Recombinant microorganism having improved l-methionine production ability through increased dihydrofolate reductase activity, and l-methionine production method using same

Abstract

The present invention relates to a recombinant microorganism having improved L-methionine production ability, and a L-methionine production method using same, and more specifically, the present invention may provide a strain, and a L-methionine production method or a composition for L-methionine production, using same, the strain inducing mutation in the transcription regulatory region of the folA gene, thereby enhancing dihydrofolate reductase activity and thus improving L-methionine production ability.

Description

Recombinant microorganism with enhanced L-methionine production capacity by increasing dihydrofolate reductase activity and method for producing L-methionine using the same

[0001] The present invention provides a recombinant microorganism having improved L-methionine production ability due to increased dihydrofolate reductase activity, a method for producing L-methionine using the same, and a composition for producing L-methionine.

[0002] L-methionine is an essential amino acid for animal cells, and is an amino acid produced from homoserine, which is derived from aspartic acid, as a precursor. It is also widely used as a feed and food additive, and as a raw material for pharmaceuticals.

[0003] Methionine is produced from DL-methionine by chemical synthesis, and there is a two-step biological process in which it is produced by enzymatic conversion from O-acetylhomoserine or O-succinylhomoserine precursors produced by microbial fermentation. There is also a microbial fermentation process in which methionine is produced from biomass carbon sources.

[0004] In production by microbial fermentation, various studies have been conducted to increase the production capacity of L-methionine.

[0005] For example, Patent Publication No. 10-2005-0034745 reported that the L-methionine production ability was increased by strengthening the metF gene encoding MetF (methylene THF reductase), which converts L-serine to glycine by GlyA (L-Serine THF methyltransferase) in a Corynebacterium glutamicum L-methionine-producing strain and reduces the methylene THF produced by transferring a methyl group to THF to methyl-THF, a substrate of L-methionine synthase.

[0006] Meanwhile, in the case of methylene-THF or methyl-THF, which is the main raw material of L-methionine, it is an essential precursor for nucleic acid production. In particular, when UMP is converted to TMP by TMP synthase, methylene-THF transfers a methyl group to UMP, THF is oxidized and converted to dihydrofolic acid, which lowers the intracellular concentration of THF and may affect L-methionine production.

[0007] Accordingly, the present inventors confirmed that the activity of dihydroxyfolate reductase encoded by the folA (dihydrofolate reductase) gene, which converts folic acid or dihydrofolic acid into THF, is important for the supply of THF, and attempted to develop a mutant strain with improved L-methionine production ability by enhancing its activity.

[0008] The purpose of the present invention is to provide a recombinant microorganism having the ability to produce L-methionine by enhancing the activity of dihydrofolate reductase, which reduces folic acid or dihydrofolic acid in the THF biosynthetic pathway to THF in order to facilitate the supply of THF.

[0009] Another object of the present invention is to provide a composition for producing L-methionine comprising the recombinant microorganism, a culture thereof, or a lysate thereof.

[0010] Another object of the present invention is to provide a method for producing L-methionine, comprising the steps of (a) culturing the recombinant microorganism in a culture medium; and (b) recovering L-methionine from the cultured culture medium.

[0011] To achieve the above purpose, the present invention provides a recombinant microorganism having enhanced dihydrofolate reductase activity and the ability to produce L-methionine.

[0012] In addition, the present invention provides a composition for producing L-methionine comprising the recombinant microorganism, a culture thereof, or a lysate thereof.

[0013] In addition, the present invention provides a method for producing L-methionine, comprising the steps of (a) culturing the recombinant microorganism in a culture medium; and (b) recovering L-methionine from the cultured culture medium.

[0014] The present invention relates to a recombinant microorganism having improved L-methionine production ability and a method for producing L-methionine using the same. More specifically, the present invention provides a strain having improved L-methionine production ability by inducing a mutation in the transcriptional regulatory region of the folA gene to enhance dihydrofolate reductase activity, and a method for producing L-methionine using the same or a composition for producing L-methionine can be provided.

[0015] Figure 1 illustrates a process for producing L-methionine in a Corynebacterium glutamicum strain according to the present invention.

[0016] Figure 2 shows the chromosomal structure of the folA gene, the transcription control region, and the mutant base sequence of the transcription control region for increasing the amount of transcription.

[0017] Figure 3 shows a vector map of pFAME75, a vector introducing mutations in the transcriptional regulatory region of the thyA-folA operon to enhance folA expression.

[0018] Hereinafter, the present invention will be described in more detail.

[0019]

[0020] The present invention provides a recombinant microorganism having enhanced dihydrofolate reductase activity and the ability to produce L-methionine.

[0021] The above recombinant microorganism is characterized by enhancing the expression of the folA gene as follows:

[0022] (i) an increase in the number of copies of a gene in a microorganism;

[0023] (ii) use of promoters that lead to high levels of expression of the gene;

[0024] (iii) attenuation of the activity or expression of a specific or non-specific transcriptional repressor of a gene, or

[0025] (iv) Use of an element that stabilizes the corresponding messenger RNA or an element that stabilizes the protein.

[0026] The above recombinant microorganism can have an enhanced protein activity encoded by the folA gene compared to the endogenous activity.

[0027] The above recombinant microorganism can cause mutations in the transcriptional regulatory region of folA, resulting in chromosomal overexpression of the folA gene.

[0028] The above recombinant microorganism may include a promoter mutation region of the thyA-folA operon.

[0029] The above promoter mutation region may be a base sequence represented by sequence number 4.

[0030] The thyA-folA operon including the above promoter mutation region may include the base sequence represented by SEQ ID NO: 2.

[0031] The recombinant microorganism may be, but is not limited to, a microorganism belonging to the genus Corynebacterium sp., the genus Escherichia sp., or the genus Lactobacillus sp. For the purposes of the present invention, the microorganism may be included without limitation as long as the intrinsic activity of the FolA protein is enhanced or the production ability of L-amino acids and / or derivatives thereof is increased by the introduction of an exogenous FolA protein.

[0032] The above genus Corynebacterium may include all microorganisms of the genus Corynebacterium. Specifically, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli pollutisoli), Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium casei, Corynebacterium glycinophilum, or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium crenatum, or Corynebacterium deserti.

[0033] The above genus Escherichia may include all Escherichia microorganisms. Specifically, it may be, but is not limited to, Escherichia coli.

[0034] The above recombinant microorganism may be Corynebacterium glutamicum deposited under the deposit number KCTC15815BP.

[0035] Meanwhile, it has already been known that the above-mentioned microorganisms of the genus Corynebacterium or Escherichia can produce L-amino acids, but their production ability is significantly low, and neither the genes nor the mechanism acting on the production mechanism have been elucidated. Therefore, the 'microorganism producing L-amino acid' of the present invention refers to a natural wild-type microorganism itself, a microorganism that has improved L-amino acid production ability by strengthening or weakening / inactivating the activity of genes related to the L-amino acid production mechanism, or a microorganism that has improved L-amino acid production ability by introducing or strengthening the activity of an external gene. The culture of the microorganism of the present invention may be prepared by culturing the microorganism of the present invention in a medium.

[0036]

[0037] In addition, the present invention provides a composition for producing L-methionine comprising the recombinant microorganism, a culture thereof, or a lysate thereof.

[0038] Another aspect of the present invention provides a composition for producing L-methionine, comprising a recombinant microorganism, a culture or a lysate thereof, in which the activity of a protein encoded by the folA gene is enhanced compared to the endogenous activity.

[0039]

[0040] In addition, the present invention provides a method for producing L-methionine, comprising the steps of (a) culturing the recombinant microorganism in a culture medium; and (b) recovering L-methionine from the cultured culture medium.

[0041] The above culture medium may include a carbon source and a sulfur source.

[0042] The carbon source may be, but is not limited to, glucose, sucrose, formic acid, or a combination thereof.

[0043] The sulfur source may be, but is not limited to, sulfate, thiosulfate, hydrogen sulfide, dithionate, dithionite, sulfite or a combination thereof.

[0044] The recombinant microorganism can be further modified by overexpressing an allele of hom encoding a homoserine oxidoreductase with reduced feedback sensitivity to methionine.

[0045] The above recombinant microorganism can be further modified by overexpressing any one or more genes selected from the group consisting of genes cysP, cysU, cysA, cysM, cysI, cysJ, cysH, serA, serB, serC, glyA, metF, hom and metH, but is not limited thereto.

[0046] The above recombinant microorganism can be further modified by suppressing the expression of any one or more genes selected from the group consisting of genes pykA, pykF, purU and mcbR, but is not limited thereto.

[0047]

[0048] The L-methionine-producing microorganism of the present invention can produce L-methionine with higher digestibility than chemical synthesis of DL-methionine in an environmentally friendly manner. The produced L-methionine can be widely used as an animal feed additive, food additive, or pharmaceutical raw material.

[0049] One aspect of the present invention provides a method for producing L-methionine using a microorganism in which the activity of a protein encoded by folA is enhanced compared to its intrinsic activity. The present invention provides a method for producing L-methionine, comprising culturing a genetically modified microorganism in an L-methionine production medium, wherein the microorganism may include a genetic modification that increases the activity of a protein encoded by the folA gene compared to the microorganism prior to the genetic modification.

[0050] In the present invention, the 'protein encoded by the folA gene' may be referred to as 'FolA' as a protein encoded by the folA gene or a protein expressed by the folA gene. (Hereinafter referred to as 'FolA protein'). The FolA protein may be defined as an enzyme that catalyzes the reduction of folic acid to dihydrofolic acid or the reduction of dihydrofolic acid to tetrahydrofolic acid using the reducing power of NAD(P)H within the cell.

[0051] In the present invention, the term "enhancing the activity of a protein compared to its intrinsic activity" can also be expressed as "increased activity", and means that the intrinsic activity of a protein of a microorganism prior to a genetic modification or a non-modified or unmodified microorganism is enhanced compared to the activity before modification. The term "intrinsic" in the present invention means the state that the parent strain originally had before the change in traits, when the traits of a microorganism are changed due to genetic mutation caused by natural or artificial factors. The activity increase may include both the introduction of a foreign protein and the enhancement of the activity of the endogenous protein. The activity increase / enhancement of the protein can be achieved by the increase / enhancement of gene expression.

[0052] Specifically, in the present invention, active enhancement is

[0053] 1) Increase in the copy number of the polynucleotide encoding the above protein,

[0054] 2) Modification of the expression control sequence to increase the expression of the above polynucleotide,

[0055] 3) Modification of the polynucleotide sequence on the chromosome to enhance the activity of the above protein;

[0056] 4) Introduction of a foreign polynucleotide exhibiting the activity of the above protein or a codon-optimized variant polynucleotide of the above polynucleotide, or

[0057] 5) It can be performed by a method of transforming it to be strengthened by a combination of these, but is not limited thereto.

[0058] The above 1) increase in the copy number of the polynucleotide may be performed in a form operably linked to a vector, or by inserting an additional homologous gene into a chromosome in a host cell, but is not particularly limited thereto. Specifically, the increase may be performed by introducing a polynucleotide encoding the protein of the present invention into a host cell by operably linking the polynucleotide to a vector capable of replicating and functioning independently of the host, or by introducing the polynucleotide into a vector capable of inserting the polynucleotide into a chromosome in the host cell by operably linking the polynucleotide to the vector, thereby increasing the copy number of the polynucleotide in the chromosome of the host cell.

[0059] Next, 2) Modification of the expression control sequence to increase the expression of the polynucleotide may be performed by, but is not particularly limited to, inducing a sequence mutation in the nucleic acid sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further enhance the activity of the expression control sequence, or by replacing the nucleic acid sequence with a nucleic acid sequence having stronger activity. The expression control sequence may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, etc. A strong heterologous promoter may be linked upstream of the above polynucleotide expression unit instead of the original promoter. Examples of the strong promoter include, but are not limited to, endogenous promoters derived from Corynebacterium such as the gapA promoter, the EF-Tu promoter, the groEL promoter, the aceA or aceB promoter, promoters derived from foreign microorganisms such as the CJ7 promoter (Korean Patent No. 0620092 and WO2006 / 065095), and synthetic promoters such as the lysCP1 promoter (WO2009 / 096689), the spl1 promoter, the spl7 promoter, and the spl13 promoter (Korean Patent No. 1783170).

[0060] In addition, 3) modification of the polynucleotide sequence on the chromosome is not particularly limited thereto, but may be performed by inducing mutations in the expression control sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of the nucleic acid sequence to further enhance the activity of the polynucleotide sequence, or by replacing it with a polynucleotide sequence that has been improved to have stronger activity.

[0061] In addition, 4) introduction of a foreign polynucleotide sequence can be performed by introducing a foreign polynucleotide encoding a protein exhibiting the same / similar activity as the protein, or a codon-optimized mutant polynucleotide thereof, into a host cell. The foreign polynucleotide can be used without limitation in its origin or sequence as long as it exhibits the same / similar activity as the protein. In addition, the introduced foreign polynucleotide can be introduced into a host cell by optimizing its codons so that optimized transcription and translation can occur within the host cell. The introduction can be performed by a person skilled in the art appropriately selecting a known transformation method, and the introduced polynucleotide can be expressed within the host cell, thereby producing a protein and increasing its activity.

[0062] Finally, 5) the method of modifying to be strengthened by a combination of the above 1) to 4) can be performed by applying at least one of the following methods together: increasing the copy number of the polynucleotide encoding the protein, modifying the expression control sequence to increase the expression thereof, modifying the polynucleotide sequence on the chromosome, and modifying a foreign polynucleotide exhibiting the activity of the protein or a codon-optimized mutant polynucleotide thereof.

[0063] The term "vector" of the present invention refers to a DNA construct containing a polynucleotide sequence encoding a target protein operably linked to a suitable regulatory sequence so as to enable expression of the target protein in a suitable host. The regulatory sequence may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. The vector, after being transformed into a suitable host cell, can replicate or function independently of the host genome, and can be integrated into the genome itself. For example, a vector for intracellular chromosomal integration can be used to replace a polynucleotide encoding a target protein in a chromosome with a mutated polynucleotide. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, such as, but not limited to, homologous recombination.

[0064] The vector of the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0065] The term "transformation" of the present invention refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed within the host cell. The transformed polynucleotide may be located within the chromosome of the host cell or outside the chromosome, as long as it can be expressed within the host cell. Furthermore, the polynucleotide includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all the elements necessary for autonomous expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0066] In addition, the term "operably linked" of the present invention means that the gene sequence is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding the target protein of the present invention. The method for transforming the vector of the present invention includes any method for introducing a nucleic acid into a cell, and can be performed by selecting an appropriate standard technique known in the art depending on the host cell. Examples thereof include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0067] The above microorganism may be a microorganism that produces L-methionine.

[0068] The term "microorganism producing L-methionine" of the present invention refers to a microorganism that naturally has the ability to produce L-methionine or a microorganism that has been given the ability to produce L-amino acids to a parent strain that does not have the ability to produce L-amino acids. For example, the microorganism producing L-methionine may be a microorganism in which the L-methionine biosynthesis pathway is strengthened or the degradation pathway is weakened. For example, the microorganism producing L-amino acids may be a microorganism in which the activity of McbR (methionine and cysteine ​​biosynthesis repressor protein) or MetJ protein is reduced or inactivated, or the activity of methionine synthase (MetH) or sulfite reductase (CysI) is strengthened, thereby strengthening and / or adding the ability to produce methionine.

[0069] Alternatively, it may be a microorganism that promotes the expression of genes encoding enzymes of other L-amino acid biosynthetic pathways or weakens / inactivates enzymes of the degradation pathway. Specifically, thiosulphate sulphurtransferases such as Rdl2p, GlpE, PspE, YgaP, ThiI, YbbB, SseA, YnjE, YceA, YibN, NCgl0671, NCgl1369, NCgl2616, NCgl0053, NCgl0054, NCGl2678, NCgl2890; sulfite reductase, cysI; thiosulphate / sulfate transport system, cysPUWA (EC 3.6.3.25); 3′-phosphoadenosine 5′-phosphosulphate reductase, cysH (EC 1.8.4.8); sulfite reductase, cysJI (EC 1.8.1.2); cysteine ​​synthase A, cysK (EC 2.5.1.47); cysteine ​​synthase B, cysM (EC 2.5.1.47); serine acetyltransferase, cysE (EC 2.3.1.30); glycine cleavage system, gcvTHPlpd (EC 2.1.2.10, EC 1.4.4.2, EC 1.8.1.4); Lipoyl synthase, lipA (EC 2.8.1.8); lipoyl protein ligase, lipB (EC 2.3.1.181); phosphoglycerate dehydrogenase, serA (EC 1.1.1.95); 3-phosphoserine phosphatase, serB (EC 3.1.3.3); 3-phosphoserine / phosphohydroxythreonine aminotransferase, serC (EC 2.6.1.52); serine hydroxymethyltransferase, glyA (EC 2.1.2.1); aspartokinase I (EC 2.7.2.4); homoserine dehydrogenase, thrA (EC 1.1.1.3); aspartate kinase, lysC (EC 2.7.2.4); homoserine dehydrogenase, hom (EC 1.1.1.3); Homoserine O-acetyltransferase, metX (EC 2.3.1.31); homoserine O-succinyltransferase, metA (EC 2.3.1.46); cystathionine gamma-synthase, metB (EC 2.5.1.48); β-CS-lyase, aecD (EC 4.4.1.8, beta-lyase); cystathionine beta-lyase, metC (EC 4.4.1.8); B12-independent homocysteine ​​S-methyltransferase, metE (EC 2.1.1.14); methionine synthetase, metH (EC 2.1.1.13); methylenetetrahydrofolate reductase, metF (EC 1.5.1.20); L-methionine exocytote, BrnFE; valine exocytote, YgaZH (B2682, B2683), ygaZH (b2682.b2683); exotransporter YjeH, b4141; pyridine nucleotide transhydrogenase PntAB, pntAB (EC 1.6.1.2); O-succinylhomoserine sulfhydrylase, MetZ (EC 2.5.1.48); and phosphoenolpyruvate carboxylase, Pyc (EC 4.1.1.31), wherein the activity of one or more proteins or some proteins constituting the system is enhanced or a polynucleotide encoding the same is overexpressed to enhance the L-amino acid biosynthetic pathway or weaken the degradation pathway. Alternatively, glucose-6-phosphate isomerase, pgi (EC 5.3.1.9); homoserine kinase, thrB (EC 2.7.1.39); S-adenosylmethionine synthase, metK (EC 2.5.1.6); dihydrodipicolinate synthase, dapA (EC 4.2.1.52); phosphoenolpyruvate carboxykinase, pck (EC 4.1.1.49); formyltetrahydrofolate hydrolase, purU (EC 3.5.1.10); pyruvate kinase I, pykF (EC 2.7.1.40); pyruvate kinase II, pykA (EC 2.7.1.40); cystathionine γ-lyase, cg3086 (EC 4.4.1.1); cystathionine β-synthase, cg2344 (EC 4.2.1.22); Regulatory protein Cg3031, cg3031; methionine and cysteine ​​biosynthesis repressor protein McbR, mcbR; L-methionine synthesis transcriptional repressor protein (Met transcriptional repressor protein), metJ; L-methionine transporter MetQNI, metQ, metN, metI; N-acyltransferase, yncA; sRNAfnrS; and L-methionine transporter, metP, the activity of one or more proteins selected from the group consisting of may be inactivated or weakened, or the expression of a gene encoding the protein may be inhibited or eliminated.

[0070] However, this is only one example, and the present invention is not limited to microorganisms that enhance the expression of genes encoding enzymes in various known L-amino acid biosynthetic pathways or weaken / inactivate enzymes in the degradation pathways. The above-mentioned L-amino acid-producing microorganisms can be produced using various known methods.

[0071] The term "attenuation / inactivation of protein activity" of the present invention means that the expression of an enzyme or protein is not expressed at all, or even if it is expressed, the activity is absent or reduced compared to a natural wild-type strain, a parent strain, or a strain in which the protein is not modified. In this case, the reduction is a concept that also includes cases in which the activity of the protein is reduced compared to the activity of the protein originally possessed by the microorganism due to mutation of the gene encoding the protein, modification of the expression regulatory sequence, deletion of part or all of the gene, etc., and cases in which the overall activity of the protein in the cell is lower than that of the natural strain or the strain before modification due to inhibition of expression or translation of the gene encoding the protein, etc., and combinations thereof. In the present invention, the inactivation / attenuation can be achieved by applying various methods well known in the art.Examples of the above method include: 1) a method of deleting all or part of the gene encoding the protein; 2) modification of an expression regulatory sequence so as to reduce expression of the gene encoding the protein; 3) modification of the gene sequence encoding the protein so as to eliminate or weaken the activity of the protein; 4) introduction of an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to a transcript of the gene encoding the protein; 5) a method of forming a secondary structure by adding a sequence complementary to the Shine-Dalgarno sequence to the front of the Shine-Dalgarno sequence of the gene encoding the protein, thereby making attachment of the ribosome impossible; 6) There is a method of adding a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence of the gene encoding the protein (Reverse transcription engineering, RTE), and a combination of these can also be achieved, but is not particularly limited thereto, and an inactivation method known in the art can be appropriately selected and applied.

[0072] The term "cultivation" of the present invention means growing the microorganism under appropriately controlled environmental conditions. The culturing process of the present invention can be performed according to an appropriate medium and culture conditions known in the art. This culturing process can be easily adjusted and used by a person skilled in the art according to the selected strain. The step of culturing the microorganism is not particularly limited thereto, but can be performed by a known batch culture method, continuous culture method, fed-batch culture method, etc. At this time, the culture conditions are not particularly limited thereto, but can be adjusted to an appropriate pH (e.g., pH 5 to 9, specifically pH 7 to 9) using a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid). In addition, during the culture, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation, and in addition, in order to maintain an aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture, or in order to maintain an anaerobic or microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected. The culture temperature can be maintained at 25°C to 40°C, specifically 30°C to 37°C, but is not limited thereto. The culture period can be continued until the desired amount of useful material is obtained, and specifically, the culture can be performed for about 0.5 to 80 hours, but is not limited thereto. In addition, the culture medium used may be used as a carbon source, including, but not limited to, sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), fats and oils (e.g., soybean oil, sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), and organic acids (e.g., acetic acid), which may be used individually or in combination.Nitrogen sources include, but are not limited to, nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea), or inorganic compounds (e.g., ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate), which may be used individually or in combination. Phosphorus sources include, but are not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts, which may be used individually or in combination. In addition, the medium may include essential growth promoting substances, such as other metal salts, amino acids, and vitamins.

[0073] The method may comprise recovering L-methionine from the microorganism or culture medium.

[0074] The above-described recovery step can recover L-methionine from the medium using a suitable method known in the art according to the culture method of the microorganism of the present invention, such as a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallization protein precipitant (staining method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC and combinations thereof can be used, but the present invention is not limited to these examples.

[0075] The above recovery step may include an additional purification process. The purification process may utilize any suitable method known in the art.

[0076] The proteins, microorganisms, and cultures encoded by the folA gene are as described above.

[0077] The above composition may further include any component that can assist in the production of L-methionine, and such component may be appropriately selected from those known in the art.

[0078]

[0079] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0080] In this specification, FolA is a protein, and folA refers to a gene encoding it.

[0081]

[0082] Example 1. Construction of a recombinant vector for enhanced FolA expression.

[0083] In order to increase the expression level of the FolA (Dihydrofolate reductase, hereinafter referred to as FolA) protein of Corynebacterium glutamicum FAME18B1, an L-methionine-producing strain, a mutation capable of increasing the transcription amount of mRNA was introduced into the folA transcriptional regulatory region, and a vector for this purpose was created.

[0084] Specifically, in order to introduce mutations in the transcriptional regulatory region to increase the transcript amount of the folA gene of sequence number 1, the folA gene and surrounding sequences of Corynebacterium glutamicum were secured based on the base sequence reported in the National Institutes of Health (NIH Genbank) of the United States. In addition, as a result of confirming with reference to a paper that confirmed the transcriptional regulatory region of all genes through analysis of the RNA transcriptome of Corynebacteria (Pfeifer-Sancar et al., BMC Genomics, 14:888, 2013), it was confirmed that it consists of one operon with the thyA gene, and as shown in Fig. 2, the transcriptional regulatory region is located from base sequence -4 to -13 from the start codon of the thyA gene as shown in sequence number 3.

[0085] Referring to the method presented in the paper (Pavla Vasicova, et al., J Bacteriol., 181(19): 6188-6191, 1999 Oct) in which the expression level of the thyA-folA operon was increased by introducing a mutation in the dapA transcriptional regulatory region, and referring to the folA gene and surrounding sequences of Corynebacterium glutamicum and the consensus sequence of the transcriptional regulatory region of Corynebacterium glutamicum, a vector having a mutation in the sequence number 4 was constructed as shown in Fig. 2.

[0086] Specifically, the above paper presented a strong transcriptional regulatory element sequence, TgTG(c / G)TATAATGG, based on the results of confirming the expression level by introducing various mutations in the nucleotide sequence of the common -10 transcriptional regulatory region of Corynebacterium glutamicum, which is T1A2(T / G)3A4A5T6, and the adjacent region. Based on this, the transcriptional regulatory element sequence of the thyA-folA operon shown in SEQ ID NO: 3 was attempted to introduce a mutation in the same sequence as SEQ ID NO: 4, based on the nucleotide sequence that showed the highest expression level experimentally presented in the above paper. To this end, a vector capable of introducing a mutation in the -10 region of the transcriptional regulatory region of thyA-folA on the chromosome was constructed.

[0087] The vector was constructed using the PCR primers and conditions presented in Table 1 below to amplify DNA fragments for mutagenesis, and the vector was constructed using the self-assembly cloning (Self Assembly Cloning, Akira Matsumoto, Biotechniques, 51(1), 2011 Jul.) method. The constructed vector was confirmed through base sequence analysis and named pFAME75. The vector map of pFAME75 is presented in Figure 3.

[0088]

[0089] Primer sequence (5'-3')pCGI-FL5'-AATTCACTGGCCGTCGTTTTAC-3'pCGI-RS5'-TGTGTGAAATTGTTATCCGCTCAC-3'pCGI-FS5'-GTCGTTTTACAACGTCGTGACTG-3'pCGI-RL5'-AGCTGTTTCCTGTGTGAAATTGTT-3'C M-06015'-GGAAACAGCTCGTTTGGTTCCGGCCATG-3'CM-06025'-TTTGGTTACATTAAGGCATGGCTAAAAG-3'CM-06035'-CGTTTGGTTCCGGCCATG-3'CM-06045'-TCAATTATAGATGTTTGGTTACATTAAGGCATGG-3'CM -06055'-CATCTAtAATTGAGAACATGACTGCTCAGATTGATG-3'CM-06065'-CGTTAGTAAGTTCCATTCTCCTCG-3'CM-06075'-GAACATGACTGCTCAGATTGATGATTCG-3'CM-06085'-GCCGCAATTATAACAGCGTTAGTAAG TTCC-3'CM-06095'-CTGTTATAATTGCGGCATGACTGTTCCAAC-3'CM-06105'-CAGAACGCTGGTAGAGCTGG-3'CM-06115'-ATGACTGTTCCAACGCCTTA-3'CM-06125'-GGCCAGTGAATTCAGAACGCTGGTAGAGCTGG-3'

[0090] Example 2. Production and cultivation of strains with enhanced FolA expression.

[0091] Using the vector produced in Example 1 above, a strain was produced in which a mutation was introduced into the transcriptional control region of the thyA-folA operon, referring to the paper (Il Kwon Kim et al., Appl. Microbiol. Biotechnol., 52:541-545, 1999) on chromosome recombination using a mutation in rpsL, a gene encoding small ribosomal protein 12 that confers streptomycin resistance to Corynebacterium glutamicum.

[0092] The pFAME75 vector, which is incapable of replicating in Corynebacterium, was transformed into the genetically recombinant L-methionine-producing strain Corynebacterium glutamicum FAME15B1 of the Corynebacterium ATCC13032 strain by electroporation through homologous recombination on the chromosome. Colonies in which the vector was introduced into the chromosome by the first homologous recombination were selected on CM agar medium containing kanamycin with the following composition.

[0093]

[0094] (CM agar medium, pH 6.8)

[0095] Glucose 10 g, polypeptone 10 g, yeast extract 10 g, urea 1.2 g, NaCl 1.5 g, agar 18 g, kanamycin 50 mg (per 1 liter of distilled water)

[0096]

[0097] After that, the colonies containing the vector in the chromosome by the first homologous recombination were cultured in CM liquid medium excluding CM agar medium, and the second recombinant strain was selected on CM agar medium containing streptomycin (50 mg / L) instead of kanamycin. The above-mentioned Corynebacterium glutamicum transformed strain that completed the second recombination was subjected to colony PCR using the PCR primers presented in Table 2 to amplify the transcriptional regulatory region, and then the strain in which a mutation was introduced into the thyA-folA operon promoter was confirmed through base sequence analysis, and this genetically recombinant strain was named Corynebacterium glutamicum FAME16C3. The constructed strain was deposited with the Biological Resource Center (International) of the Korea Research Institute of Bioscience and Biotechnology and assigned the accession number KCTC15815BP.

[0098]

[0099] Primer sequence (5'-3') pCGI-FL5'-AATTCACTGGCCGTCGTTTTAC-3'pCGI-RS5'-TGTGTGAAATTGTTATCCGCTCAC-3'

[0100] Example 3. Analysis of L-methionine production capacity of L-methionine producing strain with increased folA gene expression.

[0101] In order to analyze the L-methionine production ability of the L-methionine-producing strain Corynebacterium glutamicum FME16C3 produced in Examples 1 and 2 above, it was cultured together with the parent strain Corynebacterium glutamicum FAME15B1 using the following method.

[0102] Corynebacterium glutamicum FAME15B1 and Corynebacterium glutamicum FAME16C3 were each inoculated into a bioreactor tube containing 10 ml of the following seed medium and cultured at 30°C and 180 rpm for 16 hours. Thereafter, 1 ml of the seed culture solution was inoculated into a 150 ml corner-baffle flask containing 15 ml of the production medium and cultured at 30°C and 180 rpm for 72 hours. The compositions of the above seed medium and production medium are as follows, respectively.

[0103]

[0104] (seed medium, pH 6.8)

[0105] Glucose 10 g, polypeptone 10 g, yeast extract 10 g, urea 1.2 g, NaCl 1.5 g (per 1 liter of distilled water)

[0106]

[0107] (Production medium, pH 6.8)

[0108] Glucose 50 g, soybean meal acid hydrolyzate 20 g, (NH4)2S2O3 12 g, KH2PO4 1 g, MgSO4·7H2O 1.2 g, biotin 1.8 mg, thiamine hydrochloride 8 mg, nicotinamide 60 mg, cyanocobalamin 2 mg, pyridoxal 5 mg, CaCO3 30 g (based on 1 liter of distilled water).

[0109]

[0110] The L-methionine concentration in the culture solution was analyzed by HPLC using the above culture method and is shown in Table 3 below.

[0111]

[0112] Strain OD (600 nm) L-methionine (g / L) Average FAME15B1-156.00.63 0.64 FAME15B1-255.20.67 FAME15B1-351.00.63 FAME16C3-152.60.73 0.71 FAME16C3-256.00.71 FAME16C3-255.20.69

[0113] As a result, it was confirmed that the yield of L-methionine increased when the FolA protein was strengthened in the L-methionine producing strain.

[0114]

[0115] The foregoing description of the present invention is for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0116] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0117]

[0118]

Claims

1. A recombinant microorganism having enhanced dihydrofolate reductase activity and the ability to produce L-methionine.

2. In claim 1, The above recombinant microorganism is a recombinant microorganism characterized in that the folA (Dihydrofolate reductase) gene encoding dihydrofolate reductase is chromosomally overexpressed.

3. In claim 2, The above recombinant microorganism is characterized by enhancing the expression of the folA gene as follows: (i) an increase in the number of copies of a gene in a microorganism; (ii) use of promoters that lead to high levels of expression of the gene; (iii) attenuation of the activity or expression of a specific or non-specific transcriptional repressor of a gene, or (iv) Use of an element that stabilizes the corresponding messenger RNA or an element that stabilizes the protein.

4. In claim 3, The above recombinant microorganism is a recombinant microorganism characterized in that the protein activity encoded by the folA gene is enhanced compared to the intrinsic activity.

5. In claim 3, The above recombinant microorganism is a recombinant microorganism characterized in that a mutation is induced in the transcriptional regulatory region of folA, resulting in chromosomal overexpression of the folA gene.

6. In claim 5, The above recombinant microorganism is a recombinant microorganism characterized in that it includes a promoter mutation region of the thyA-folA operon.

7. In claim 6, A recombinant microorganism characterized in that the promoter mutation region is a base sequence represented by sequence number 4.

8. In claim 6, A recombinant microorganism characterized in that the thyA-folA operon including the above promoter mutation region comprises a base sequence represented by sequence number 2.

9. In claim 1, A recombinant microorganism characterized in that the recombinant microorganism is Corynebacterium sp., Escherichia sp. or Lactobacillus sp.

10. In claim 9, The above Corynebacterium genus includes Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, and Corynebacterium. A recombinant microorganism characterized by being at least one selected from the group consisting of Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium casei, Corynebacterium glycinophilum, and Corynebacterium flavescens.

11. In claim 9, A recombinant microorganism characterized in that the above Escherichia genus is Escherichia coli.

12. In claim 1, The recombinant microorganism is characterized in that the recombinant microorganism is Corynebacterium glutamicum deposited under the deposit number KCTC15815BP.

13. A composition for producing L-methionine comprising a recombinant microorganism according to any one of claims 1 to 12, a culture thereof, or a lysate thereof. 14.(a) A step of culturing a recombinant microorganism according to any one of claims 1 to 12 in a culture medium; and (b) A method for producing L-methionine, comprising a step of recovering L-methionine from the cultured culture medium.

15. In claim 14, A method for producing L-methionine, characterized in that the above culture medium contains a carbon source and a sulfur source.

16. In claim 15, A method for producing L-methionine, characterized in that the carbon source is glucose, sucrose, formic acid or a combination thereof.

17. In claim 15, A method for producing L-methionine, characterized in that the sulfur source is sulfate, thiosulfate, hydrogen sulfide, dithionate, dithionite, sulfite or a combination thereof.

18. In claim 14, A method for producing L-methionine, wherein the recombinant microorganism is further modified by overexpressing an allele of hom encoding a homoserine oxidoreductase having reduced feedback sensitivity to methionine.

19. In claim 14, A method for producing L-methionine, characterized in that the recombinant microorganism is further modified by overexpressing at least one gene selected from the group consisting of genes cysP, cysU, cysA, cysM, cysI, cysJ, cysH, serA, serB, serC, glyA, metF, hom, and metH.

20. In claim 14, A method for producing L-methionine, characterized in that the recombinant microorganism is further modified by suppressing the expression of any one or more genes selected from the group consisting of genes pykA, pykF, purU, and mcbR.

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