e.g., an activity to increase L-alanine production in a host cell) of the present application.

[0032] Thus, the polynucleotide of the present invention can be operably linked to a gene encoding a protein of interest.

[0033] As used herein, the term "gene expression control sequence" refers to a sequence that can induce expression of a gene of interest that comprises a polynucleotide of the present invention and is operably linked thereto.

[0034] As used herein, the term "operatively linked" means that the polynucleotide having promoter activity of the present invention is functionally linked to a gene sequence of interest so as to initiate and mediate transcription of the gene. Operable linkage can be achieved using recombinant DNA techniques known in the art, and site-specific DNA cleavage and ligation can be achieved using cleavage and ligation enzymes known in the art, but is not limited thereto.

[0035] Furthermore, the gene expression control sequence of the present invention may further include, in addition to a promoter for transcribing the gene, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and DNA for regulating the termination of transcription and decoding.

[0036] For example, a regulatory sequence suitable for prokaryotes may further include, but is not limited to, a ribosome binding site in addition to a promoter. A polynucleotide having promoter activity of the present invention can be constructed by one of ordinary skill in the art as needed to regulate gene expression as described above.

[0037] In the present invention, the target gene refers to a gene encoding a target protein whose expression is to be regulated in a microorganism.

[0038] For example, the gene may be, but is not limited to, a gene involved in amino acid production. Specifically, the gene may be, but is not limited to, a gene encoding an enzyme involved in amino acid biosynthesis. More specifically, the gene may be, but is not limited to, a gene encoding an aminotransferase.

[0039] The present invention also provides a vector comprising a polynucleotide in which the nucleotide at position 47 of the nucleotide sequence represented by SEQ ID NO: 2 is substituted with T, and the nucleotides at positions 48 and 49 are each substituted with A.

[0040] The polynucleotide is as described above.

[0041] The vector may further comprise a gene encoding a protein of interest operably linked to the polynucleotide.

[0042] As used herein, the term "vector" refers to a DNA construct containing a polynucleotide sequence encoding a protein of interest operably linked to a suitable regulatory sequence to enable expression of the protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosomal binding site, and / or a sequence regulating the termination of transcription and / or translation. After transformation into an appropriate host cell, the vector may be expressed independently of the host cell's genome (genetic body) or integrated into the host cell's genome.

[0043] The vectors usable herein are not particularly limited as long as they are replicable in host cells and can be selected from any commonly used vector. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, phage or cosmid vectors such as pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used. Plasmid vectors that can be used include pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specific examples include, but are not limited to, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC.

[0044] The vectors usable herein may be known expression vectors and / or vectors for inserting a polynucleotide into a host cell chromosome. The polynucleotide may be inserted into a host cell chromosome by any method known in the art, including, but not limited to, homologous recombination or the CRISPR system. The vector may additionally contain a selection marker for detecting the presence or absence of the insertion into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to detect the presence or absence of the polynucleotide insertion. The selection marker is selected from genes that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0045] The present invention also provides a host cell containing the polynucleotide and a gene encoding a protein of interest operably linked to the polynucleotide.

[0046] The polynucleotide and the gene encoding the target protein operably linked to the polynucleotide are as described above.

[0047] The host cell may be, but is not limited to, a microorganism.

[0048] As used herein, the term "microorganism" includes wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and is a concept that includes all microorganisms in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or weakening the activity of an endogenous gene.

[0049] In the present invention, the microorganism may contain the polynucleotide, specifically, the polynucleotide and / or a gene operably linked to the polynucleotide and encoding a target protein. Alternatively, the microorganism may contain, but is not limited to, the polynucleotide or a vector containing a gene expression regulatory sequence and a gene encoding a target protein. Furthermore, the polynucleotide, the gene encoding the target protein, and the vector may be introduced into the microorganism by, but is not limited to, transformation. Furthermore, as long as the gene can be expressed in the microorganism, it does not matter whether the polynucleotide and the gene encoding the target protein are located on or off the chromosome.

[0050] The microorganism containing the polynucleotide and the gene encoding the target protein may have an increased ability to produce amino acids, specifically, an increased ability to produce L-alanine.

[0051] For example, the microorganism may be enriched for aminotransferase.

[0052] In the present invention, the microorganism is not limited as long as it can function as a promoter when the polynucleotide having promoter activity of the present invention is introduced therein.

[0053] Specifically, the microorganism may be a Corynebacterium microorganism, more specifically, Corynebacterium stationis, Corynebacterium thermoaminogenes, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, and strains produced therefrom, but is not limited thereto. Specifically, the microorganism may be a Corynebacterium stationis or Corynebacterium glutamicum strain.

[0054] The present invention also provides a method for producing an amino acid, comprising culturing in a medium a host cell containing a polynucleotide in which the nucleotide at position 47 of the nucleotide sequence set forth in SEQ ID NO: 2 is substituted with T and the nucleotides at positions 48 and 49 of the nucleotide sequence are substituted with A.

[0055] The polynucleotide and the host cell are as described above.

[0056] The method may further include, after the culturing step, recovering amino acids from the cultured microorganism, the culture, or both.

[0057] The amino acid may be, but is not limited to, L-alanine.

[0058] As used herein, the term "cultivation" refers to growing a microorganism under suitable artificially controlled environmental conditions. The method for culturing the microorganism of the present invention can be carried out using a culture method for Corynebacterium glutamicum that is widely known in the art. Specific examples of the culture method include, but are not limited to, batch culture, continuous culture, and fed-batch culture. Such various methods are disclosed, for example, in "Biochemical Engineering" (James M. Lee, Prentice-Hall International Editions, pp. 138-176, 1991).

[0059] As used herein, the term "culture" refers to a substance containing a medium in which microorganisms are growing or have completed growing under appropriately artificially controlled environmental conditions. In the narrow sense, the term "culture" does not include the grown microorganisms, but in the broad sense, it does. The "culture" includes medium components formulated for microbial cultivation as well as various substances secreted into the medium by the microorganisms during growth, specifically the target substance L-alanine.

[0060] The culture medium used for cultivation should be appropriately tailored to meet the requirements of the specific strain. Culture media for Corynebacterium strains are known. For example, the microorganism of the present application can be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., while adjusting the temperature and pH. Carbon sources include carbohydrates such as glucose, fructose, and sucrose, and amino acids such as glutamic acid and cysteine. Natural organic nutrient sources such as starch hydrolysates and molasses can be used, with carbohydrates such as glucose, fructose, and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) being preferred. Various other carbon sources can be used in appropriate amounts without limitation, but are not limited to these. Nitrogen sources include inorganic nitrogen sources such as ammonia; amino acids such as glutamic acid and cysteine; and organic nitrogen sources such as peptone, meat extract, and yeast extract. These nitrogen sources can be used alone or in combination, but are not limited to these. The medium may contain, but is not limited to, phosphoric acid, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or the corresponding sodium-containing salts as a phosphorus source. Inorganic compounds may include magnesium sulfate, iron sulfate, manganese sulfate, and calcium chloride, and may also contain amino acids, vitamins, and appropriate precursors. These media or precursors may be added to the culture in a batch or continuous manner, but are not limited to these.

[0061] During cultivation, compounds such as potassium hydroxide, ammonia, and phosphoric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. Additionally, antifoaming agents such as fatty acid polyglycol esters can be used to suppress foam formation during cultivation. Oxygen or oxygen-containing gas can be injected into the culture to maintain an aerobic state. The culture temperature is 27°C to 37°C, specifically 30°C to 33°C. The cultivation period can be continued until the desired amount of useful substances is produced, specifically 20 to 120 hours.

[0062] The amino acid recovery step may involve collecting the target amino acid from the culture medium, culture solution, or microorganism using a suitable method known in the art depending on the culture method. For example, the recovery step may be performed by one or more methods selected from, but not limited to, centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc. The method for producing the amino acid may additionally include a purification step before, during, or after the recovery step. [Effects of the Invention]

[0063] 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 of the present invention, in which a specific site in the promoter region of the gene encoding AvtA has been mutated, has significantly increased L-alanine-producing ability, and therefore the novel polynucleotide can be usefully used for efficient L-alanine production. DETAILED DESCRIPTION OF THE INVENTION

[0064] The present invention will be described in more detail below with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. [Example]

[0065] Example 1. Construction of a strain with a mutation introduced into the promoter region of the avtA gene We attempted to create a mutant strain in which the 47th nucleotide G was replaced with T, the 48th nucleotide G with A, and the 49th nucleotide C with A in the promoter sequence of the avtA gene.

[0066] Specifically, to introduce the mutation into wild-type Corynebacterium glutamicum (ATCC13869) (substituting nucleotide G at position 47 with T, nucleotide G at position 48 with A, and nucleotide C at position 49 with A in the promoter region sequence represented by SEQ ID NO: 2), a gene fragment containing the mutation was obtained by PCR using primer pairs represented by SEQ ID NO: 3 and SEQ ID NO: 4, and primer pairs represented by SEQ ID NO: 5 and SEQ ID NO: 6. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, 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 501 bp polynucleotide was obtained by amplified using primers represented by SEQ ID NO: 3 and SEQ ID NO: 4, and a 501 bp polynucleotide was obtained by amplified using primers represented by SEQ ID NO: 5 and SEQ ID NO: 6. The two resulting gene fragments were ligated using infusion enzyme into pDCM2 (Korea Publication No. 10-2020-0136813), which had been digested with the restriction enzymes BamHI and SalI, to create a gene replacement vector containing the avtA promoter mutation, which was named pDCM2-Pm_avtA. The primer sequences used to construct this vector are listed in Table 1 below.

[0067] [Table 1]

[0068] The vector was transformed into Corynebacterium glutamicum ATCC13869 by electroporation (Appl. Microbiol. Biotechnol., 1999), and then plated on complex plates containing 25 mg / L kanamycin to obtain colonies. A secondary crossover process was then performed to obtain the final strain containing the mutation and the introduced vector, from which the vector was removed. The strain containing the mutation was finally confirmed by gene sequence analysis using the primer pair of SEQ ID NO: 7 (5'-CGTACACCACCATCAAGGACA-3') and SEQ ID NO: 8 (5'-CAAAGGACCCGAGCGAAGAGC-3'). The strain containing the targeted mutation was designated CJ0011.

[0069] Example 2. Confirmation of L-alanine production in strains into which novel promoter genes have been introduced To compare the L-alanine producing ability of the mutant CJ0011 strain prepared in Example 1 and the wild-type Corynebacterium glutamicum ATCC13869 strain, they were cultured as follows.

[0070] Specifically, the parent strain Corynebacterium glutamicum ATCC13869 and the mutant strain CJ0011 were inoculated into a 250 mm corner-baffle flask containing 25 ml of production medium, and then cultured at 30° C. for 46 hours at 200 rpm.

[0071] After the cultivation was completed, the L-alanine concentration in each medium was measured using high performance liquid chromatography, and the L-alanine concentration in the culture medium of each strain tested is shown in Table 2 below.

[0072] [Table 2]

[0073] As a result, as shown in Table 2, the mutant Corynebacterium glutamicum CJ0011 strain produced L-alanine at a concentration of 7.5 g / L, demonstrating approximately 132% L-productivity compared to the parent strain. The above results indicate that the mutations in which the 47th nucleotide G is substituted with T, the 48th nucleotide G with A, and the 49th nucleotide C with A in the promoter region sequence shown in SEQ ID NO: 2 significantly increase the L-alanine producing ability of the microorganism, and therefore the promoter in which the 47th nucleotide G is substituted with T, the 48th nucleotide G with A, and the 49th nucleotide C with A in the promoter region sequence shown in SEQ ID NO: 2 can be useful in methods for producing L-alanine.

[0074] The composition of the medium used in Example 2 is as follows: <Activation medium> 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) 100μl / L

[0075] <Seed medium> Glucose (anhydrous glucose) 20g / L, polypeptone 10g / L, yeast extract 10g / L, ammonium sulfate [(NH4)2SO4] 10g / L, urea 1.5g / L, potassium phosphate monobasic (KH2PO4) 5.2g / L, potassium phosphate dibasic (K2HPO4) 10.7g / L, d-biotin 1.8mg / L, thiamine-HCl 9mg / L, CAPA 9mg / L, NCA 60mg / L, magnesium sulfate (MgSO4) 0.5g / L

[0076] <Production medium> 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-HCl 0.1mg / L, MnSO4 6.7mg / L, FeSO4 10mg / L

Claims

1. A polynucleotide in which the nucleotide at position 47 of the nucleotide sequence represented by SEQ ID NO: 2 is replaced with T, and the nucleotides at positions 48 and 49 are each replaced with A.

2. The polynucleotide of claim 1, wherein the polynucleotide consists of the nucleotide sequence of SEQ ID NO:

1.

3. The polynucleotide according to claim 1 or 2, which has promoter activity.

4. A recombinant vector comprising the polynucleotide of claim 3 and a gene encoding a target protein operably linked to said polynucleotide.

5. 5. The recombinant vector according to claim 4, wherein the target protein is an aminotransferase.

6. A Corynebacterium microorganism comprising the polynucleotide of claim 3 and a gene encoding a target protein operably linked to said polynucleotide.

7. The Corynebacterium microorganism according to claim 6 , wherein the polynucleotide consists of the nucleotide sequence of SEQ ID NO:

1.

8. The Corynebacterium microorganism according to claim 6, wherein the target protein is an aminotransferase.

9. The microorganism of the genus Corynebacterium according to any one of claims 6 to 8, wherein the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.

10. A method for producing an amino acid, comprising culturing the Corynebacterium microorganism according to any one of claims 6 to 8 in a medium.

11. 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 any one of claims 6 to 8.

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

Images