Method for producing L-glutamic acid from nitrogen molecule by fermentation

Abstract

The purpose of the present invention is to develop a method for the fermentative production of L-glutamic acid based on nitrogen molecules in the atmosphere using nitrogen-immobilized bacteria. The present invention develops a culture method for maintaining a state in which nitrogen-immobilizing ability is increased in nitrogen-immobilizing bacteria. This culture method is characterized by mixing an organic acid such as citric acid into a nitrogen-restricted medium placed in an open container. A method for producing L-glutamic acid by fermentation depending on nitrogen immobilization can be developed by culturing a gene-altered strain that highly expresses a citrate synthase gene and a citrate transporter gene using this culture method. In this process, a method for producing L-glutamic acid by fermentation that depends on nitrogen immobilization by using a modified strain that highly expresses a citrate synthase gene or a 2-methylcitrate synthase gene can be developed. The present invention relates to a method for producing the microorganism and a method for culturing the microorganism.

Description

Technical field

[0001] The present invention relates to a culture medium capable of maximizing the nitrogen-fixing ability of nitrogen-fixing bacteria, a culture method using the culture medium, and further relates to fermentative production of L-glutamic acid using atmospheric nitrogen molecules using the culture medium and culture method, as well as microorganisms capable of fermenting L-glutamic acid using atmospheric nitrogen molecules. [Background Technology]

[0002] Some microorganisms have the ability to convert atmospheric nitrogen molecules into ammonia. This ability is called nitrogen fixation. Numerous studies have been conducted on microorganisms with nitrogen fixation, and hundreds of species have been discovered. However, since all of these belong to the kingdoms Eubacteria (eubacteria) or Archaea (archaea), they are collectively referred to as nitrogen-fixing bacteria. Nitrogen-fixing bacteria can grow using nitrogen molecules as their sole nitrogen source.

[0003] The enzyme involved in nitrogen fixation is nitrogenase, which carries out the following reaction under ideal reaction conditions in a test tube. N2+8H + +8e - +16ATP→2NH3+H2+16ADP+16Pi Nitrogenase is a complex enzyme composed of dinitrogenase, which reduces nitrogen molecules, and dinitrogenase reductase, which transfers electrons to dinitrogenase. There are three types of dinitrogenase: Mo, V, and Fe. All nitrogen-fixing bacteria possess the Mo type, which has an iron-molybdenum cofactor in its active center. Therefore, nitrogen-fixing bacteria cannot exert their nitrogen-fixing ability when their surrounding environment lacks sufficient molybdenum and iron. Furthermore, nitrogen-fixing bacteria do not exert their nitrogen-fixing ability when their surrounding environment contains a usable nitrogen source (ammonium salts, nitrates, protein decomposition products such as peptones), but instead utilize this nitrogen source to proliferate. In other words, nitrogen-fixing bacteria only fix nitrogen when the nitrogen source in their surrounding environment is depleted.

[0004] Among nitrogen-fixing bacteria, cyanobacteria can independently fix nitrogen by producing electrons and ATP for nitrogenase reductase through photosynthesis. In contrast, auxotrophic nitrogen-fixing bacteria naturally produce electrons and ATP for nitrogenase reductase through dissimilatory metabolism from carbon sources supplied by plants. Nitrogen-fixing bacteria are classified based on their relationship with plants into three types: root-nodule-forming, rhizosphere-dwelling, and endophytic. Some nodule-forming nitrogen-fixing bacteria can only perform nitrogen fixation within root nodules, while others can be cultured in vitro using nitrogen-free medium (synthetic minimal medium completely devoid of nitrogen). However, the preferred sugar or organic acid as a carbon source varies among nitrogen-fixing bacteria. Furthermore, the pathway through which electrons are delivered to nitrogenase reductase, and whether nitrogen fixation occurs under anaerobic, microaerobic, or aerobic conditions, also varies among nitrogen-fixing bacteria. These combinations lead to a wide variety of methods for cultivating nitrogen-fixing bacteria to achieve nitrogen fixation. For example, under normal circumstances, Klebsiella oxytoca can only be cultured anaerobically with sugar, Azospirillum lipoferum can only be cultured microaerobically with organic acids, and Azotobacter vinelandii can only exhibit nitrogen fixation ability in aerobic culture with sugar.

[0005] When nitrogen-fixing bacteria are inoculated (e.g., at 10 6When the culture is started by transferring the culture medium (cells / mL level) to a liquid nitrogen-free medium, the proliferation accompanied by the exertion of nitrogen fixation ability begins after a long delay time. If you want to observe the various phenomena that depend on the nitrogen fixation ability, you need a culture period of several weeks. On the one hand, it is known that when nitrogen-fixing bacteria are applied to a nitrogen-limited medium solidified with agar to which yeast extract is added at 100 mg / L to a nitrogen-free medium, rapid colony formation that depends on the nitrogen fixation ability is caused. Moreover, sometimes even if nitrogen-fixing bacteria are cultured on a solid medium under the atmosphere, colonies that exert nitrogen fixation ability are obtained. One of its nitrogen-fixing bacteria is a nitrogen-fixing bacterium of the genus Klebsiella (Non-patent Document 1: Rennie, Can. J. Microbiol. 27 8-14 1981). Therefore, we investigated whether this method, supplemented with 100 mg / L of yeast extract, could also be applied to nitrogen-free liquid culture media. This led to the development of a culture method that stably demonstrated nitrogen fixation even in nitrogen-restricted liquid culture media containing Klebsiella oxytoca placed in an open container and then statically cultured under atmospheric pressure. This culture method resulted in nitrogen-fixing bacteria proliferating almost without any lag time, exhibiting nitrogen fixation 24 hours after the start of culture, enabling observation of various phenomena dependent on nitrogen fixation within a culture period of less than a week.

[0006] A simple method for measuring the extent of nitrogen fixation ability of cultured nitrogen-fixing bacteria is the acetylene reduction method. Due to the low substrate specificity of nitrogenase, in addition to nitrogen fixation activity, it also has the activity of reducing acetylene to ethylene (acetylene reduction activity). When the aerosol of a culture vessel placed in a nitrogen-restricted medium is filled with a mixed gas of nitrogen molecules and acetylene to cultivate nitrogen-fixing bacteria, the nitrogen-fixing bacteria reduce both and simultaneously generate ammonia and ethylene. Since ammonia is consumed by nitrogen assimilation, it is impossible to measure the nitrogen fixation activity in real time. On the one hand, ethylene is not further metabolized and released into the aerosol. Thus, the acetylene reduction activity of nitrogenase is determined by measuring the amount of ethylene accumulated in the aerosol. Generally, the nitrogen fixation ability of nitrogen-fixing bacteria is represented by the value of acetylene reduction activity.

[0007] On the one hand, L-glutamic acid is an important amino acid as a food or pharmaceutical. Microorganisms have the ability to produce L-glutamic acid from ammonium ions and 2-oxoglutaric acid using L-glutamate dehydrogenase or a glutamine synthetase / glutamate synthetase complex. This metabolism is called nitrogen assimilation. Methods have been developed for producing L-glutamic acid by fermentation using genetically modified microorganisms of various types. These genetic modifications essentially enhance the production of 2-oxoglutaric acid, which serves as an ammonium ion acceptor in nitrogen assimilation. For example, Patent Document 1, Patent Publication No. 4144131, describes a Klebsiella bacterium modified to overexpress citrate synthase (CS) derived from rod-shaped bacteria. These microorganisms are then aerobically cultured in a medium containing a large amount of ammonium ions (20 g / L ammonium sulfate) to produce L-glutamic acid by fermentation. Furthermore, Patent Document 2, Japanese Patent Application Laid-Open No. 2009-254323, discloses a method for producing L-glutamic acid by modifying Klebsiella bacteria to overexpress methylcitrate synthase (MCS) derived from Escherichia coli or rod-shaped bacteria, and then anaerobic culturing the microorganism in a medium containing a large amount of ammonium ions (10 g / L ammonium sulfate). In these examples, ammonium ions are supplied to the culture medium in large quantities for nitrogen assimilation. Currently, the most popular method for producing ammonia is the Haber-Bosch process. This process produces ammonia by reacting hydrogen molecules produced by steam reforming fossil fuels (coal, petroleum, methane gas) with nitrogen molecules in the atmosphere, and its operation is quite costly.

Prior art literature

[0008] [Patent Document 1] Patent No. 4144131 [Patent Document 2] Japanese Patent Application Laid-Open No. 2009-254323

Non-patent literature

[0009] [Non-patent document 1] Rennie, Can. J. Microbiol. 27 8-14 1981 [Non-patent document 2] Bott, Mol. Microbiol. 18 533-546 1995 [Non-patent document 3] Weitzman, Adv. Microb. Physiol. 22 185-244 1981 [Non-patent document 4] Saier, Microbiol. Mol. Biol. Rev. 64, 354-411, 2000 [Non-patent document 5] Soares-Silva et al., FEMS Microbiol. Letter. 367 1-15 2020 [Non-patent document 6] Kaestner et al., Arch. Microbiol. 177 500-506 2002 [Non-patent document 7] Son HF et al., PLoS ONE. 11(6): e0158402 [Non-patent document 8] Choi, J. Wet al., Microb Cell Fact 14, 21 2015 [Summary of the Invention] [Problems to be solved by the invention]

[0010] In light of the above, upon discovering the culture conditions under which nitrogen-fixing bacteria exhibit maximum nitrogen-fixing capacity, a novel fermentation production method has been devised in which the nitrogen-fixing bacteria cultured thereunder are modified strains for enhanced 2-oxoglutaric acid production, as described in Patent Documents 1 and 2 (Patent No. 4144131 and JP-A-2009-254323). Thus, the aim is to provide a novel fermentation production method for L-glutamic acid that efficiently converts ammonia generated at maximum nitrogen-fixing capacity into L-glutamic acid.

Methods for solving the problem

[0011] As mentioned above, since the culture conditions required to maximize nitrogen fixation vary greatly among nitrogen-fixing bacteria, the culture medium composition and culture methods for Klebsiella microorganisms were investigated. It was discovered that the nitrogen fixation capacity of Klebsiella microorganisms is enhanced in nitrogen-restricted culture medium supplemented with organic acids such as citric acid. Consequently, it was discovered that by introducing the citrate synthase (CS) gene and the 2-methylcitrate synthase (2-MCS) gene into Klebsiella microorganisms and culturing them in nitrogen-restricted culture medium supplemented with organic acids such as citric acid, L-glutamic acid or its salts can be produced from atmospheric nitrogen molecules. Furthermore, a citrate transporter gene was discovered as a novel gene that, when introduced into Klebsiella microorganisms and cultured in nitrogen-restricted culture medium supplemented with organic acids such as citric acid, enables the production of L-glutamic acid or its salts from atmospheric nitrogen molecules. Furthermore, Klebsiella microorganisms introduced with both the citrate synthase gene and the citrate transporter gene produced larger amounts of L-glutamic acid than those introduced with only one gene. Consequently, a method for fermentative production of L-glutamic acid based on the nitrogen-fixing ability of nitrogen-fixing bacteria using modified strains different from those described in Patent Documents 1 and 2 (Patent No. 4144131 and JP-A-2009-254323) was established, thereby completing the present invention. Therefore, the present invention relates to the following technical solutions: [1] A method for fermenting L-glutamate or a salt thereof from atmospheric nitrogen molecules, comprising: culturing nitrogen-fixing bacteria into which at least one gene selected from the group consisting of a citrate transporter, a citrate synthase, and a 2-methylcitrate synthase is introduced in a nitrogen-limited medium containing an organic acid or a salt thereof constituting a TCA pathway. [2] The method according to item 1, wherein the nitrogen-limited medium contains 3 mg / L to 1500 mg / L of nitrogen source in terms of yeast extract and / or 0.02 mM to 15 mM of nitrogen source in terms of ammonium ions. [3] The method according to item 1 or 2, wherein the organic acid or its salt has a concentration of 0.5 g / L to 100 g / L. [4] The method according to any one of items 1 to 3, wherein the organic acid is citric acid, succinic acid, malic acid, fumaric acid, or 2-oxoglutaric acid. [5] The method according to any one of items 1 to 4, wherein the nitrogen-fixing bacteria are microorganisms belonging to the family Enterobacteriaceae. [6] The method according to item 5, wherein the nitrogen-fixing bacteria are microorganisms selected from the genera Klebsiella, Rahnella, Raoultella and Kosakonia. [7] A nitrogen-fixing bacterium into which a gene for a citrate transporter is introduced, the bacterium being selected from the genera Klebsiella, Rahnella, Kosakonia and Raoultella. [8] The nitrogen-fixing bacteria according to item 7, wherein the nitrogen-fixing bacteria of the genus Klebsiella are selected from Klebsiella oxytoca, Klebsiella michiganensis, Klebsiella grimontii, Klebsiella pasteurii, Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella indica, the nitrogen-fixing bacteria of the genus Rahnella are Rahnella aquatilis, or the nitrogen-fixing bacteria of the genus Raoultella are selected from Raoultella terrigena, Raoultella ornithinolytica, and Raoultella planticola. [9] The nitrogen-fixing bacteria described in item 7, wherein the nitrogen-fixing bacteria are Klebsiella oxytoca strain NG13 (deposit number: NITE BP-03721), Klebsiella indica (JCM33718), Klebsiella variicola (JCM12419), Klebsiella sp. (NBRC100048, NBRC100441, NBRC109911), Raoultella terrigena (JCM1687=ATCC33257), Rahnella aquatilis (JCM1683=ATCC33071), and Klebsiella planticola (JCM20069=ATCC8329).

[10] The nitrogen-fixing bacterium according to any one of items 7 to 9, wherein the nitrogen-fixing bacterium ferments nitrogen molecules in the atmosphere under nitrogen limitation to produce L-glutamic acid or a salt thereof. Effects of the Invention

[0012] The microorganisms produced by the production method of the present invention fix nitrogen in the atmosphere and simultaneously produce L-glutamic acid, making it possible to ferment L-glutamic acid independently of ammonia produced by the Haber-Bosch process. [Brief description of the accompanying drawings]

[0013]

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[0014] One embodiment of the present invention relates to a method for culturing nitrogen-fixing bacteria while allowing them to exert their nitrogen-fixing ability in the atmosphere, and a culture medium for maximizing the nitrogen-fixing ability of nitrogen-fixing bacteria. Another embodiment of the present invention relates to nitrogen-fixing bacteria into which a gene has been introduced to promote the production of L-glutamic acid, and further relates to a method for producing L-glutamic acid from atmospheric nitrogen molecules by fermentation, comprising culturing nitrogen-fixing bacteria into which a gene has been introduced to promote the production of L-glutamic acid in a nitrogen-limited medium containing an organic acid such as citric acid. [Nitrogen-limited medium]

[0015] In the present invention, a nitrogen-limited medium refers to a culture medium in which the nitrogen content is adjusted to allow nitrogen-fixing bacteria to exert their nitrogen-fixing ability. The nitrogen-limited medium may contain a nitrogen source at a concentration that promotes the initial growth of nitrogen-fixing bacteria without inhibiting nitrogen-fixing activity. The nitrogen concentration of the nitrogen-limited medium can vary depending on the type of nitrogen-fixing bacteria and the type of nitrogen source. For example, it can be expressed in terms of yeast extract. The required amount of nitrogen can vary depending on the type of nitrogen-fixing bacteria, the type of nitrogen source, and the concentration of the carbon source. A person skilled in the art can appropriately determine an amount that promotes the initial growth of nitrogen-fixing bacteria without inhibiting nitrogen-fixing activity, depending on individual culture conditions. For example, the nitrogen-limited medium of the present invention contains an organic acid such as citric acid or a salt thereof and a nitrogen source at a concentration of 50 mg / L to 300 mg / L, preferably 50 mg / L to 200 mg / L, in terms of yeast extract. Furthermore, the carbon source may be contained separately from the organic acid such as citric acid or a salt thereof. The nitrogen-limited culture medium of the present invention is suitable for culturing nitrogen-fixing bacteria that fix nitrogen molecules in the atmosphere, and by containing citric acid or a salt thereof, it is possible to enhance the nitrogen-fixing ability.

[0016] The amount of nitrogen source contained in the nitrogen-limited medium can be changed by the nitrogen-fixing bacteria used. The nitrogen-limited medium can be specified by the amount of nitrogen source converted into yeast extract and / or ammonium ion. As an example, the nitrogen-limited medium can be prepared in a manner that contains 3 mg / L to 1500 mg / L of nitrogen source converted into yeast extract. As a more preferred amount of nitrogen source, it can be prepared in a manner that contains 25 mg / L to 500 mg / L of nitrogen source converted into yeast extract. As the most preferred amount of nitrogen source, it can be prepared in a manner that contains 50 mg / L to 300 mg / L of nitrogen source converted into yeast extract. In addition, as an example, when ammonium salt is used as the nitrogen source of the nitrogen-limited medium, it can be prepared in a manner that contains 0.02 mM to 15 mM as an ammonium ion concentration. The ammonium ion concentration of the nitrogen source of the nitrogen-limited medium is preferably 0.2 mM to 5 mM, and the most preferred amount of nitrogen source is 0.35 mM to 3 mM. The nitrogen contained in the nitrogen-limited medium is taken up by the nitrogen-fixing bacteria during their initial growth and consumed. When the nitrogen concentration in the medium falls below a certain level, the nitrogen-fixing bacteria begin to fix atmospheric nitrogen, thereby producing L-glutamic acid or its salt derived from atmospheric nitrogen molecules. Specifically, the present invention is characterized in that the nitrogen atoms that constitute the L-glutamic acid produced by fermentation contain nitrogen molecules derived from the atmosphere.

[0017] Nitrogen sources include extracts such as yeast extract, mixtures of various amino acids such as casamino acids or peptones, and ammonium salts such as ammonium chloride and ammonium sulfate. The concentration of the nitrogen source used in nitrogen-limited culture media can be expressed as a yeast extract concentration. Since yeast extract contains approximately 10% total Kjeldahl nitrogen, the yeast extract-equivalent concentration can be calculated based on the total Kjeldahl nitrogen content of the nitrogen source used. The nitrogen-limited culture media used in the present invention typically contain 3 mg / L to 1500 mg / L of nitrogen source as yeast extract at the start of culture, i.e., 0.3 mg / L to 150 mg / L of total Kjeldahl nitrogen. From the viewpoint of maximizing the nitrogen fixation capacity, a nitrogen source containing 25 mg / L or more as yeast extract, i.e., 2.5 mg / L or more as total Kjeldahl nitrogen, is preferred, and 50 mg / L or more as yeast extract, i.e., 5 mg / L or more as total Kjeldahl nitrogen, is more preferred. 500 mg / L or less as yeast extract, i.e., 50 mg / L or less as total Kjeldahl nitrogen, is more preferred, and 300 mg / L or less as yeast extract, i.e., 30 mg / L or less as total Kjeldahl nitrogen, is more preferred.

[0018] The mechanism by which nitrogen-fixing bacteria's nitrogen-fixing capacity is enhanced by culturing in nitrogen-limited medium supplemented with organic acids or their salts is not yet understood, but the following is a possible explanation. Nitrogen-fixing bacteria sense the intracellular 2-oxoglutarate / L-glutamine ratio. When this ratio is high, nitrogen-fixing capacity is enhanced, while when it is low, nitrogen-fixing capacity is suppressed. 2-Oxoglutarate is generated by harvesting organic acids from the medium, and nitrogen-fixing capacity may be enhanced by maintaining a high intracellular 2-oxoglutarate / L-glutamine ratio. The concentration of the organic acid or its salt in the nitrogen-limited medium is typically 0.5 g / L to 100 g / L. To maximize nitrogen-fixing capacity, concentrations of 1 g / L, 2.5 g / L, 5 g / L, 6 g / L, or 7 g / L are preferred. Alternatively, concentrations of 15 g / L, 12 g / L, 10 g / L, 9 g / L, or 8 g / L are preferred. As the salt of the organic acid, any salt can be used, and as an example, potassium salt, sodium salt, magnesium salt, etc. As the organic acid, an organic acid involved in the TCA pathway, such as oxaloacetic acid, citric acid, isocitric acid, 2-oxoglutaric acid, succinic acid, fumaric acid, malic acid, or salts thereof, can be added to the nitrogen-restricted medium.

[0019] As a carbon source, a nitrogen-restricted medium containing a combination of sugars such as glucose and organic acids or their salts can also be used. As an example, the amount of carbon source contained in the nitrogen-restricted medium can be 0.5g / L to 300g / L in terms of glucose, preferably 5g / L to 100g / L. The ratio of sugars such as glucose and organic acids or their salts can be appropriately selected according to the experimental conditions. As an example, the weight ratio of glucose and citric acid or its salt can be 1:5 to 5:1, preferably 1:4 to 4:1, more preferably 1:3 to 3:1, and even more preferably 1:2 to 2:1. In addition, by using an amount of sugar such as glucose that is about twice the amount of citric acid or its salt, the amount of glutamate produced can be increased. As an example, when adding glucose and citric acid or its salt to the nitrogen-restricted medium, glucose can be used in the range of 0.5g / L to 100g / L, and citric acid or its salt can be used in the range of 0.5g / L to 50g / L. Glucose and citric acid or its salts may each be used at a concentration of 2.5 g / L or more, 5 g / L or more, 6 g / L or more, or 7 g / L or less, or 15 g / L or less, 12 g / L or less, 10 g / L or less, 9 g / L or less, or 8 g / L or less, in the aforementioned mixing ratio. From the perspective of using nitrogen-fixing bacteria introduced with a citrate transporter gene, a culture medium containing a sugar such as glucose in an amount multiple of that in the concentration of 5 to 10 g / L of citric acid or its salt may be used.

[0020] As a carbon source, sugars such as monosaccharides, disaccharides, sugar alcohols, polysaccharides, organic acids, etc. can also be used. As the sugars involved, glucose, mannose, galactose, fructose, xylose, sucrose, maltose, cellobiose, trehalose, mannitol, inositol, sorbitol, glycerol, starch hydrolyzate, etc. can be used, and glucose is usually used. As an organic acid, any organic acid can be used as long as it is an organic acid that bacteria can utilize. As an example of an organic acid, an organic acid contained in the TCA pathway, such as oxaloacetic acid, citric acid, isocitric acid, 2-oxoglutaric acid, succinic acid, fumaric acid, malic acid, etc. can be used as a carbon source. As the concentration of the carbon source, 0.5g / L to 300g / L can be set in terms of glucose. From the perspective of maximizing the nitrogen fixation capacity, it is preferably 10g / L or more, more preferably 15g / L or more, and even more preferably 22.5g / L or more. The organic acid such as citric acid added to the nitrogen-limited medium of the present invention is blended as one of the carbon sources, and the total of the organic acid such as citric acid and other carbon sources, for example, glucose, is used as the concentration of the carbon source.

[0021] When nitrogen, particularly inorganic nitrogen, is present in the culture medium during the cultivation of nitrogen-fixing bacteria, the nitrogen fixation reaction is strongly inhibited. On the one hand, in the initial stages of cultivation, nitrogen is required to the extent necessary for the proliferation of nitrogen-fixing bacteria and the production of nitrogenase. Therefore, in nitrogen-restricted culture media used to cultivate nitrogen-fixing bacteria that produce L-glutamic acid or its salts by fixing atmospheric nitrogen molecules, the nitrogen source in the culture medium is consumed during cultivation, and when the nitrogen source concentration falls below a certain level, atmospheric nitrogen molecules are fixed. Therefore, when culturing using the nitrogen-restricted culture medium according to the present invention, no additional gaseous nitrogen, such as nitrogen sources other than atmospheric nitrogen molecules, is typically added.

[0022] In the nitrogen-limited medium, a synthetic minimal medium containing the necessary components can also be used according to the type of nitrogen-fixing bacteria. From the perspective of culturing microorganisms of the Enterobacteriaceae family, such components include inorganic salts, buffer substances, and vitamins. As inorganic salts, phosphates, sodium salts, magnesium salts, calcium salts, iron salts, manganese salts, molybdenum salts, etc. can be added. More specifically, sodium chloride, magnesium sulfate, calcium chloride, molybdate, and inorganic salts such as Na2MoO4·2H2O can be added. As buffer substances, potassium dihydrogen phosphate and dipotassium hydrogen phosphate can be mentioned. As vitamins, p-aminobenzoic acid, biotin, etc. can be used. The pH of the nitrogen-limited medium can be arbitrarily selected within a range that does not inhibit the growth of nitrogen-fixing bacteria, and can be adjusted to, for example, pH 6.8 to 7.5. [Nitrogen-fixing bacteria]

[0023] Examples of nitrogen-fixing bacteria that can be used in the present invention include microorganisms belonging to the family Enterobacteriaceae. For example, microorganisms belonging to the family Enterobacteriaceae can be selected from the genera Klebsiella, Rahnella, Raoultella, and Kosakonia. Preferred microorganisms include Klebsiella oxytoca, Klebsiella michiganensis, Klebsiella grimontii, Klebsiella pasteurii, Klebsiella pneumoniae, Klebsiella variicola, Klebsiella indica, Rahnella aquatilis, Raoultella terrigena, Raoultella ornithinolytica, and Raoultella planticola. More specifically, Klebsiella oxytoca, a nitrogen-fixing bacterium isolated from rice roots, can be used, particularly the NG13 strain (Accession Number: NITE BP-03721, Accession Number: NITE ABP-03721, Application Receipt Date: July 21, 2023). The NG13 strain is deposited with the National Institute for Technology Evaluation and Research, Patent Microorganism Depository (National Institute for Technology Evaluation and Research, Patent Microorganism Depository). Alternatively, microorganisms such as Escherichia coli into which a genome associated with nitrogen fixation has been introduced can be used as nitrogen-fixing bacteria. Furthermore, as nitrogen-fixing bacteria other than those belonging to the Enterobacteriaceae family, microorganisms such as Xanthobacter and Azorhizobium can be used. Among these microorganisms, microorganisms belonging to the genera Klebsiella, Rahnella, and Raoultella are particularly preferred. High production of L-glutamic acid can be achieved by specifically introducing a glutamate synthetase gene and a citrate transporter gene into these microorganisms.

[0024] In the present invention, nitrogen-fixing bacteria can be introduced with genes that promote the production of L-glutamic acid. Nitrogen-fixing bacteria that have been introduced with genes that promote the production of L-glutamic acid are referred to as nitrogen-fixing bacteria according to the present invention. As an example, the introduced genes are genes encoding proteins selected from citrate synthase, 2-methylcitrate synthase, and citrate transporter. Furthermore, preferably, both the citrate synthase gene and the citrate transporter gene are introduced, or both the 2-methylcitrate synthase gene and the citrate transporter gene are introduced. Citrate synthase, 2-methylcitrate synthase, or the citrate transporter is introduced into the nitrogen-fixing bacteria with the goal of increasing the intracellular concentration of citric acid. Furthermore, the citrate transporter functions independently of the citrate synthase and 2-methylcitrate synthase. As a result, the production of 2-oxoglutarate increases as the intracellular concentration of citric acid in the nitrogen-fixing bacteria increases, thereby converting ammonia fixed from atmospheric nitrogen molecules into L-glutamic acid or its salts through the nitrogen assimilation pathway, thereby producing L-glutamic acid or its salts.

[0025] Citrate synthase (CS) is an enzyme that plays a central role in the sugar oxidation process and is present in almost all organisms. It contributes to the initial stage of the TCA pathway. Specifically, it catalyzes the reaction of converting acetyl CoA, oxaloacetate, and water generated by pyruvate produced by glycolysis to form citrate and CoA. Figure 1). In the present invention, the CS gene introduced into nitrogen-fixing bacteria can also be derived from any bacteria. Among the CS of bacteria, there are "small" CS possessed by Gram-positive bacteria and "large" CS possessed by Gram-negative bacteria. The activity of "small" CS is not inhibited by NADH, while the activity of "large" CS is inhibited by NADH (Non-patent Document 3: Weitzman, Adv. Microb. Physiol. 22 185-244 1981). Therefore, as the CS gene introduced into nitrogen-fixing bacteria, a CS gene obtained from a Gram-positive bacterium is preferred. For example, a CS gene obtained from bacteria of the genus Arthrobacter, bacteria of the genus Corynebacterium, bacteria of the genus Bacillus, bacteria of the genus Mycobacterium, and bacteria of the genus Streptomyces. In particular, the CS gene of Corynebacterium glutamicum can be used. As an example, the CS gene of Corynebacterium glutamicum ATCC13869 (SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 30) can be used. On the other hand, even CS from Gram-negative bacteria may not be inhibited by NADH, and these CS genes are also suitable as CS genes for introduction into nitrogen-fixing bacteria. For example, CS genes obtained from bacteria belonging to the genera Acetobacter, Halobacterium, Thermus, and Thiobacillus can be used. In particular, the CS gene from Thermus aquaticus can be used. CS genes obtained from the bacteria to be introduced can also be used. For example, the CS gene of Klebsiella oxytoca strain NG13 is encoded by the base sequence of SEQ ID NO: 1 (amino acid sequence: SEQ ID NO: 28). By introducing the CS gene into nitrogen-fixing bacteria, the concentration of citric acid in the cells of the microorganism can be increased. L-glutamic acid or a salt thereof can be produced by culturing nitrogen-fixing bacteria into which a gene for citrate synthase has been introduced in a nitrogen-limited medium containing citric acid.

[0026] 2-Methylcitrate synthase (2-MCS) is an enzyme possessed by most bacteria. It is an enzyme that catalyzes the reaction of generating 2-methylcitrate and CoA from propionyl-CoA, oxaloacetic acid and water. On the one hand, 2-MCS catalyzes the same reaction as CS as a side activity. 2-MCS can also be simply referred to as methylcitrate synthase (MCS). In the present invention, the 2-MCS gene introduced into nitrogen-fixing bacteria can be a 2-MCS gene of any bacterial origin as long as the 2-MCS encoded by the gene catalyzes the same reaction as CS. For example, the 2-MCS genes of Escherichia coli and Corynebacterium glutamicum can be used. For example, the 2-MCS gene from Escherichia coli strain W3110 (SEQ ID NO: 5, amino acid sequence: SEQ ID NO: 32) or the 2-MCS gene from Corynebacterium glutamicum ATCC13869 (SEQ ID NO: 4, amino acid sequence: SEQ ID NO: 31) can be used. Furthermore, the 2-MCS gene of Escherichia coli 2-MCS and the 2-MCS genes of Citrobacter, Serratia, Stenotrophomonas, Chromobacterium, Ralstonia, Cupriavidus, Burkholderia, Achromobacter, and Herbaspirilla bacteria that show more than 73% identity in amino acid sequence can also be used. In addition, among bacteria of the genus Corynebacterium, 2-MCS genes of Corynebacterium comens, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium testudinoris, and Brevibacterium flavum can also be used. 2-MCS genes obtained from bacteria at the site of introduction can also be used. By introducing the 2-methylcitrate synthase gene into nitrogen-fixing bacteria, the concentration of citric acid in the cells of the microorganism can be increased. By culturing nitrogen-fixing bacteria into which the 2-MCS gene has been introduced in a nitrogen-limited medium containing citric acid, L-glutamic acid or a salt thereof can be produced.

[0027] Citric acid transporters are transporters that transport citric acid outside the body of a microorganism into the body. Bacterial citric acid transporters are classified into five types according to the Transporter Classification (TC) system: 2.A.1.6, 2.A.11, 2.A.24, 2.A.47, and 2.A.80 (Non-patent Document 4: Saier, Microbiol. Mol. Biol. Rev. 64 354-411 2000, Non-patent Document 5: Soares-Silva et al., FEMS Microbiol. Letter. 367 1-15 2020). TC No. 2.A.1.6 transporters are represented by CitH of Klebsiella pneumoniae. + This transporter is present in many bacteria. The transporter of TC No.2.A.11 contains citric acid and divalent metal ions, represented by CitM of Bacillus subtilis: H + Co-transporter, citric acid and divalent metal ions represented by CitH of Bacillus subtilis: H + Co-transporter, citrate:H represented by YRAO of Bacillus subtilis + The transporter proteins of TC No. 2.A.24 include citrate:Na transporters represented by CitS of Klebsiella pneumoniae. + co-transporter, citric acid:Na represented by MleP of Lactococcus lactis + Co-transporter, represented by CimH of Bacillus subtilis, which transports electroneutrally neutral citrate:H +The transporter protein of TC No. 2.A.47 is a citric acid: succinic acid countertransporter represented by CitT of Escherichia coli. The transporter protein of TC No. 2.A.80 is a trimolecular complex-type citric acid transporter represented by TctCBA of Corynebacterium glutamicum. In the present invention, the gene encoding the citric acid transporter introduced into nitrogen-fixing bacteria can be any bacterial gene encoding the various citric acid transporters mentioned above. The gene encoding the citric acid transporter obtained from the bacteria to be introduced can also be used. Particularly preferably, citric acid:Na + cotransporter genes and citrate:H + Since Klebsiella oxytoca has the same gene as the citrate:acetate exchange transporter gene (citW) possessed by Klebsiella pneumoniae (Non-Patent Document 6: Kaestner et al., Arch. Microbiol. 177 500-506 2002), the citrate:acetate exchange transporter gene possessed by Klebsiella oxytoca can also be introduced. Citrate·Na of Klebsiella oxytoca NG13 strain + The co-transporter is encoded by the base sequence of SEQ ID NO: 2 (amino acid sequence: SEQ ID NO: 29). By culturing nitrogen-fixing bacteria that highly express the citrate transporter gene in a nitrogen-limited medium containing citric acid, the citric acid concentration in the bacteria can be increased, allowing the production of L-glutamic acid or its salt.

[0028] When genes in various metabolic pathways are altered, the nitrogen-fixing capacity of nitrogen-fixing bacteria often decreases. Therefore, even in nitrogen-fixing bacteria with genetically altered metabolic pathways, a culture medium is needed that can maximize nitrogen-fixing capacity. Nitrogen-limited culture media containing organic acids or their salts can enhance nitrogen-fixing capacity. By culturing nitrogen-fixing bacteria with genes introduced to enable L-glutamic acid production using these media, it is possible to ferment L-glutamic acid or its salts from atmospheric nitrogen molecules.

[0029] The gene encoding the protein selected from citrate synthase, 2-methylcitrate synthase and citrate transporter may also have a promoter and terminator sequence that controls expression. More preferably, a promoter that enables transient expression is preferably utilized. As a promoter capable of transient expression, the trc promoter that can be controlled by IPTG addition can be used. In a conventional method, a vector containing a gene having a promoter and terminator can be used to introduce the gene.

[0030] For the nitrogen-fixing bacteria used in the present invention, the genes for at least one gene selected from citrate synthase, 2-methylcitrate synthase and citrate transporter can be further introduced for the purpose of producing amino acids other than L-glutamic acid. As amino acids other than L-glutamic acid, essential amino acids such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine, arginine, aspartic acid, glutamine, asparagine, alanine, glycine, cysteine, aspartic acid, proline, glutamine, tyrosine, and γ-aminobutyric acid can be enumerated, usually L-form amino acids. For amino acids other than L-glutamic acid, it is possible to produce them by selecting a suitable gene for introduction based on the well-known techniques in the art. As an example, by introducing the gene for glutamate-pyruvate aminotransferase (SEQ ID NO: 33, amino acid sequence: SEQ ID NO: 37) and the gene for L-alanine transporter (SEQ ID NO: 34, amino acid sequence: SEQ ID NO: 38), L-alanine and L-valine can be produced. Furthermore, as an example, it is believed that by introducing the gene for glutamate-oxaloacetate aminotransferase (SEQ ID NO: 35, amino acid sequence: SEQ ID NO: 39), L-aspartic acid can be produced (Non-Patent Document 7: Son HF et al., PLoS ONE. 11(6): e0158402). Furthermore, as an example, it is believed that by introducing the gene for glutamate decarboxylase (SEQ ID NO: 36, amino acid sequence: SEQ ID NO: 40), γ-aminobutyric acid can be produced (Non-Patent Document 8: Choi, JW et al., Microb Cell Fact 14, 21 2015). Thus, another embodiment of the present invention relates to nitrogen-fixing bacteria that may be introduced with additional genes, in addition to at least one gene selected from citrate synthase, 2-methylcitrate synthase, and citrate transporter, for the purpose of producing amino acids other than L-glutamate. Furthermore, another embodiment of the present invention also relates to a method for fermentatively producing amino acids other than L-glutamate, such as L-alanine and / or L-valine, comprising culturing the nitrogen-fixing bacteria in a nitrogen-limited medium containing an organic acid or a salt thereof. The nitrogen-limited medium used in the method for fermentatively producing amino acids other than L-glutamate, such as L-alanine and / or L-valine, can be the same medium used in the method for fermentatively producing L-glutamate.

[0031] Glutamate-pyruvate aminotransferase is an enzyme that catalyzes the reaction of producing L-alanine and 2-oxoglutarate from L-glutamate and pyruvate, contributing to the biosynthesis of L-alanine. It is believed that the glutamate-pyruvate aminotransferase gene can be derived from any bacteria. By introducing the glutamate-pyruvate aminotransferase gene into nitrogen-fixing bacteria, the amino group of L-glutamate produced using atmospheric nitrogen molecules can be transferred to pyruvate, producing L-alanine or a salt thereof.

[0032] The L-alanine transporter protein functions to excrete L-alanine out of cells. The L-alanine transporter gene can be derived from any bacteria. By introducing the L-alanine transporter gene into nitrogen-fixing bacteria along with the glutamate-pyruvate aminotransferase gene, it is believed that the extracellular transport of produced L-alanine can be promoted, thereby improving the productivity of L-alanine or its salts.

[0033] Glutamate-oxaloacetate aminotransferase is an enzyme that catalyzes the reaction of producing L-aspartate and 2-oxoglutarate from L-glutamate and oxaloacetate, contributing to the biosynthesis of L-aspartate. It is believed that the glutamate-oxaloacetate aminotransferase gene can be derived from any bacteria. By introducing the glutamate-oxaloacetate aminotransferase gene into nitrogen-fixing bacteria, the amino group of L-glutamate produced using atmospheric nitrogen molecules can be transferred to oxaloacetate, producing L-aspartate or its salts.

[0034] Glutamate decarboxylase is an enzyme that catalyzes the reaction of producing γ-aminobutyric acid from L-glutamate, contributing to the biosynthesis of γ-aminobutyric acid. It is believed that the glutamate decarboxylase gene can be derived from any bacteria. By introducing the glutamate decarboxylase gene into nitrogen-fixing bacteria, L-glutamate produced by atmospheric nitrogen molecules can be decarboxylated to produce γ-aminobutyric acid or its salts.

[0035] The gene introduced in the present invention can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity or sequence identity relative to the sequence of the respective original genes, and can introduce a gene that has the same or the same enzymatic activity when the gene is expressed. Identity can be determined using the NCBI blastn program using a database registered with NCBI. Specifically, a gene having citrate synthase activity can be introduced that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity relative to the sequence of the CS gene of SEQ ID NOs: 1 and 3. In another example, a citrate synthase activity can be introduced relative to the citrate·Na gene of SEQ ID NO: 2. +The sequence of the gene for the co-transporter has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity, and when the gene is expressed, a citrate·Na + Co-delivery of an active gene. In another embodiment, a gene having 2-methylcitrate synthase activity can be introduced during gene expression by having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the 2-methylcitrate synthase gene of SEQ ID NOs: 4 and 5.

[0036] The genes introduced in the present invention can have at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity or sequence identity relative to the amino acid sequence of the respective original genes, and when expressed, encode an amino acid sequence that has the same or similar enzymatic activity. Identity can be determined using the NCBI blastp program using databases registered with NCBI. Specifically, a gene encoding an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity relative to the amino acid sequence of CS of SEQ ID NOs: 28 and 30 and has citrate synthase activity can be introduced. In another example, a gene encoding a citrate·Na-1-3-hydroxy-1 ... + The amino acid sequence of the co-transporter has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity and has citrate·Na + Furthermore, in another embodiment, a gene encoding an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of 2-methylcitrate synthase of SEQ ID NOs: 31 and 32 and having 2-methylcitrate synthase activity can be introduced. [Method for producing L-glutamic acid by fermentation]

[0037] As another embodiment of the present invention, a method for fermentatively producing L-glutamic acid or a salt thereof from atmospheric nitrogen molecules comprises culturing nitrogen-fixing bacteria into which genes have been introduced to promote L-glutamic acid production in the nitrogen-restricted medium of the present invention, i.e., a nitrogen-restricted medium containing an organic acid. By using a nitrogen-restricted medium containing an organic acid, nitrogen fixation ability is enhanced, and by culturing the nitrogen-fixing bacteria into which genes have been introduced to promote L-glutamic acid production, fermentation of L-glutamic acid or a salt thereof from atmospheric nitrogen molecules can be enhanced.

[0038] In the culture process involved in the present invention, instead of using an open container for static culture under the atmosphere, the culture conditions corresponding to the nitrogen-fixing bacteria used can be appropriately selected. For example, if you want to increase the degree of anaerobicity, you can also put a nitrogen-restricted culture medium and air into a sealed container, and culture in a manner in which the dissolved oxygen contained at the beginning of the culture is consumed in the culture process and the anaerobic state is strengthened. Furthermore, nitrogen gas and a nitrogen-restricted culture medium can be put into a sealed container and cultured under a completely anaerobic state. Alternatively, when using an open container for static culture under the atmosphere, by expanding the contact area between the culture solution and the air, a more aerobic culture can be performed. From the perspective of culturing microorganisms of the enterobacteriaceae family, the culture temperature can be 20°C to 37°C, more preferably 20°C to 30°C.

[0039] In the cultivation process of the present invention, the nitrogen source contained in the nitrogen-limited culture medium is essential for the initial growth of nitrogen-fixing bacteria. The nitrogen source contained in the nitrogen-limited culture medium is taken up by the bacteria and consumed during the initial growth. During the cultivation process, when the nitrogen concentration in the culture medium falls below a certain level, the nitrogen-fixing bacteria begin to fix atmospheric nitrogen. At this time, nitrogen-fixing bacteria that have been genetically modified to promote L-glutamic acid production have increased intracellular citric acid concentrations. The fixed ammoniacal nitrogen is converted into L-glutamic acid or its salts through the nitrogen assimilation pathway, thereby producing L-glutamic acid or its salts.

[0040] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0041] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited solely by the claims. Modifications to the present invention, such as additions, deletions, and substitutions of constituent elements, may be made without departing from the spirit of the present invention. [Example] [Example 1: Discussion of culture conditions]

[0042] The NG13 strain (Accession Number: NITE BP-03721, Accession Number: NITE ABP-03721, Application Receipt Date: July 21, 2023) was previously thought to exhibit nitrogen fixation capabilities only through anaerobic culture. Anaerobic culture involves sealing a culture vessel and filling the atmosphere with nitrogen or a mixture of nitrogen and argon, a complex and laborious process. Therefore, the goal was to establish a novel culture method that utilizes an open culture vessel to freely utilize atmospheric nitrogen molecules to exhibit nitrogen fixation capabilities. Furthermore, the culture medium used was a nitrogen-limited medium containing 100 mg / L of yeast extract supplemented with a nitrogen-free medium.

[0043] 5 mL of nitrogen-limited medium (KD medium containing only 7.5 g / L glucose, see Table 1) was placed in a φ18×180 mm test tube and 10 7 The NG13 strain of cells was statically cultured at 25°C. The mouth of the test tube was covered with only an aluminum cap, and the air outside the test tube freely entered the inside of the test tube through the gap between the test tube and the aluminum cap. During the culture, the cell density in the culture solution was measured, and the nitrogen fixation ability of the culture solution was measured by the acetylene reduction method (described later). The NG13 strain began to proliferate after a short delay time after the start of culture, and exerted a nitrogen fixation ability of 0.63μmol / 4-h / tube (see Table 2) 24 hours after the start of culture. The intensity of this nitrogen fixation ability is equivalent to the nitrogen fixation ability exerted in anaerobic culture in which the mouth of the test tube was sealed with a butyl double stopper and the air layer was filled with nitrogen. Furthermore, even if the glucose in the KD medium was replaced with the same amount of fructose or sucrose, the maximum nitrogen fixation ability was the same. The above established a new and simple culture method for enabling the NG13 strain to exert nitrogen fixation ability.

[0044] The acetylene reduction method follows the following procedure. First, the mouth of a test tube is sealed with a butyl double stopper. At this point, the gas layer is air. Subsequently, 15% of the gas layer is replaced with acetylene, and the mixture is incubated at 25°C for 4 hours. After incubation, 0.5 mL of the gas layer is withdrawn with a syringe, and the amount of ethylene is measured using a gas chromatograph. This is converted to the amount of ethylene produced per test tube and used as the nitrogen fixation capacity.

[0045] Next, to explore nitrogen-limited media that enhance the nitrogen fixation ability of the NG13 strain through culture in KD medium, nitrogen-limited media were prepared by varying the carbon source composition of the KD medium as shown in Table 1. The NG13 strain was statically cultured at 25°C for 24 hours using each medium to measure its nitrogen fixation ability. The results are shown in Table 2.

Table 1 (unit is mg / L)

Table 2

[0046] Nitrogen fixation capacity was not enhanced in KDD medium, which doubled the glucose content (15 g / L) by adding 7.5 g / L glucose to KD medium, or in KDP medium, which added sodium pyruvate (7.5 g / L). However, nitrogen fixation capacity was enhanced to varying degrees in other media containing sodium salts of various organic acids (7.5 g / L) added to KD medium. In particular, the combination with sodium citrate (KDC medium) had the greatest effect (up to 2.6-fold). However, accumulation of L-glutamate in the culture medium was not observed in any of the cultures.

[0047] To determine the sodium citrate concentration that enhances the nitrogen fixation capacity of the NG13 and NG13-pCCS strains in KDC medium, nitrogen-limited media were prepared with the sodium citrate composition of the KDC medium modified as shown in Table 3. The two strains were statically cultured at 25°C for 24 hours in each medium, and their nitrogen fixation capacity was measured by the acetylene reduction method. The results are shown in Table 3.

Table 3

[0048] Genomic DNA was extracted from the cells of NG13 strain, Corynebacterium glutamicum ATCC13869 (hereinafter referred to as ATCC13869 strain), and Escherichia coli W3110 (hereinafter referred to as W3110 strain) using Wizard Genomic DNA Purification Kit (Prω) or InstaGene (Bio-Rad). Gene fragments encoding citrate synthase (CS, ATCC13869: BBD29_04605) and 2-methylcitrate synthase (2-MCS, ATCC13869 strain: BBD29_03720, and W3110 strain: PrpC) were prepared by PCR using the genomic DNA of each bacterium as a template and the primers listed in Table 4 below in the combination described in Table 5. Citric acid of NG13 strain: Na + After the co-transporter gene was cloned based on the genomic information of Klebsiella oxytoca M5a1 strain and sequence information was obtained, PCR was used to make gene fragments. It was designed to give the same sequence of about 20bp as the imported vector to both ends of each primer, and the imported gene was expressed using a vector-derived expression promoter and ribosome binding site (RBS) (Table 4). The production of the vector side fragment was carried out using PCR. The sequence and combination of the primers used are shown below. The target gene fragment and the fragment on the vector side were combined with Gibson Assembly. 10 μL of the reaction solution was used to transform the Escherichia coli DH5α strain, and the drug selection marker possessed by the plasmid was used to select colonies. Plasmids were extracted from the presented colonies to confirm whether the plasmids designed by sequence analysis could be obtained (Table 5). [Table 4: 5'→3' sequences of primers used (underlined sequences are used in Gibson Assembly)]

[0049] Table 5: List of names of various plasmids produced, information on proteins encoded by the genes expressed, and information on templates and primers used in plasmid production. (2) Introduction of plasmid into NG13 strain

[0050] The NG13 strain was grown in LB liquid medium (Bacto Tryptone 1%, Bacto Yeast Extract 0.5%, NaCl 1%) until OD 600 = 0.5, collect the bacteria, wash with 40 mL of ice-cold sterile water and 40 mL of 10% glycerol, and then suspend with 10% glycerol until OD 600 = Near 100, with 60 μL each as competent cells. After mixing with about 200 ng of each plasmid, use GENE PULSER II (BioRad) to introduce with an electric field strength of 25 kV / cm, a capacitance of 25 μF, and a resistance of 200 Ω. The competent cells after introduction are mixed with 1 mL of LB liquid culture medium without antibiotics, cultured at 30°C for 1 hour and 300 rpm, and then spread onto LB agar plates containing antibiotics for drug selection markers maintained by plasmids (spectinomycin: 100 μg / mL, tetracycline: 15 μg / mL), and cultured at 30°C overnight. The plasmid is extracted from the colonies that appear, and the agarose gel electrophoresis after restriction endonuclease treatment confirms whether the target plasmid is just retained. The target transformant is stored at -80°C as a 25% glycerol suspension. [Example 3: Fermentation production of L-glutamic acid by NG13 strain into which the 2-methylcitrate synthase gene was introduced]

[0051] Since the NG13 strain exhibits nitrogen fixation under anaerobic conditions, based on the description of Patent Document 2 (Japanese Patent Application Laid-Open No. 2009-254323), an NG13 strain (NG13-pCMCS) was introduced into the plasmid pCMCS expressing the 2-MCS gene of Corynebacterium glutamicum, and an NG13 strain (NG13-pEMCS) was introduced into the plasmid pEMCS expressing the 2-MCS gene of Escherichia coli. These modified strains were statically cultured at 25°C for 4 days in KDC medium supplemented with IPTG at a final concentration of 0.1 mM to investigate L-glutamic acid productivity. The results are shown in Table 6.

[0052]

Table 6

[0053] Patent Document 1 (Patent Publication No. 4144131) states that when a Klebsiella microorganism, introduced with the CS gene from coryneform bacteria, is aerobically cultured, it produces 2.85 g / L of L-glutamic acid by fermentation. Thus, similar to the disclosure in Patent Document 1, the NG13 strain (NG13-pCCS strain) was prepared by introducing the plasmid pCCS expressing the CS gene from Corynebacterium glutamicum. First, this modified strain was cultured with shaking at 30°C in KDC medium supplemented with 10 g / L of ammonium chloride and a final concentration of 0.1 mM IPTG. 28 hours after the start of culture, the bacterial count reached 8 × 10 10 cells / tube, accumulating 1.4 g / L of L-glutamic acid in the culture medium. Separately, an NG13 strain (NG13-pKCS strain) was constructed by introducing the plasmid pKCS, which expresses the CS gene of the NG13 strain itself. This modified strain was cultured with shaking at 30°C in KDC medium supplemented with 1 g / L of ammonium chloride and a final concentration of 0.1 mM IPTG. Eighteen hours after the start of culture, the bacterial count reached 0.5 × 10 10 cells / tube, and accumulate 20 mg / L of L-glutamic acid in the culture medium.

[0054] Next, the NG13-pCCS and NG13-pKCS strains were statically cultured for 5 days at 25° C. in a KDC medium supplemented with IPTG at a final concentration of 0.1 mM to examine L-glutamic acid productivity.

Table 7

[0055] To confirm that the nitrogen element absorbed by the produced L-glutamic acid is derived from nitrogen molecules in the atmosphere, the NG13-pCCS strain was cultured in a KDC medium without Na2MoO4·2H2O to eliminate its nitrogen fixation ability. Although the maximum bacterial count in the culture medium remained the same as when cultured in a normal KDC medium, no L-glutamic acid accumulation was observed in the culture medium. Furthermore, when the NG13-pCCS strain was cultured in a KDC medium, the mouth of the test tube was sealed with a butyl double stopper and the air layer was sealed with a 15 Mixed gas of N2 and O2 ( 15When the culture medium was filled with N2:O2=82:18), 120 mg / L of L-glutamic acid accumulated in the culture medium on the 6th day of culture. 15 N molecular weight 149 L-glutamic acid cation ([C5H 10 15 NO4] + ) accounts for the majority ( Figure 2 ). This indicates that L-glutamic acid produced in the KDC culture medium was generated by nitrogen fixation ability, which was derived from nitrogen molecules in the atmosphere.

[0056] Next, the NG13-pCCS strain was statically cultured at 25°C for 6 days in KD, KDD, KDC, KDM, KDS, and KDO media to investigate whether the presence of organic acids other than citric acid in the nitrogen-limited medium affected L-glutamic acid production. The results are shown in Table 8.

[0057]

Table 8

[0058] When the NG13 strain is statically cultured in KDC medium, it first consumes glucose, with minimal citric acid consumption. Then, after glucose consumption ends, citric acid consumption begins. It is believed that the NG13 strain has a citric acid sensing gene in its genome: Na + The gene for the co-transporter gene and the two-component control system for the presence of citrate (composed of CitA as a sensor kinase and CitB as a response regulator), citrate:Na + The expression of the cotransporter gene is promoted by phosphorylated CitB (Non-Patent Document 2: Bott, Mol. Microbiol. 18 533-546 1995). When the NG13 strain is cultured in KDC medium, CitA, which senses the high concentration of citric acid in the medium, phosphorylates CitB, which promotes the conversion of citric acid:Na + Without intending to be bound by a particular theory, this is believed to result in minimal utilization of citric acid in the presence of glucose. +The expression of co-transporter genes is also promoted by the expression of cyclic AMP receptor protein (CRP). This is believed to be related to the utilization of citric acid after glucose consumption. + The co-transporter gene was cloned and the plasmid pKCT was constructed for high expression in the NG13 strain. The NG13 strain (NG13-pKCT strain) introduced with this plasmid was cultured statically in KDC medium at 25°C for 5 days to assess L-glutamic acid productivity. The results are shown in Table 9.

[0059]

Table 9

[0060] The NG13-pKCT and NG13-pCCS strains were 13 The NG13-pCCS strain was cultured in a KDC medium containing C-labeled glucose. When L-glutamic acid accumulated in the medium was analyzed by LC-MS, it was found that all L-glutamic acid accumulated in the NG13-pCCS strain contained 13 C, in contrast, the L-glutamic acid accumulated by the NG13-pKCT strain contained all 12 C. That is, the L-glutamic acid produced by the NG13-pKCT strain is thought to be converted from citric acid collected from the KDC culture medium.

[0061] The above results show that CS and citric acid:Na + The cotransporter independently contributes to L-glutamate production. Therefore, the NG13-pCCS-pKCT strain was constructed by introducing both the plasmids pCCS and pKCT into the NG13 strain. Additionally, as a control, the NG13-pCCS-pCDF strain was constructed by introducing both the plasmids pCCS and pCDFDuet-1 (empty vector) into the NG13 strain. Both strains were statically cultured in KDC medium at 25°C for 5 days to assess L-glutamate productivity. The results are shown in Table 10.

[0062]

Table 10

[0063] Based on the composition of the KDC medium, a culture medium was prepared in which the amount of glucose or sodium citrate was doubled (KDDC medium containing 15 g / L glucose, KDCC medium containing 15 g / L sodium citrate, and KDDCC medium containing 15 g / L glucose and 15 g / L sodium citrate). 25 mL of each was placed in a sterilized petri dish of φ90×15 mm, and 100 microorganisms were inoculated therein. 7 The NG13-pCCS-pKCT cell line was cultured statically at 25°C for 17 days in an environment where humidity was maintained to prevent evaporation of the culture medium, and the L-glutamic acid productivity was evaluated. The results are shown in Table 11.

Table 11

[0064] By adding citric acid:Na + The method of producing L-glutamic acid by fermentation by directly utilizing nitrogen molecules in the atmosphere and citric acid in the culture medium is an invention based on a completely new understanding. In addition, by adding citric acid:Na to the known technology of L-glutamic acid production promoted by the expression of CS, +The invention also presents a method for producing L-glutamic acid by fermentation, which utilizes the technical feature of promoting co-transporter expression and enhances L-glutamic acid productivity through the synergistic effect of the two, based on novel insights. Since citric acid is inexpensive among organic acids, the present invention offers a method for producing L-glutamic acid that can compete with current fermentative production methods that rely on the Haber-Bosch process. [Example 7. Enhancement of Nitrogen Fixation Ability in Nitrogen-Fixing Bacteria Other than NG13]

[0065] A culture medium containing 3.75 g / L glucose and 3.75 g / L fructose was prepared by replacing the 7.5 g / L glucose in KD and KDC culture medium with 3.75 g / L glucose and 3.75 g / L fructose. Nitrogen-fixing bacteria from the Proteobacteria phylum listed in Table 12 were cultured. After 24 hours of static culture at 25°C, acetylene was sealed in the culture chamber. Nitrogen-fixing capacity was calculated from ethylene production after 168 hours of culture. The results are shown in Table 12.

Table 12

[0066] Similar to the NG13 strain, a Raoultella terrigena JCM1687 strain (R. terrigena-pCCS strain) was constructed by introducing the plasmid pCCS expressing the CS gene of Corynebacterium glutamicum. This modified strain was statically cultured at 25°C for 6 days in KD medium and KDC medium supplemented with IPTG at a final concentration of 0.02 mM to assess L-glutamic acid productivity. The results are shown in Table 13.

Table 13

[0067] Preparation of plasmid pCCS expressing the CS gene of Corynebacterium glutamicum and the citric acid:Na expression vector of NG13 strain + Raoultella terrigena JCM1687 strain (R. terrigena-pCCS-pKCT strain) carrying the symporter gene plasmid pKCT was used. This modified strain was statically cultured at 25°C for 6 days in KDD and KDC medium supplemented with IPTG at a final concentration of 0.02 mM to assess L-glutamic acid productivity. The results are shown in Table 14.

Table 14

[0068] KDC and KDDC media were prepared, and 25 mL of each was placed in sterilized 90 x 15 mm petri dishes. The strain Raoultella terrigena (R. terrigena) -pCCS-pKCT was inoculated. The cultures were statically cultured at 25°C for 6 days in an environment where humidity was maintained to prevent evaporation of the culture medium. L-glutamic acid productivity was evaluated. The results are shown in Table 15.

Table 15

[0069] Similar to the NG13 strain, a Klebsiella variicola JCM12419 strain (Klebsiella variicola-pCCS) was prepared by introducing the plasmid pCCS expressing the CS gene of Corynebacterium glutamicum, and introducing the citric acid:Na + Klebsiella variicola JCM12419 strain harboring the symporter gene plasmid pKCT (K. variicola-pKCT) and Klebsiella variicola JCM12419 strain harboring both plasmids pCCS and pKCT (K. variicola-pCCS-pKCT) were tested for L-glutamic acid productivity by static culture at 25°C for 6 days in KDD and KDC media supplemented with IPTG at a final concentration of 0.02 mM. The results are shown in Table 16. Table 16 When the pCCS strain into which citrate synthase was introduced, the pKCT strain into which citrate transporter was introduced, and the pCCS-pKCT strain into which citrate synthase and citrate transporter were introduced were cultured in KDC medium, production of L-glutamic acid was observed.

[0070] Since it was confirmed that the nitrogen element of L-glutamic acid produced was derived from nitrogen molecules in the atmosphere, when K. variicola-pCCS strain was cultured in KDC medium, the mouth of the test tube was sealed with a butyl double stopper and the air layer was sealed with a 15 Mixed gas of N2 and O2 ( 15 When the culture medium was filled with N2:O2=82:18), 92 mg / L of L-glutamic acid accumulated in the culture medium on the 6th day of culture. 15 N molecular weight 149 L-glutamic acid cation ([C5H 1015 NO4] + This indicates that L-glutamic acid produced in KDC medium, like the NG13 strain, is produced by nitrogen fixation, which takes up nitrogen from atmospheric nitrogen molecules.

[0071] KDC and KDDC media were prepared, and 20 mL of each was placed in sterilized 90 x 15 mm petri dishes. Klebsiella variicola (K. variicola) -pCCS-pKCT strain was then inoculated. The cultures were statically cultured at 25°C for 14 days in an environment where humidity was maintained to prevent evaporation of the culture medium. L-glutamic acid productivity was evaluated. The results are shown in Table 17.

Table 17

[0072] Preparation of plasmid pCCS expressing the CS gene of Corynebacterium glutamicum and the citric acid:Na expression vector of NG13 strain + Klebsiella sp. NBRC100048 strain (Klebsiella sp. NBRC100048-pCCS-pKCT) and Klebsiella sp. NBRC109911 strain (Klebsiella sp. NBRC109911-pCCS-pKCT) containing the symporter gene plasmid pKCT were tested for L-glutamic acid productivity by static culture at 25°C for 6 days in KDD and KDC media supplemented with IPTG at a final concentration of 0.02 mM. The results are shown in Table 18.

Table 18

[0073] Preparation of plasmid pCCS expressing the CS gene of Corynebacterium glutamicum and the citric acid:Na expression vector of NG13 strain + The Rahnella aquatilis JCM1683 strain (R. aquatilis-pCCS-pKCT strain) carrying the cotransporter gene plasmid pKCT was cultured statically at 25°C for 6 days in KDC medium supplemented with IPTG at a final concentration of 0.02 mM to examine L-glutamic acid productivity. The results are shown in Table 19.

Table 19

[0074] Since it is confirmed that the nitrogen element of L-glutamic acid produced is derived from nitrogen molecules in the atmosphere, when the aquatic R. aquatilis-pCCS-pKCT strain is cultured in KDC medium, the mouth of the test tube is sealed with a butyl double stopper and the air layer is sealed with a 15 Mixed gas of N2 and O2 ( 15 When the culture medium was filled with N2:O2=82:18), 41 mg / L of L-glutamic acid accumulated in the culture medium on the 6th day of culture. 15 N molecular weight 149 L-glutamic acid cation ([C5H 10 15 NO4] + ) accounts for the majority ( Figure 12 This indicates that L-glutamic acid produced in KDC medium, like the NG13 strain, is generated by taking up nitrogen elements brought from nitrogen molecules in the atmosphere through its nitrogen fixation ability.

[0075] The above results indicate that the method of the present invention for producing L-glutamic acid by fermentation using atmospheric nitrogen molecules can also be performed using nitrogen-fixing bacteria belonging to the family Enterobacteriaceae other than the NG13 strain. Example 12. Fermentation Production of L-Alanine and L-Valine by the NG13 Strain Introduced with the Citrate Synthase Gene, the Glutamate-Pyruvate Aminotransferase Gene, and the L-Alanine Transporter Gene

[0076] Glutamate-pyruvate aminotransferase is an enzyme that catalyzes the reaction of generating L-alanine and 2-oxoglutaric acid from L-glutamate and pyruvate, contributing to the biosynthesis of L-alanine. It is believed that by introducing the glutamate-pyruvate aminotransferase gene into the NG13-pCCS strain, L-alanine or its salts can be generated by transferring the amino group of L-glutamate produced by utilizing atmospheric nitrogen molecules to pyruvate. Furthermore, it is believed that by also introducing an L-alanine transporter gene that has the function of expelling L-alanine to the extracellular space, the extracellular transport of the generated L-alanine can be promoted, thereby improving the productivity of L-alanine or its salts. Thus, a strain into which both genes were introduced was produced, and the L-amino acids present in the culture fluid of this strain were studied. (1) Preparation of plasmid pKACE

[0077] The glutamate-pyruvate aminotransferase and L-alanine transporter genes of the NG13 strain were cloned based on the genome of Klebsiella oxytoca M5a1, and sequence information was obtained (SEQ ID NO: 33 and SEQ ID NO: 34). The procedure for preparing the plasmid pKACE, which co-expresses the glutamate-pyruvate aminotransferase and L-alanine transporter genes, is as follows. PCR was performed using the primers listed in Table 4 and the template and primer combinations listed in Table 5 to produce both gene fragments. Each primer was assigned an approximately 20-bp sequence identical to that of the vector to be introduced, and the introduced gene was designed to be expressed using the vector-derived expression promoter and ribosome binding site (RBS). The target gene fragment and the vector-side fragment were combined using Gibson Assembly. 10 μL of the reaction solution was used to transform Escherichia coli DH5α strain, and colonies were selected using the drug selection marker contained in the plasmid. Plasmids were extracted from the colonies and confirmed to be the designed plasmids through sequence analysis. (2) Introducing plasmid pKACE into NG13-pCCS strain The NG13-pCCS strain was transformed with the plasmid pKACE by electroporation, and transformants were isolated based on their chloramphenicol resistance. Plasmids were adjusted from this strain, and the retention of the target plasmid was confirmed by agarose gel electrophoresis after restriction enzyme treatment. (3) Cultivation of NG13-pCCS-pKACE strain The NG13 strain (NG13-pCCS), which was introduced with the plasmid pCCS expressing the citrate synthase gene from Corynebacterium glutamicum, and the NG13-pCCS strain (NG13-pCCS-pKACE), which was introduced with the plasmid pKACE expressing both the NG13 strain's glutamate-pyruvate aminotransferase gene and the L-alanine transporter gene, were statically cultured at 25°C for 7 days in KDC medium supplemented with IPTG at a final concentration of 0.1 mM to examine L-amino acid productivity. The results are shown in Table 20. Table 20 When L-amino acids accumulated in the culture medium of the NG13 strain (NG13-pCCS-pKACE strain), into which the plasmid pKACE was introduced along with the plasmid pCCS, L-alanine, the target substance, was detected in addition to L-glutamate. Furthermore, L-valine was also detected. Klebsiella bacteria possess L-valine-pyruvate aminotransferase, which performs the reaction "3-methyl-2-oxobutyrate + L-alanine = L-valine + pyruvate." When nitrogen-fixing Klebsiella bacteria grow by nitrogen fixation, they often convert L-glutamate derived from atmospheric nitrogen into L-alanine by glutamate-pyruvate aminotransferase, and then convert this L-alanine into L-valine by L-valine-pyruvate aminotransferase. Therefore, it is believed that in the NG13-pCCS strain, the increased L-alanine production by glutamate-pyruvate aminotransferase, which produces L-glutamate, and the increased L-valine production by L-valine-pyruvate aminotransferase, do not reach the level excreted into the culture medium. In contrast, it is believed that in the NG13-pCCS strain, L-alanine production becomes extremely enhanced due to overproduction of glutamate-pyruvate aminotransferase, and L-valine production by L-valine-pyruvate aminotransferase is also enhanced, with both being excreted into the culture medium. This demonstrates that the NG13-pCCS-pKACE strain can produce L-valine from atmospheric nitrogen, along with L-alanine, through fermentation.

[0078] The above results indicate that the method of the present invention for producing L-glutamic acid by fermentation using atmospheric nitrogen molecules can also be used for the fermentation production of amino acids other than L-glutamic acid.

Industrial Applicability

[0079] The microorganisms of the present invention are also used in the fermentation production of L-glutamic acid or its salts. By using nitrogen from the atmosphere, it is possible to eliminate the large amount of fixed nitrogen compounds that were previously consumed by the Haber-Bosch process.

Accession number

[0080] Accession number: NITE BP-03721 (Accession number: NITE ABP-03721, Date of receipt of application: July 21, 2023, Name and address of depository institution: National Institute for Technology Evaluation and Research, National Patent Microorganism Depository (NPMD), Room 122, 2-5-8 Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan, 292-0818)

Claims

1. A method for producing L-glutamic acid or a salt thereof from atmospheric nitrogen molecules by fermentation, comprising: The process of culturing nitrogen-fixing bacteria in a nitrogen-limited medium, wherein The nitrogen-fixing bacteria are introduced with at least one gene selected from the group consisting of a citrate transporter, a citrate synthase, and a 2-methylcitrate synthase. The nitrogen-limited medium contains an organic acid or a salt thereof that constitutes the TCA pathway.

2. The method of claim 1, wherein the nitrogen-limited culture medium A nitrogen source containing 3 mg / L to 1500 mg / L calculated as yeast extract, and / or Contains 0.02mM to 15mM nitrogen source calculated as ammonium ion.

3. The method according to claim 1, wherein the organic acid or its salt has a concentration of 0.5 g / L to 100 g / L.

4. The method of claim 3, wherein the organic acid is citric acid, succinic acid, malic acid, fumaric acid, or 2-oxoglutaric acid.

5. The method according to any one of claims 1 to 4, wherein the nitrogen-fixing bacteria are microorganisms of the family Enterobacteriaceae.

6. The method of claim 5, wherein the nitrogen-fixing bacteria is a microorganism selected from the group consisting of Klebsiella, Rahnella, Raoultella, and Kosakonia.

7. A nitrogen-fixing bacterium into which a gene for a citrate transporter has been introduced, the bacterium being selected from the group consisting of the genera Klebsiella, Rahnella, Kosakonia and Raoultella.

8. The nitrogen-fixing bacteria according to claim 7, wherein the nitrogen-fixing bacteria of the genus Klebsiella are selected from the group consisting of Klebsiella oxytoca, Klebsiella michiganensis, Klebsiella grimontii, Klebsiella pasteurii, Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella indica, The nitrogen-fixing bacteria of the genus Rahnella are Rahnella aquatilis, or The nitrogen-fixing bacteria of the genus Raoultella are selected from the group consisting of Raoultella terrigena, Raoultella ornithinolytica and Raoultella planticola.

9. The nitrogen-fixing bacterium according to claim 7, wherein the nitrogen-fixing bacterium is Klebsiella oxytoca strain NG13 (Accession No.: NITE BP-03721), Klebsiella indica (JCM33718), Klebsiella variicola (JCM12419), Klebsiella sp. (NBRC100048, NBRC100441, NBRC109911), Raoultella terrigena (JCM1687=ATCC33257), Rahnella aquatilis (JCM1683=ATCC33071), and Klebsiella planticola (JCM20069=ATCC8329).

10. The nitrogen-fixing bacterium according to any one of claims 7 to 9, wherein the nitrogen-fixing bacterium ferments nitrogen molecules in the atmosphere under nitrogen limitation to produce L-glutamic acid or a salt thereof.

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