Method for producing target substance

Abstract

The present disclosure relates to a novel technique capable of highly efficiently producing a high-concentration preparation of a target substance in a production process for the target substance utilizing a microorganism under anaerobic conditions or microaerobic conditions, wherein a necessary substrate is efficiently supplied to a microbial metabolic pathway involved in the biosynthesis of the target substance and the dilution of the target substance in a reaction solution is suppressed. Specifically, the present disclosure relates to a method for producing a target substance, the method including (q) culturing or reacting cells of a microorganism having a urease activity or a treatment product of the cells in a culture medium or reaction medium (X) under anaerobic conditions or microaerobic conditions to produce the target substance, wherein, during the step (q), a urea-containing solution (Y) is added to the culture medium or reaction medium (X).

Description

Method for producing a target substance REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority based on Japanese Patent Application No. 2024-092549, filed on June 6, 2024, the entire disclosure of which is incorporated herein by reference for all purposes and constitutes a part of this specification.

[0002] The present invention relates to a method for producing a target substance in a microbial bioprocess under anaerobic or microaerobic conditions by adding a feed solution containing urea to a microbial culture or reaction system.

[0003] In substance production using microorganisms, various techniques are known for controlling the pH of the medium within a predetermined range and supplying substrates necessary for the biosynthesis of the target substance, by adding components that serve as carbon sources and nitrogen sources to the medium in advance or by supplying a feed solution containing the same components to the medium during the production reaction of the target substance.

[0004] For example, a glutamic acid production technique is known, which utilizes Corynebacterium glutamicum, a bacterium that requires biotin for growth and produces glutamic acid efficiently under aerobic conditions. More specifically, glutamic acid fermentation generally suffers from the problems of a decrease in the pH of the medium as glutamic acid is produced, thereby inhibiting the progress of glutamic acid fermentation, and the depletion of the nitrogen source in the medium necessary for glutamic acid production as the fermentation reaction progresses. To address these problems simultaneously, a technique has been used in which glutamic acid is produced while continuously supplementing the fermentation broth with urea during the fermentation reaction (Non-Patent Document 1).

[0005] Food Hygiene Journal, Vol. 8 (1967), No. 3, pp. 197-206 "Amino Acid Fermentation"

[0006] The applicant has been developing and improving efficient target substance production technologies using coryneform bacteria and Escherichia coli, which have the property of not substantially growing under anaerobic or microaerobic conditions. These technologies utilize the property of these bacteria that, while they do not substantially grow under anaerobic or microaerobic conditions, certain metabolic pathways, including the incomplete reductive TCA pathway, are still active within the cells. Furthermore, during microbial reactions, a pH adjuster must be continuously supplied to the reaction solution to maintain an optimal pH for target substance biosynthesis. However, this often results in a significant amount of pH adjuster being carried into the reaction solution, diluting the target substance concentration in the culture or reaction solution obtained after the target substance production reaction. Furthermore, even in the above-mentioned technologies for producing target substances under anaerobic or microaerobic conditions, the development of technologies that can more efficiently supply the substrates required in microbial substance production reactions and achieve even more efficient target substance production is anticipated.

[0007] Therefore, the object of the present invention is to provide a new technology that can efficiently supply the necessary substrates to the microbial metabolic pathway involved in the biosynthesis of a target substance in a target substance production process using microorganisms under anaerobic or microaerobic conditions, while suppressing dilution of the target substance in the reaction solution, thereby efficiently producing a highly concentrated preparation of the target substance.

[0008] In order to solve the above-mentioned problems, the present inventors conducted extensive research into the components of a pH-adjusting solution to be supplied to a reaction solution during a microbial reaction. As a result, they discovered that adding urea, which has traditionally been used to supply a nitrogen source, to a pH-adjusting solution under anaerobic or microaerobic conditions can maintain the pH of the reaction solution within an optimal range while efficiently supplying not only a nitrogen source but also carbon sources such as bicarbonate ions as substrates to the microbial metabolic pathway involved in the biosynthesis of the target substance, while also suppressing dilution of the target substance in the reaction solution, thereby enabling the efficient production of a highly concentrated preparation of the target substance. The present invention was made based on this discovery.

[0009] As mentioned above, Non-Patent Document 1 describes a technique for producing glutamic acid while continuously supplying urea to the fermentation broth to address the problems of the decrease in medium pH associated with glutamic acid production, which inhibits the progress of glutamic acid fermentation, and the depletion of the nitrogen source in the medium necessary for glutamic acid production as the fermentation reaction progresses. In this regard, since the glutamic acid fermentation described in Non-Patent Document 1 proceeds under aerobic conditions, the urea supplied to the fermentation broth is utilized as a nitrogen source by the microorganisms, but the carbon dioxide generated is not effectively utilized as a carbonate source as in the present invention, which employs anaerobic or microaerobic conditions. In other words, the technical problems and technical effects of the present invention, in which urea is utilized by microorganisms under anaerobic or microaerobic conditions as not only a nitrogen source but also a carbonate source, and Non-Patent Document 1, in which urea is utilized substantially only as a nitrogen source in fermentation under aerobic conditions, are different from each other. The above-mentioned problems solved by the present invention are novel and have not been found in prior art, and the technical effects achieved by the present invention could not have been predicted from Non-Patent Document 1.

[0010] According to an aspect of the present invention, there is provided a method for producing a target substance as follows.

[0011] [1] A method for producing a target substance, comprising: (q) culturing or reacting cells of a microorganism having urease activity or a treated product of the cells in a culture medium or reaction medium (X) under anaerobic or microaerobic conditions to produce a target substance; and during the step (q), adding a solution (Y) containing urea to the culture medium or reaction medium (X).

[0012] [2] The method according to [1], wherein the solution (Y) further contains at least one selected from the group consisting of ammonium bicarbonate, potassium carbonate, and ammonia.

[0013] [3] The method according to [1] or [2], wherein in step (q), the pH of the culture medium or reaction medium (X) is monitored and a predetermined amount of solution (Y) is added to the culture medium or reaction medium (X) to control the pH of the culture medium or reaction medium (X) to a predetermined value or within a predetermined range.

[0014] [4] The method according to any one of [1] to [3], wherein the microorganism has a predetermined metabolic pathway, and the predetermined metabolic pathway includes a metabolic reaction that utilizes at least one selected from the group consisting of carbon dioxide, carbonate ions, bicarbonate ions, ammonia, and ammonium ions as a reaction substrate.

[0015] [5] The method according to any one of [1] to [4], wherein the microorganism has a predetermined metabolic pathway, and the predetermined metabolic pathway includes a metabolic reaction that utilizes bicarbonate ions and ammonium ions as reaction substrates.

[0016] [6] The method according to any one of [1] to [5], wherein the microorganism is a microorganism having a reductive TCA cycle or an incomplete reductive TCA pathway.

[0017] [7] The method according to any one of [1] to [6], wherein the microorganism is a recombinant microorganism that satisfies all of the following conditions (I) to (III): condition (I) fumarate reductase activity, which catalyzes the conversion of fumarate to succinate in the reductive TCA cycle or the incomplete reductive TCA pathway, is reduced or inactivated; condition (II) lactate dehydrogenase activity is reduced or inactivated compared to that of the wild-type microorganism; and condition (III) the microorganism has a modified phosphoenolpyruvate carboxylase activity that is resistant to feedback inhibition by aspartate in wild-type phosphoenolpyruvate carboxylase activity, or an exogenous phosphoenolpyruvate carboxylase activity that is more resistant to feedback inhibition by aspartate than the wild-type phosphoenolpyruvate carboxylase activity exhibited by the wild-type microorganism.

[0018] [8] The method according to any one of [1] to [7], wherein the microorganism is a coryneform bacterium or an Escherichia bacterium.

[0019] [9] The method according to any one of [1] to [8], wherein the microorganism is Corynebacterium glutamicum or Escherichia coli.

[0020]

[10] The method according to any one of [1] to [9], wherein the target substance is at least one selected from the group consisting of amino acids, organic acids, and salts thereof.

[0021]

[11] The method according to any one of [1] to

[10] , wherein the intermediate metabolite is at least one selected from the group consisting of downstream metabolites biosynthesized via oxaloacetic acid or fumaric acid and salts thereof.

[0022]

[12] The method according to any one of [1] to

[11] , wherein the target substance is at least one selected from the group consisting of aspartic acid, downstream metabolites biosynthesized via aspartic acid as an intermediate metabolite, and salts thereof.

[0023]

[13] The method according to any one of [1] to

[12] , wherein the target substance is at least one selected from aspartic acid, beta-alanine, asparagine, and salts thereof.

[0024]

[14] The method according to any one of [1] to

[13] , wherein in step (q), the redox potential of the culture medium or reaction medium (X) is controlled within the range of +60 mV to −550 mV (preferably −200 mV to −550 mV).

[0025]

[15] The method according to any one of [1] to

[14] , wherein the solution (Y) contains 1 to 12.5 M urea, and in the step (q), the solution (Y) is added at a ratio of 2 to 1100 (v / v)% relative to the total volume of the culture medium or reaction medium (X).

[0026]

[16] The method according to any one of [1] to

[15] , wherein the solution (Y) contains 1 to 12.5 M or 1 to 10 M urea, and during the step (q), the solution (Y) is added to the culture medium or reaction medium (X) so as to satisfy all of the following conditions (a) to (d): (a) amount of the solution (Y) added / volume of the culture medium or reaction medium (X)=2 to 1100 (v / v)%; (b) amount of the solution (Y) added / total amount of glucose consumed=0.08 to 0.40 mL / mmol; (c) CO ３ ２－ or HCO ３ －(d) NH ３ Amount of glucose supplied / amount of glucose consumed = 2.00 to 3.50 mmol / mmol, provided that CO ３ ２－ or HCO ３ － Supply amount and NH under condition (d) ３ The amount of urea supplied is determined so that all of the urea contained in the solution (Y) supplied to the culture medium or reaction medium (X) is converted into CO ２ and N.H. ３ The CO ２ All of these are CO ３ ２－ or HCO ３ － The theoretical value is calculated assuming that the

[0027]

[17] The method according to any one of [1] to

[16] , wherein the target substance is biosynthesized through a metabolic reaction that utilizes the reductive TCA cycle or an incomplete reductive TCA pathway and bicarbonate ions (bicarbonate) as a reaction substrate.

[0028] According to an embodiment of the present invention, in a substance production process using microorganisms, the pH of the reaction solution can be maintained within an optimal range, while efficiently supplying the necessary substrates to the microbial metabolic pathway involved in the biosynthesis of the target substance, and dilution of the target substance in the reaction solution can be suppressed, resulting in efficient production of a highly concentrated preparation of the target substance.

[0029] FIG. 1 is a schematic diagram showing an example of a metabolic pathway (incomplete reductive TCA pathway) possessed by a microorganism that can be used in the present invention. FIG. 2 is a schematic diagram showing another example of a metabolic pathway (anaplerotic pathway) possessed by a microorganism that can be used in the present invention. FIG. 3 is a schematic diagram showing yet another example of a metabolic pathway possessed by a microorganism that can be used in the present invention. FIG. 4 is a schematic diagram showing yet another example of a metabolic pathway possessed by a microorganism that can be used in the present invention [L-arginine biosynthesis I (via L-ornithine)]. FIG. 5 is a schematic diagram showing yet another example of a metabolic pathway possessed by a microorganism that can be used in the present invention [L-arginine biosynthesis II (acetyl cycle)]. FIG. 6 is a schematic diagram showing yet another example of a metabolic pathway (UMP biosynthesis) possessed by a microorganism that can be used in the present invention. FIG. 7 is a schematic diagram showing yet another example of a metabolic pathway (inosine-5'-phosphate biosynthesis I) possessed by a microorganism that can be used in the present invention. FIG. 8 is a diagram showing the results of Test Examples 1 and 2 in the Examples. FIG. 9 is a diagram showing the results of Test Examples 1 and 2 in the Examples. FIG. 1 is a diagram showing the results of Test Examples 3 to 6 in the Examples. FIG. 2 is a diagram showing the results of Test Examples 3 to 6 in the Examples. FIG. 3 is a diagram showing the results of Test Examples 3 to 6 in the Examples. FIG. 4 is a diagram showing the results of Test Examples 3 to 6 in the Examples.

[0030] According to an aspect of the present invention, there is provided a method for producing a target substance, comprising: (q) culturing or reacting cells of a microorganism having urease activity or a treated product of the cells in a culture medium or reaction medium (X) under anaerobic or microaerobic conditions to produce a target substance, wherein a solution (Y) containing urea is added to the culture medium or reaction medium (X) during the step (q).

[0031] Below, each term will be explained and specific embodiments that can be employed in the present invention will be shown, but the present invention is not limited to the following embodiments.

[0032] <Culture medium or reaction medium (X) under anaerobic or microaerobic conditions> The term "anaerobic or microaerobic conditions" is a general scientific and technical term, and its meaning should be understood without any particular limitation.

[0033] In some embodiments, the "culture medium or reaction medium (X) under anaerobic or microaerobic conditions" in step (q) refers to a culture medium or reaction medium (X) whose dissolved oxygen concentration has been adjusted or controlled to a range in which the cells of the microorganism to be tested in step (q) do not substantially grow. In certain embodiments, the dissolved oxygen concentration of the culture medium or reaction medium (X) can be adjusted or controlled to a range of, for example, 0 to about 2.0 mg / L, preferably 0 to about 1.0 mg / L, and more preferably 0 to about 0.50 mg / L. The dissolved oxygen concentration of the culture medium or reaction medium (X) can be appropriately measured using a measurement method well known to those skilled in the art, and can be easily measured, for example, using a dissolved oxygen meter. Additionally, in certain embodiments, step (q) may be carried out using a culture apparatus or a bioreactor. In this case, the culture apparatus or the bioreactor may be equipped with a dissolved oxygen meter, and may be configured to monitor the dissolved oxygen concentration in the culture medium or reaction medium (X) during step (q) and control the dissolved oxygen concentration at a predetermined value or range.

[0034] Furthermore, strictly speaking, "anaerobic or microaerobic conditions" can be said to be a state in which dissolved oxygen is substantially absent or scarce, but it can also be said to be a technical term meaning a state in which electron acceptors, including oxides such as nitrate, are scarce or substantially absent.

[0035] Therefore, in some embodiments, in step (q), the target substance may be produced by culturing or reacting the microbial cells or a treated product thereof under reducing conditions to an extent that the microorganism does not substantially grow. In a preferred embodiment, the upper limit of the oxidation-reduction potential (ORP) of the culture medium or reaction medium (X) can be adjusted to, for example, about +60 mV or less, preferably about +55 mV or less, more preferably about +50 mV or less, even more preferably about +40 mV or less, about +30 mV or less, about +20 mV or less, about +10 mV or less, or about 0 mV or less, for example, about −50 mV or less, about −100 mV or less, about −150 mV or less, about −200 mV or less, or about −250 mV or less. In this case, the lower limit of the oxidation-reduction potential of the culture medium or reaction medium (X) is sufficient as long as microorganisms do not substantially grow therein. If the oxidation-reduction potential of the culture medium or reaction medium (X) is adjusted or controlled using the above-mentioned upper limit as a guide, the ORP value will be determined automatically, and therefore, is not particularly limited, and can be adjusted to, for example, about −550 mV or more, about −500 mV or more, about −450 mV or more, or about −400 mV or more.

[0036] In a specific embodiment, in step (q), the oxidation-reduction potential (ORP) of the culture medium or reaction medium (X) may be adjusted or controlled to fall within any range that is a consistent combination of any one of the upper and lower limit values ​​of the oxidation-reduction potential described above, and each of these numerical ranges is expressly defined in this specification as an embodiment that can be employed in the present invention.

[0037] The oxidation-reduction potential of the culture medium or reaction medium (X) can generally be measured using an oxidation-reduction potentiometer, and may be measured using a commercially available oxidation-reduction potentiometer (e.g., ORP Electrodes, manufactured by Broadley James). Alternatively, as a simple method, the reduction state of the culture medium or reaction medium (X) may be confirmed using a resazurin indicator (which decolorizes from blue to colorless in the reduced state).

[0038] In some embodiments, the "culture medium or reaction medium (X) under anaerobic or microaerobic conditions" can be achieved by not bubbling an oxygen-containing gas into the culture medium or reaction medium (X), or by bubbling an inert gas such as carbon dioxide, nitrogen, helium, neon, argon, krypton, or xenon into the culture medium or reaction medium (X).

[0039] Furthermore, in an embodiment employing reducing conditions under which microorganisms do not substantially grow, the method for preparing a culture medium or reaction medium (X) under the reducing conditions is not particularly limited, and various methods can be used. For example, the following known methods for preparing an aqueous solution for a reaction medium can be used.

[0040] That is, as a method for preparing an aqueous solution for a reaction medium as a culture medium or reaction medium (X), for example, a method for preparing a culture medium for an obligate anaerobic microorganism such as a sulfate-reducing microorganism (Pfenning, N. et al., (1981): The dissimilatory sulfate-reducing bacteria, In The Prokaryotes, A Handbook on Habitats, Isolation and Identification of Bacteria, Ed. by Starr, M.P. et al., pp. 926-940, Berlin, Springer Verlag) or "Agricultural Chemistry Experiment Book, Vol. 3, Kyoto University Faculty of Agriculture" can be used. The method described in "Agricultural Chemistry Class, ed., 26th edition, 1990, published by Sangyo Tosho Co., Ltd." can be adopted.

[0041] More specifically, a reduced state distilled water or aqueous solution for use as a reaction medium can be obtained by employing a step of removing dissolved gases by heating or reducing the pressure of distilled water or an aqueous solution containing a predetermined component. In this case, the distilled water or aqueous solution containing the predetermined component can be treated under a reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, and more preferably about 3 mmHg or less for about 1 to 60 minutes, preferably about 5 to 40 minutes. This removes dissolved gases, particularly dissolved oxygen, from the distilled water or the aqueous solution, and prepares an aqueous solution for use as a reaction medium under reducing conditions (anaerobic or microaerobic conditions).

[0042] Furthermore, in some embodiments, a culture medium or reaction medium (X) to which an appropriate reducing agent (e.g., thioglycolic acid, ascorbic acid, cysteine ​​hydrochloride, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, etc.) has been added may be used.

[0043] The culture medium or reaction medium (X) under anaerobic, microaerobic or reducing conditions may be prepared by any combination of the above-mentioned various techniques and component additions.

[0044] The composition of the culture medium or reaction medium (X) is not particularly limited, as long as it allows the target substance to be produced by the culture or reaction of the microorganism in step (q). Examples of the culture medium or reaction medium (X) that can be used include A medium [Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)], BT medium [Omumasaba, CA et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)], and various culture media or reaction media described in the Examples below.

[0045] Furthermore, in another embodiment, various known media typically used for culturing microorganisms may be used, such as LB medium, NB medium, SCD medium, YPD medium, PSY medium, ISP medium, MRS medium, SW medium, and SWS medium.

[0046] The type and composition of the culture medium or reaction medium (X) in the present invention are not particularly limited and may be appropriately selected in consideration of various factors such as the type and properties of the microorganism used, suitability for target substance production, etc. More specifically, the components (substrates) constituting the culture medium or reaction medium (X) can generally be divided into three component categories: carbon source, nitrogen source, and inorganic salts (minerals), and the composition of the culture medium or reaction medium (X) may be determined using these component categories as indicators, and any components or materials may be appropriately adopted and designed in consideration of various factors such as the type and properties of the microorganism and suitability for target substance production, as described above.

[0047] Examples of carbon sources are: CO ２ Gas (aeration), NaHCO ３ , KHCO ３ , Na ２ CO ３ , K. ２ CO ３ In addition to inorganic carbon sources such as those mentioned above, carbohydrates, more specifically sugars including polysaccharides and monosaccharides, and various materials containing these, for example, the following components can be mentioned.

[0048] monosaccharides such as glucose, fructose, mannose, xylose, arabinose, and galactose; disaccharides such as sucrose, maltose, lactose, cellobiose, xylobiose, and trehalose; polysaccharides such as cellulose, starch, glycogen, agarose, pectin, and alginic acid; molasses (blackstrap molasses, molasses), etc.; non-edible agricultural waste and non-edible biomass (resources made from non-edible herbaceous and woody plants) such as rice straw, forestry residues, bagasse, and corn stover; saccharified solutions containing multiple sugars such as glucose and xylose obtained by saccharifying energy crops such as switchgrass, napier grass, and miscanthus using saccharifying enzymes; sugar alcohols such as mannitol, sorbitol, xylitol, and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; alcohols such as ethanol, propanol, and butanol; and hydrocarbons such as normal paraffin.

[0049] In some embodiments, among these, monosaccharides are preferably used, and glucose is particularly more preferably used. In addition, in certain embodiments, sugars (e.g., disaccharides, oligosaccharides, polysaccharides) containing monosaccharides (glucose) are preferably used. The carbon source can be used alone or in combination of two or more. In addition, the concentration of the carbon source in the culture medium or reaction medium (X) is preferably about 1 to 20 w / v%, more preferably about 2 to 10 w / v%. In addition, the concentration of the sugar in the culture medium or reaction medium (X) is, for example, about 1 to 20 w / v%, more preferably about 2 to 10 w / v%, and even more preferably about 2 to 8 w / v%.

[0050] The nitrogen source is ammonium bicarbonate (NH ４ HCO ３ ), ammonium carbonate ((NH ４ ) ２ CO ３ ), inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, urea, aqueous ammonia, sodium nitrate, and potassium nitrate, etc. Furthermore, nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolysates, casamino acids, and amino acids can also be used.

[0051] The nitrogen source may be used alone or in combination of two or more. The concentration of the nitrogen source in the culture medium or reaction medium (X) may be appropriately adjusted depending on various conditions such as the type of microorganism, the type and properties of the desired target substance, reaction conditions, and the type of nitrogen compound, and is not particularly limited, but may be adjusted to, for example, about 0.1 to 20 w / v %, or about 0.1 to 10 w / v %.

[0052] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate (hydrate), sodium chloride, iron(II) sulfate heptahydrate, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. These inorganic salts can be used singly or in combination of two or more. The concentration of inorganic salts in the culture medium or reaction medium (X) can be adjusted appropriately depending on various conditions, such as the type of genetically modified microorganism used, the type and properties of the desired target substance, reaction conditions, and the type of inorganic salt. The concentration is not particularly limited, but may be approximately 0.01 to 5 w / v%, for example, approximately 0.01 to 1 w / v%.

[0053] Furthermore, the culture medium or reaction medium (X) may contain vitamins as needed. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, etc. The concentration of vitamins may be adjusted appropriately depending on the type and properties of the microorganism used, the type and properties of the desired target substance, reaction conditions, the type of each vitamin, and other conditions. The concentration is not particularly limited, but may be, for example, about 0.01 to 1 w / v %, preferably about 0.01 to 0.08 w / v %, or about 0.01 to 0.06 w / v %.

[0054] Furthermore, the culture medium or reaction medium (X) may optionally contain an antifoaming agent such as a polyalkylene glycol-based, silicone-based, animal fat-based, vegetable fat-based, etc. Furthermore, when using a microorganism that has acquired drug resistance by genetic recombination technology, for example, a corresponding drug (e.g., antibiotics such as hygromycin, chloramphenicol, kanamycin, ampicillin, penicillin, etc.) may be added.

[0055] <Microorganisms> In the present invention, "microorganisms" include microalgae (e.g., diatoms, dinoflagellates, cyanobacteria, green algae, and red algae), protists (e.g., Euglena), fungi (e.g., yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe), prokaryotes (e.g., bacteria and archaea), and the like.

[0056] In some embodiments, the microorganism is a chemoautotrophic bacterium (e.g., nitrifying bacteria such as Nitrosomonas europaea and Nitrobacter winogradskyi; hydrogen-oxidizing bacteria; sulfur-oxidizing bacteria such as Acidithiobacillus thiooxidans; iron-oxidizing bacteria such as Acidithiobacillus ferrooxidans), cyanobacteria (e.g., Synechocystis sp. PCC 6803, Thermosynechococcus elongatus BP-1, Anabaena sp. PCC 7120), or photosynthetic bacteria (e.g., purple bacteria, green sulfur bacteria, green gliding bacteria, heliobacteria). In certain embodiments, the microorganism is a hydrogen-oxidizing bacterium, such as Acidovorax facilis, Alcaligenes latus, Alcaligenes latus, Aquaspirillum autotrophicum, Arthrobacter sp. , Pseudonocardia autotrophica, Rhodococcus opacus, Hydrogenibacillus schlegelii, Hydrogenophilus thermoluteolus (TH-1 strain), Hydrogenobacter thermophilus, Hydrogenobacter halophilus, Hydrogenobacter acidophilus, Aquifex pyrophilus, and Hydrogenovibrio marinus.

[0057] In another embodiment, the microorganism is selected from the group consisting of Escherichia (e.g., Escherichia coli), Enterococcus (e.g., Enterococcus faecalis), Bacillus (e.g., Bacillus subtilis), Lactobacillus (e.g., Lactobacillus plantarum, Lactobacillus acidophilus), Clostridium (e.g., Clostridium thermocellum, Clostridium acetobutylicum), Rhodopseudomonas (e.g., Rhodopseudomonas The bacteria may be bacteria belonging to the genus Rhodobacter (e.g., Rhodobacter capsulatus), the genus Pantoea (e.g., Pantoea ananatis), or even bacteria belonging to the coryneform bacteria described in detail below (Bargey's Manual of Determinative Bacteriology, Vol. 8, p. 599, 1974).

[0058] More specifically, examples of coryneform bacteria include bacteria of the genus Corynebacterium (including the former genus Brevibacterium), bacteria of the genus Arthrobacter, bacteria of the genus Mycobacterium, bacteria of the genus Micrococcus, bacteria of the genus Microbacterium, etc. Examples of species and strains belonging to coryneform bacteria are shown below.

[0059] Corynebacterium: Corynebacterium glutamicum (e.g., FERM P-18976 strain, ATCC13032 strain, ATCC31831 strain, ATCC13058 strain, ATCC13059 strain, ATCC13060 strain, ATCC13232 strain, ATCC13286 strain, ATCC13287 strain, ATCC13655 strain, ATCC13745 strain, ATCC13746 strain, ATCC13761 strain, ATCC14020 strain); Corynebacterium acetoglutamicum (Corynebacterium Corynebacterium acetoglutamicum (e.g., ATCC 15806 strain); Corynebacterium acetacidophilum (e.g., ATCC 13870 strain); Corynebacterium melassecola (e.g., ATCC 17965 strain); Corynebacterium efficiens (e.g., YS-314 strain, YS-314 Ｔ strain (NBRC100395 ＴCorynebacterium alkanolyticum (e.g., ATCC 21511 strain); Corynebacterium callunae (e.g., ATCC 15991 strain, NBRC 15359 strain, DSM 20147 strain); Corynebacterium lilium (e.g., ATCC 15990 strain); Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium efficiens) (e.g., strain AJ12340, strain FERM BP1539); Corynebacterium herculis (e.g., strain ATCC13868); Corynebacterium ammoniagenes (Brevibacterium ammoniagenes) (e.g., strain ATCC6871, strain ATCC6872, strain DSM20306, strain NBRC12071); Ｔ strain, NBRC12072 strain, NBRC12612 Ｔ strain); Corynebacterium pollutisoli; Corynebacterium marinum (e.g., strain DSM44953); Corynebacterium humireducens (e.g., strain NBRC106098); Corynebacterium halotolerans (e.g., strain YIM70093); Corynebacterium desartii (Corynebacterium Corynebacterium doosanense (e.g., strain CAU212, strain DSM45436); Corynebacterium maris (e.g., strain DSM45190).

[0060] Bacteria of the genus Arthrobacter: Arthrobacter globiformis (e.g., ATCC8010 strain, ATCC4336 strain, ATCC21056 strain, ATCC31250 strain, ATCC31738 strain, ATCC35698 strain, NBRC3062 strain, NBRC12137T strain).

[0061] Bacteria of the genus Micrococcus: Micrococcus freudenreichii [e.g., strain No. 239 (FERM P-13221)]; Micrococcus luteus [e.g., strain NCTC2665, strain No. 240 (FERM P-13222)]; Micrococcus ureae (e.g., strain IAM1010); Micrococcus roseus (e.g., strain IFO3764).

[0062] Microbacterium: Microbacterium ammoniaphilum (e.g., ATCC 15354 strain).

[0063] In some embodiments, the microorganism is a microorganism having a reductive TCA cycle or an incomplete reductive TCA pathway (e.g., an archaea or a bacterium, preferably a green sulfur bacterium such as Chlorobium limicola, a hydrogen-oxidizing bacterium such as Hydrogenophilus thermoluteolus, a coryneform bacterium, a Corynebacterium sp., an Escherichia sp. such as Escherichia coli, or a Lactobacillus sp. such as Lactobacillus plantarum).

[0064] In the present invention, the microorganism has urease activity (urease activity, EC 3.5.1.5) that catalyzes the following reaction formula (I): urea (NH ２ ) ２CO is converted into carbon dioxide (CO ) by the urease activity of the microorganisms, as shown in reaction formula (I). ２ ) and ammonia (NH ３ ) which are used as substrates in microbial metabolism, enabling more efficient production of the target substance.

[0065] (NH ２ ) ２ CO + H ２ O → CO ２ + 2NH ３ ...(I)

[0066] In microbial fermentation under aerobic conditions, a large amount of carbon dioxide is released within the system through aerobic respiration via the oxidative TCA cycle. However, in microbial reactions under anaerobic or microaerobic conditions, although predetermined metabolic pathways such as the reductive TCA cycle and the incomplete reductive TCA pathway are activated as described below, aerobic respiration via the oxidative TCA cycle does not occur. Therefore, carbon dioxide is not inherently generated via microbial metabolism. In this regard, according to the present invention, as shown in reaction formula (I), urea ((NH ２ ) ２ The urea-containing solution (Y) is added to the culture medium or reaction medium (X) during step (q) in the method according to the present invention.

[0067] The urease activity possessed by the microorganism may be realized by at least one of the following (i) and (ii): (i) wild-type urease activity inherently possessed by the microorganism; (ii) exogenous urease activity introduced into the microorganism in an expressible manner.

[0068] In an embodiment of (i) above, for example, bacteria inherently having urease activity (e.g., coryneform bacteria, more specifically, bacteria of the genus Corynebacterium or Corynebacterium glutamicum) may be used as the microorganism to be tested in step (q). In this case, the bacteria may not have been subjected to the introduction of a nucleic acid (gene) encoding urease. However, the bacteria may be genetically engineered for the purpose of constructing or enhancing a biosynthetic pathway of a target substance, and may be wild-type or recombinant.

[0069] Furthermore, the urease activity according to (ii) above may be realized by introducing into a host microorganism a nucleic acid (gene) encoding at least one of the wild-type urease (homologous urease), heterologous urease, and mutant urease according to (i) above. More specifically, an example of an embodiment is one in which the microorganism tested in step (q) contains a nucleic acid encoding a homologous or heterologous wild-type urease and / or mutant urease in an expressible form, regardless of whether the microorganism inherently has wild-type urease activity or not.

[0070] For example, microorganisms that inherently have urease activity, such as coryneform bacteria, may be subjected to step (q) without introducing a nucleic acid encoding urease, or may be subjected to introduction of a nucleic acid encoding at least one of wild-type urease, heterologous urease, and mutant urease in order to enhance or modify the urease activity.

[0071] The urease-encoding gene that can be introduced into a host microorganism can be a wild-type urease isolated from any biological species or a mutant urease-encoding gene thereof. Examples of the urease-encoding gene that can be introduced into a host microorganism include, but are not limited to, urease genes derived from various bacteria, more specifically, ureA, B, C, D, E, F, and G isolated and sequenced from Corynebacterium glutamicum (FEMS Microbiology Letters 189 (2000) pp. 305-310, Gene Accession No. AJ251883), or homologs, orthologs, or paralogs thereof. In a specific embodiment, a genetically modified microorganism [preferably a genetically modified bacterium, more specifically a coryneform bacterium (e.g., a bacterium of the genus Corynebacterium) or a bacterium of the genus Escherichia (e.g., Escherichia coli)] containing a nucleic acid encoding the bacterial urease as described above in an expressible form may be subjected to step (q).

[0072] As described above, in the bioprocess system in step (q), CO is produced from urea in the solution (Y) added to the culture medium or reaction medium (X) by the urease activity of the microorganism. ２ When this occurs, the CO ２ Theoretically, the gas-liquid equilibrium shown in the following formula (II-1) and the chemical equilibrium shown in (II-2) to (II-4) in the culture medium or reaction medium (X) are reached.

[0073]

[0074] That is, in the bioprocess system, carbon dioxide CO in the gas phase ２ (g) and carbon dioxide CO in a culture medium or reaction medium (X). ２ (aq) means a gas-liquid equilibrium state represented by formula (II-1) and further, the culture medium or reaction medium (X) contains hydrated carbon dioxide CO ２ (aq) (i.e., dissolved CO ２ ), carbonate H ２ CO ３ (aq), bicarbonate ion HCO ３ － (aq), and carbonate ion CO ３２－ These four carbonate substances in the culture medium or reaction medium (X) reach a state of chemical equilibrium as shown in formulas (II-2) to (II-4) in Table 1. However, the CO ２ (aq) and H ２ CO ３ The ratio of carbon dioxide to carbon dioxide (aq) is approximately 1000:1. ２ CO ３ (aq) has little significance, and from the viewpoint of microbial metabolism, carbon dioxide CO ２ (aq) (i.e., dissolved CO ２ ), bicarbonate ion HCO ３ － (aq), and carbonate ion CO ３ ２－ It can be said that attention should be paid to the following three types of carbonate substances.

[0075] Thus, in some embodiments, the microorganisms in step (q) are activated by carbon dioxide CO ２ (aq) (i.e., dissolved CO ２ ), bicarbonate ion HCO ３ － (aq), and carbonate ion CO ３ ２－ (In particular, carbon dioxide CO ２ (aq) and bicarbonate ion HCO ３ － (aq)) as a substrate.

[0076] In particular, HCO ３ － (bicarbonate ion) is CO held by microorganisms ２ It can be a substrate used by some of the various enzymes in fixed metabolic pathways. ３ － After being taken up into the microbial cell, bicarbonate ions are converted into CO by carbonic anhydrases possessed by the microorganism (for example, CAH1, CAH3, CAH6, CAH9, etc. possessed by the green alga Chlamydomonas reinhardtii). ２ and the CO thus converted ２can be used in metabolic reactions catalyzed by certain enzymes that can use carbon dioxide (CO ２ ) and water (H ２ O) to carbonic acid (H ２ CO ３ ) and its reverse reaction, and the carbonic acid (H ２ CO ３ ) is bicarbonate ion (HCO ３ － ) and hydrogen ions (H ＋ These ions can be used as substrates in certain metabolic reactions. Carbonic anhydrases are known to exist in a wide range of organisms, from prokaryotes such as bacteria to eukaryotes.

[0077] Thus, in certain embodiments, the microorganism is ２ and / or HCO ３ － As a substrate, the microorganism may include a metabolic pathway that utilizes the enzyme as a substrate. Additionally or alternatively, the microorganism may include the above-mentioned carbonic anhydrase (EC 4.2.1.1). These embodiments may be realized by expression of a native enzyme gene possessed by a wild-type microorganism, by forced expression of a native enzyme gene using genetic engineering techniques, or by forced expression of a corresponding recombinant enzyme gene.

[0078] In the present invention, "HCO ３ － Unless otherwise specified, when we say "a metabolic pathway that uses HCO as a substrate," we mean HCO present in microbial cells. ３ － is directly utilized or incorporated into metabolic reactions catalyzed by specific enzymes that can utilize it as a substrate, and / or ３ － However, CO ２ and then the CO ２On the other hand, in the present invention, "HCO ３ － When referring to a reaction or metabolic reaction that utilizes HCO as a substrate, the term should be interpreted literally. ３ － refers to a specific metabolic reaction in which a protein is directly utilized or incorporated as a reaction substrate.

[0079] As described above, the metabolic pathways of microorganisms are specifically CO ２ or HCO ３ － The metabolic pathway may be a metabolic pathway inherently possessed by wild-type microorganisms, or may be a metabolic pathway artificially constructed by genetic engineering techniques. ３ － Preferably, the metabolic reaction involves the use of the enzyme as a substrate.

[0080] Examples of metabolic pathways found in microorganisms include the glycolytic pathway and TCA cycle found in many microorganisms; the reductive pentose phosphate cycle found in photosynthetic bacteria (cyanobacteria) and chemosynthetic bacteria; the reductive TCA cycle found in purple nonsulfur bacteria, purple sulfur bacteria (a type of photosynthetic bacteria), and hydrogen-oxidizing bacteria; the incomplete reductive TCA pathway found in coryneform bacteria and the like [Figure 1(a)]; the 3-hydroxypropionic acid cycle found in green nonsulfur bacteria and the like; the acetyl-CoA pathway found in methanotrophs and Moorella thermoacetica; the anaplerotic pathway found in many microorganisms, which replenishes specific intermediates in the TCA cycle and the incomplete TCA pathway [Figure 1(b)]; and the glyoxylate cycle found in some microorganisms such as yeast.

[0081] Thus, in some embodiments, the microorganism comprises at least one selected from the group consisting of a glycolytic pathway, a TCA cycle, a reductive pentose phosphate cycle, a reductive TCA cycle, an incomplete reductive TCA pathway, a 3-hydroxypropionic acid cycle, an acetyl-CoA pathway, an anaplerotic pathway, and a glyoxylate cycle. Furthermore, in certain embodiments, the metabolic pathway possessed by the microorganism comprises a glycolytic pathway and at least one selected from the group consisting of a TCA cycle, a reductive pentose phosphate cycle, a reductive TCA cycle, an incomplete reductive TCA pathway, a 3-hydroxypropionic acid cycle, an acetyl-CoA pathway, an anaplerotic pathway, and a glyoxylate cycle. Furthermore, in another embodiment, the metabolic pathway possessed by the microorganism comprises a glycolytic pathway and at least one selected from the group consisting of a TCA cycle, a reductive TCA cycle, an incomplete reductive TCA pathway, and an anaplerotic pathway.

[0082] In particular, anaplerotic pathways that replenish certain intermediates in the TCA cycle, or the reductive TCA cycle or an incomplete reductive TCA cycle, include HCO ３ － This pathway incorporates HCO into intermediate metabolites of C3 compounds in glycolysis to produce C4 compounds such as oxaloacetate and malate [Figure 1(b)]. ３ － as a substrate, preferably HCO ３ － More specifically, the anaplerotic pathway may include pyruvate carboxylase (PCx, EC 6.4.1.1), phosphoenolpyruvate carboxylase (PEPCx, EC 4.1.1.31), phosphoenolpyruvate carboxykinase [PCK; EC 4.1.1.49, EC 4.1.1.32, or EC 4.1.1.38], and optionally malic enzyme (ME, EC 1.1.1.40, bicarbonate ion HCO ３ －In an environment where the concentration is relatively high, pyruvate carboxylase (PC, EC 6.4.1.1) reversibly catalyzes the production of malic acid by carboxylation of pyruvate [Figure 1(b)]. Among these anaplerotic enzymes, pyruvate carboxylase (PC, EC 6.4.1.1) catalyzes the production of malic acid by carboxylation of HCO ３ － It is an enzyme that uses HCO as a substrate and incorporates it into the C3 compound pyruvate to produce the C4 compound oxaloacetate. ３ － The method of the present invention employs a configuration in which a solution (Y) containing urea is added to the culture medium or reaction medium (X) during step (q), and a sufficient amount of HCO ３ － is supplied, and HCO ３ － Since the enzyme is utilized by the above-mentioned group of enzymes that utilize the above as a substrate, the target substance can be produced efficiently.

[0083] In some embodiments, the microorganism comprises a TCA cycle and / or a reductive TCA cycle or an incomplete reductive TCA cycle, and an anaplerotic pathway that replenishes certain intermediates in the TCA cycle or the reductive TCA cycle or the incomplete reductive TCA cycle. Furthermore, in certain embodiments, the microorganism comprises a glycolytic pathway and an anaplerotic pathway, and also comprises a reductive TCA cycle or an incomplete reductive TCA pathway. In another embodiment, the microorganism comprises a glycolytic pathway and an anaplerotic pathway, and also comprises a reductive TCA cycle or an incomplete reductive TCA pathway. ３ － The present invention comprises, in an expressible manner, a gene encoding a wild-type or recombinant phosphoenolpyruvate carboxylase (PEPC) and / or pyruvate carboxylase (PC) that utilizes the above as a substrate.

[0084] In some embodiments, the microorganism is CO ２ or HCO ３ －The gene contains at least one of a group of metabolic pathway control enzymes (encoding genes for each enzyme) including enzymes that utilize the above as a substrate.

[0085] For example, the microorganism contains at least one of the following enzyme groups (i) to (v) (respective enzyme-encoding genes): (i) At least one enzyme (enzyme-encoding gene) from a group of enzymes controlling the reductive pentose phosphate pathway, which includes the enzyme identified by EC 4.1.1.39 (ribulose-1,5-bisphosphate carboxylase / oxygenase).

[0086] (ii) an enzyme specified by EC 1.2.7.3 (2-oxoglutarate synthase); an isocitrate dehydrogenase (e.g., an enzyme specified by EC 1.1.1.41 (isocitrate dehydrogenase (NAD ＋ )) and / or the enzyme specified by EC 1.1.1.42 (isocitrate dehydrogenase (NADP ＋ )))] and / or at least one enzyme (enzyme-encoding gene) of a group of enzymes controlling the reductive TCA cycle, including an enzyme specified by EC 6.4.1.7 (2-oxoglutarate carboxylase); and an enzyme specified by EC 1.2.7.1 (pyruvate synthase).

[0087] (iii) at least one of the enzymes specified by EC 6.4.1.1 (pyruvate carboxylase); the enzymes specified by EC 4.1.1.31 (phosphoenolpyruvate carboxylase, PEPC); the enzymes specified by EC 4.1.1.49 (phosphoenolpyruvate carboxykinase (ATP)), the enzymes specified by EC 4.1.1.32 (phosphoenolpyruvate carboxykinase (GTP)), and the enzymes specified by EC 4.1.1.38 (phosphoenolpyruvate carboxykinase (diphosphate)); and optionally an enzyme specified by EC 1.1.1.40 (malic enzyme, bicarbonate ion HCO ３ －At least one enzyme (enzyme-encoding gene) from a group of enzymes that control the anaplerotic pathway, including ATP, which reversibly catalyzes the carboxylation of pyruvate to produce malic acid in an environment where the concentration is relatively high.

[0088] (iv) an enzyme specified by EC 1.1.1.37 (malate dehydrogenase (NAD+)) and / or an enzyme specified by EC 1.1.1.82 (malate dehydrogenase (NADP ＋ )) and / or the enzyme specified by EC 1.1.1.299 (malate dehydrogenase [NAD(q) ＋ ] and / or 1.1.5.4 (malate dehydrogenase (quinone)); enzymes specified by EC 4.2.1.2 (fumarate hydratase); and at least one enzyme (enzyme-encoding gene) from a group of enzymes that control the incomplete reductive TCA pathway, including an enzyme specified by EC 1.3.5.1 (succinate dehydrogenase).

[0089] (v) At least one enzyme (enzyme-encoding gene) of a group of enzymes controlling the 3-hydroxypropionic acid cycle, including the enzyme specified by EC 6.4.1.2 (acetyl-CoA carboxylase); and the enzyme specified by EC 6.4.1.3 (propionyl-CoA carboxylase).

[0090] Additionally, in some embodiments, the microorganisms are capable of producing ammonia (NH ３ The enzymes include at least one of a group of enzymes (encoding genes for each enzyme) that control each metabolic pathway including metabolic reactions that utilize (aq)) as a substrate.

[0091] For example, the microorganism may contain at least one enzyme (enzyme-encoding gene) among the enzymes shown in Table 2 below. More specifically, the microorganism may contain an enzyme (enzyme-encoding gene) that can catalyze at least one of the reactions represented by the reaction formulas shown in Table 2. Furthermore, in certain embodiments, the microorganism may contain an enzyme (enzyme-encoding gene) identified by at least one of the EC numbers shown in Table 2.

[0092]

[0093] In particular, in the enzymatic reactions represented by reaction formulas (vi-1), (vi-2), (vii), and (viii), when the reverse reaction proceeds from the right side to the left side, an L-amino acid or L-aspartic acid can be produced. In particular, in reaction formulas (vi-1), (vi-2), and (viii), NH produced from urea contained in solution (Y) ３ can be used as a substrate.

[0094] Furthermore, in a specific embodiment, the microorganism may be a genetically modified microorganism that satisfies at least one of the following conditions (I) to (IV): Condition (I) Succinate dehydrogenase activity or fumarate reductase activity is reduced or inactivated compared to a wild-type microorganism corresponding to the genetically modified microorganism [preferably, fumarate reductase activity that catalyzes the conversion reaction from fumarate to succinate under reducing conditions, anaerobic conditions, or microaerobic conditions is reduced or inactivated compared to a wild-type microorganism, more preferably, an sdh gene (e.g., at least one of the CAB subunits) or an frd gene (e.g., at least one of the DCBA subunits) is inactivated or deleted]. Condition (II) lactate dehydrogenase activity is reduced or inactivated compared to a wild-type microorganism (preferably, the ldh gene is inactivated or deleted); Condition (III) a modified phosphoenolpyruvate carboxylase activity that is resistant to feedback inhibition by aspartate in the wild-type phosphoenolpyruvate carboxylase activity, or an exogenous phosphoenolpyruvate carboxylase activity that is more resistant to feedback inhibition by aspartate than the wild-type phosphoenolpyruvate carboxylase activity exhibited by a wild-type microorganism [preferably, Corynebacterium glutamicum a gene encoding a mutant phosphoenolpyruvate carboxylase derived from a bacterium (preferably a coryneform bacterium, more preferably the genus Corynebacterium, even more preferably Corynebacterium glutamicum) having an amino acid substitution corresponding to D299N and / or K813S based on the amino acid sequence of wild-type PEPC derived from the Corynebacterium glutamicum strain ATCC13032 (SEQ ID NO: 2 disclosed in WO 2020 / 208842 A1) has been introduced in an expressible manner, with the proviso that the mutant phosphoenolpyruvate carboxylase is more resistant to feedback inhibition by aspartate than the corresponding wild-type enzyme; and condition (IV) that pyruvate:quinone oxidoreductase activity is reduced or inactivated compared to the wild-type microorganism.

[0095] In addition, in some embodiments, at least one of conditions (I), (II), and (IV) may be satisfied, and the following condition (III) may also be satisfied, preferably at least two of conditions (I), (II), and (IV), and in certain embodiments, both conditions (I) and (II), both conditions (I) and (IV), or both conditions (II) and (IV) may be satisfied.

[0096] By using a genetically modified microorganism that satisfies at least one of the conditions (I) to (IV), it is possible to efficiently produce target substances via the TCA cycle, the reductive TCA cycle, or the incomplete reductive TCA pathway, as well as downstream metabolic pathways, such as the metabolic pathways and downstream metabolic products of these metabolic pathways.

[0097] Furthermore, in a particularly preferred embodiment, a microorganism (preferably a coryneform bacterium, a bacterium of the genus Corynebacterium, or a bacterium of the genus Escherichia) that satisfies all of the following conditions (I) to (IV) is subjected to the method of the present invention. Such a microorganism configuration allows the construction of an aspartic acid biosynthetic pathway as shown in Figure 2, enabling efficient production of aspartic acid or further downstream metabolites that use aspartic acid as an intermediate metabolite as a target substance.

[0098] The above-mentioned genetically modified microorganisms, together with various genetically modified configurations and embodiments, are disclosed in International Publication WO 2020 / 208842 A1, the contents of which are incorporated herein by reference.

[0099] In some embodiments, the microorganism may contain at least one enzyme (enzyme-encoding gene) among the enzymes shown in Table 3 below. More specifically, the microorganism may contain an enzyme (enzyme-encoding gene) that can catalyze at least one of the reactions represented by the reaction formulas shown in Table 3. Furthermore, in certain embodiments, the microorganism may contain an enzyme (enzyme-encoding gene) identified by at least one of the EC numbers shown in Table 3.

[0100]

[0101] Furthermore, in certain embodiments, the microorganism may have at least one of the metabolic pathways or biosynthetic pathways shown in (a) to (n) below, or may have each enzyme activity identified by each EC number shown in at least one of (a) to (n) below.

[0102] (a) L-arginine biosynthetic pathway I (via L-ornithine, Figure 3(a)): EC 2.3.1.1, EC 2.7.2.8, EC 1.2.1.38, EC 2.6.1.11, EC 3.5.1.16, EC 6.3.5.5, EC 2.1.3.3, EC 6.3.4.5, and EC 4.3.2.1;

[0103] (b) L-arginine biosynthetic pathway II (acetyl cycle, Figure 3(b)): EC 2.3.1.1, EC 2.7.2.8, EC 1.2.1.38, EC 2.6.1.11, EC 2.3.1.35, EC 6.3.5.5, EC 2.1.3.3, EC 6.3.4.5, and EC 4.3.2.1;

[0104] (c) UMP biosynthetic pathway ( Figure 4 ): EC 6.3.5.5, EC 2.1.3.2, EC 3.5.2.3, EC 1.3.5.2, EC 2.4.2.10, and EC 4.1.1.23;

[0105] (d) UTP and CTP de novo biosynthetic pathways (UMP → UDP → UTP → CTP) starting from UMP, a product of the UMP biosynthetic pathway ( FIG. 4 ): EC 2.7.4.14 (or EC 2.7.4.22), EC 2.7.4.6, and EC 6.3.4.2;

[0106] (e) Inosine-5′-phosphate biosynthetic pathway I ( Figure 5 ): EC 6.3.1.48, EC 5.4.99.18, EC 6.3.2.6, EC 4.3.2.2, EC 2.1.2.30, and EC 3.5.4.10;

[0107] (f) Guanosine nucleotide de novo biosynthetic superpathway (IMP → XMP → GMP → GDP → GTP / dGDP → GTP) originating from the product IMP in the inosine-5′-phosphate biosynthetic pathway I ( FIG. 5 ): EC 1.1.1.205, EC 6.3.5.2, EC 2.7.4.8, EC 2.7.4.8, EC 1.17.4.1, EC 2.7.4.6, and EC 1.17.4.2;

[0108] (g) adenosine nucleotide de novo biosynthetic superpathway (L-aspartate + IMP → adenylosuccinate → AMP → ADP → dADP / ATP → dATP) originating from the product IMP in inosine-5′-phosphate biosynthetic pathway I ( FIG. 5 ): EC 6.3.4.4, EC 4.3.2.2, EC 2.7.4.3, EC 1.17.4.1, EC 2.7.4.6, EC 3.6.3.14, and EC 1.17.4.2;

[0109] (h) L-asparagine biosynthetic pathway I (L-aspartic acid → L-asparagine): EC 6.3.5.4;

[0110] (i) L-asparagine biosynthetic pathway II (L-aspartic acid → L-asparagine): EC 6.3.1.1;

[0111] (j) L-aspartate biosynthesis superpathway (L-asparagine → L-aspartate): EC 3.5.1.1 / EC 3.5.1.38;

[0112] (k) L-glutamate degradation pathway II (L-glutamate⇔L-aspartate⇔fumarate / TCA cycle) or a part thereof (L-glutamate⇔L-aspartate, or fumarate⇔L-aspartate): EC 2.6.1.1 (aspartate transaminase / aspB) and EC 4.3.1.1 (aspartate ammonia-lyase, aspA);

[0113] (l) L-aspartate biosynthetic pathway [oxaloacetate (TCA cycle) + L-glutamate → L-aspartate]: EC 2.6.1.1, EC 4.3.1.1;

[0114] (m) NAD biosynthesis pathway I from aspartic acid [L-aspartic acid → 2-iminosuccinic acid → quinolinic acid → β-nicotinic acid D-ribonucleic acid [1-(5-O-phosphono-β-D-ribofuranosyl)-3-carboxypyridinium] → nicotinic acid adenine dinucleotide → NAD ＋ ]: EC1.4.3.16, EC2.5.1.72, EC2.4.2.19, EC2.7.7.18, and EC6.3.5.1 / EC6.3.1.5;

[0115] (n) β-alanine biosynthetic pathway III (L-aspartate → β-alanine): EC 4.1.1.11 (aspartate 1-decarboxylase, panD);

[0116] (o) Phosphopantothenic acid biosynthesis pathway I [(L-valine biosynthesis →) 3-methyl-2-oxobutanoic acid → 2-dehydropantoic acid → (R) pantoic acid → (R) pantoic acid + β-alanine → (R)-pantothenic acid → (R)-4'-phosphopantothenic acid (→ coenzyme A biosynthesis pathway I)]: EC 2.1.2.11, EC 1.1.1.169, EC 6.3.2.1, and EC 2.7.1.33.

[0117] At least a part of the metabolic pathways or biosynthetic pathways shown in each of (a) to (o) above, or at least a part of each enzyme activity identified by each EC number shown in at least one of (a) to (o) above, may be inherently possessed by a wild-type microorganism used as a host, or may be artificially imparted, enhanced, or modified by genetic engineering.

[0118] In the present invention, the specific form of the "treated bacterial cell" is not particularly limited as long as it can realize the production of the target substance in step (q). Examples of the "treated bacterial cell" include microbial cells treated with various chemicals, and microbial cells immobilized on a carrier such as acrylamide, carrageenan, or other suitable polymers. Any form that maintains active bacterial cells in terms of having the ability to produce the target substance can be used without particular limitation.

[0119] <Solution (Y)> In the method according to the present invention, a solution (Y) containing urea is added to the culture medium or reaction medium (X) during step (q). When urea is supplied to a target substance production process using a microorganism by adding solution (Y), carbon dioxide (carbonic acid gas) and ammonia are produced from urea due to the urease activity of the microorganism, as shown in the above reaction formula (I). Furthermore, dissolved carbon dioxide, carbonate ions, bicarbonate ions, dissolved ammonia, and ammonium ions may be produced in the culture medium or reaction medium (X). These components are then taken up into the microbial cells and efficiently used in metabolic reactions that use these components as substrates, thereby promoting the growth and metabolism of the microorganisms and, as a result, enabling efficient production of the target substance. Furthermore, urea has a solubility in water of 108 g / 100 mL (20°C), making it relatively soluble in solvents such as water. Therefore, even when a relatively small amount of solution (Y) is added to the culture medium or reaction medium (X), the substrate components (dissolved carbon dioxide, carbonate ions, bicarbonate ions, dissolved ammonia, and ammonium ions) can be supplied to the microbial bioprocess performed in step (q). This allows for efficient and high-yield production of the target substance. Furthermore, in step (q), excessive dilution of metabolites released from microbial cells into the culture medium or reaction medium (X) due to the addition of solution (Y) can be suppressed. As a result, the method of the present invention makes it possible to obtain a preparation (e.g., culture solution, reaction solution, etc.) containing a high concentration of the target substance. Obtaining such a preparation containing a high concentration of the target substance avoids the problem of target substance loss or spillage during the subsequent target substance extraction and purification steps, thereby enabling the efficient production of a relatively large amount of extracted and purified target substance.

[0120] The concentration (final concentration) of urea in solution (Y) is not particularly limited, and is, for example, 0.5 to 12.5 M, preferably 1 to 12 M, more preferably 1.5 to 11 M or 1.5 to 10 M, and even more preferably 1.8 to 10 M. In some embodiments, the concentration of urea in solution (Y) is 6 to 15 M, preferably 6.5 to 12 M, more preferably 7 to 10 M, for example, 7.5 to 9 M.

[0121] Furthermore, in a specific embodiment, the solution (Y) essentially consists of urea and water in the above-mentioned concentration ranges. Note that, in this application, the term "essentially consists" does not exclude the inclusion of impurities.

[0122] Furthermore, in another embodiment, the solution (Y) contains, in addition to urea, ammonium bicarbonate (NH ４ HCO ３ ), ammonium bicarbonate and ammonium carbamate (NH ２ COONH ４ ), a mixture with potassium carbonate (K ２ CO ３ ), sodium carbonate (Na ２ CO ３ ), sodium bicarbonate (NaHCO ３ ), and ammonia (NH ３ Furthermore, in some embodiments, the solution (Y) may contain at least one selected from the group consisting of ammonium bicarbonate (NH ４ HCO ３ ), potassium carbonate (K ２ CO ３ ) and ammonia (NH ３ (aq)) and water as a solvent.

[0123] Furthermore, in certain embodiments, solution (Y) may consist essentially of the following components: (i) 0.5-4 M, 1-4 M, or 1.5-3 M urea; (ii) 0.2-2 M, 0.5-1.5 M, or 0.5-1 M ammonium bicarbonate; (iii) 0.2-2 M, 0.2-1.5 M, or 0.5-1 M potassium carbonate; (iv) 1-4 N (mol / L), 2-4 N (mol / L), or 2.5-3.5 N (mol / L) ammonia; and (v) the remainder, water.

[0124] In some embodiments, during step (q), a predetermined amount of solution (Y) may be added to the culture medium or reaction medium (X) once, while in other embodiments, a predetermined amount of solution (Y) may be added to the culture medium or reaction medium (X) intermittently or multiple times at regular intervals. In still other embodiments, during step (q), solution (Y) may be continuously supplied and added to the culture medium or reaction medium (X) at a predetermined supply rate.

[0125] Furthermore, in certain embodiments, during step (q), the pH of the culture medium or reaction medium (X) may be monitored and solution (Y) may be added to the culture medium or reaction medium (X) as a pH adjuster to control the pH of the culture medium or reaction medium (X) within a predetermined value or range. In step (q), there is concern that the pH of the culture medium or reaction medium (X) may deviate from the optimal range for the growth or metabolic reaction of the microorganism as the microorganism grows or produces the target substance. Therefore, by employing an embodiment in which solution (Y) is used not only as a source of substrate components but also as a pH adjuster, deviation of the pH from the optimal range can be avoided, enabling more efficient production of the target substance, and therefore such an embodiment is advantageously employed in the present invention. Furthermore, monitoring the pH of the culture medium or reaction medium (X), adding solution (Y) to the culture medium or reaction medium (X), and controlling the pH to a predetermined value or range may be performed using a microbial culture apparatus or bioreactor capable of automatically controlling these operations and functions.

[0126] In step (q), the total amount of solution (Y) added relative to the total volume of the culture medium or reaction medium (X) at the start of the reaction may be appropriately set depending on various conditions, such as the degree of dilution of the target substance of interest, pH control of the culture medium or reaction medium (X), the amount of the substrate component to be supplied to the microbial bioprocess, etc., and is not particularly limited. In some embodiments, the total amount of solution (Y) added in step (q) may be adjusted to a ratio of 2 to 1100 (v / v)% relative to the total volume of the culture medium or reaction medium (X).

[0127] Furthermore, in another embodiment, during step (q), solution (Y) may be added to the culture medium or reaction medium (X) so as to satisfy all of the following conditions (a) to (d): (a) amount of solution (Y) added / volume of culture medium or reaction medium (X)=2 to 1100 (v / v)%; (b) amount of solution (Y) added / total amount of glucose consumed=0.08 to 0.40 mL / mmol; (c) CO ３ ２－ or HCO ３ － (d) NH ３ Amount of glucose supplied / amount of glucose consumed = 2.00 to 3.50 mmol / mmol, provided that CO ３ ２－ or HCO ３ － Supply amount and NH under condition (d) ３ The amount of urea supplied is determined so that all of the urea contained in the solution (Y) supplied to the culture medium or reaction medium (X) is converted into CO ２ and N.H. ３ The CO ２ All of these are CO ３ ２－ or HCO ３ － The theoretical value is calculated assuming that the

[0128] <Culture / Reaction Conditions> The culture temperature or reaction temperature of the microorganism is not particularly limited and may be appropriately set in consideration of various conditions such as the type and properties of the microorganism used. Generally, when psychrophilic bacteria are used as the microorganism, the suitable temperature range can be set to about 8°C to about 23°C, preferably about 10°C to about 20°C, and more preferably about 12°C to about 18°C; when mesophilic bacteria are used, the suitable temperature range can be set to about 20°C to about 48°C, preferably about 25°C to about 47°C, and more preferably about 28°C to about 40°C; and when thermophilic bacteria (thermophiles) are used, the suitable temperature range can be set to about 40°C to 80°C, preferably about 50°C to about 70°C, and more preferably about 55°C to 65°C.

[0129] The culture time or reaction time of the microorganism is not particularly limited and may be adjusted as needed to achieve the desired microbial growth and target substance production amount. Generally, the culture time is, for example, about 1 hour to about 7 days, and from the viewpoint of more efficient target substance production, it is preferably about 1 hour to about 3 days, for example, about 1 hour to 48 hours.

[0130] Furthermore, the cultivation or reaction of microorganisms may be carried out by any of batch, fed-batch, and continuous methods, with the batch method being preferred.

[0131] In a specific embodiment, in step (q), microbial cells or a treated product thereof are cultured or reacted in a culture medium or reaction medium (X) to produce a target substance, and then the microbial cells or a treated product thereof are recovered from the culture medium or reaction medium (X) by an appropriate procedure such as centrifugation, and the recovered microbial cells or a treated product thereof may be reused to repeat step (q) multiple times. Such an embodiment in which step (q) is repeated multiple times by reusing the microbial cells leads to cost reduction in the production and treatment steps and enables efficient production and treatment processes of the target substance, and is therefore an embodiment that can be preferably employed in the present invention.

[0132] Furthermore, in a preferred embodiment, step (q) may be carried out in a state in which the microbial cells are suspended at a high density in the culture medium or reaction medium (X). When promoting metabolism or production of substances without growth using Coryneform bacteria, Escherichia bacteria, or the like, by culturing or reacting the microbial cells in such a high-density state, the microbial cells act as if they were a chemical catalyst, allowing the desired metabolism or production of the target substance to proceed efficiently.

[0133] Examples of "a state in which microbial cells are suspended at a high density" include a state in which microbial cells are suspended in a culture medium or reaction medium (X) so that the mass volume percent concentration of wet microbial cells is within the range of about 1.0 to about 50.0 w / v%, preferably about 3.0 to about 30.0 w / v%. Furthermore, in some embodiments, step (q) may be carried out in a state in which the microbial cells are suspended in a culture medium or reaction medium (X) so that the mass volume percent concentration of wet microbial cells is within the range of about 1.0 to about 20.0 w / v%, about 2.0 to about 15.0 w / v%, about 3.0 to about 14.0 w / v%, about 4.0 to about 13.0 w / v%, 4.5 to about 12.0 w / v%, or 4.5 to about 12.5 w / v%. Furthermore, in another embodiment, the mass volume percentage concentration of the above-mentioned microbial wet cells may be a numerical range that combines any of the lower and upper limit values ​​of each of the above-mentioned numerical ranges in a manner that does not cause any contradictions.

[0134] Furthermore, the pH of the culture medium or reaction medium (X) is not particularly limited as long as it is within a range in which the reaction for producing the desired target substance proceeds, and it is sufficient to adopt an optimal range for the growth, metabolism, etc. of the microorganism. For example, when using a microorganism that generally grows in a neutral range, such as a coryneform bacterium, the pH of the culture medium or reaction medium (X) is preferably about 6.0 to 8.0, more preferably 6.5 to 8.0, for example, around 7.5, and may be adjusted in advance and controlled within this range or value during step (q).

[0135] <Target Substances> In the method of the present invention, various target substances can be produced in high yields using microbial organisms or processed products thereof. More specifically, metabolites produced via metabolic pathways or biosynthetic pathways possessed by microbial organisms can be obtained as target substances. However, unless otherwise specified, not only metabolites in the metabolic pathways or biosynthetic pathways possessed by microbial organisms, but also substances produced by secondary chemical reactions or chemical modifications of the metabolites within the microbial organisms or in the culture or reaction system can be included in the target substances of the present invention.

[0136] The types of target substances vary depending on the type of microorganism used, but specific examples include nucleic acid-related compounds (e.g., adenine, guanine, cytosine, thymine, uracil, 5'-guanylic acid, adenosine, ATP, CDP-choline); various physiologically active substances such as hormone-like substances; carbohydrates and sugars; vitamin-related substances and coenzymes (e.g., vitamin C, vitamin B2, B12, sorbose, NAD, FAD, coenzyme A); proteins, peptides, amino acids; L-3,4-dihydro Examples of the organic compounds include amino acid derivatives such as hydroxyphenylalanine (L-DOAP), 5-hydroxytryptophan, and pyrrolidonecarboxylic acid; alcohols such as ethanol, butanol, and isopropanol; phenol, catechol, 4-hydroxybenzoic acid, 4-aminobenzoic acid, anthranilic acid, gallic acid, succinic acid, fumaric acid, malic acid, shikimic acid, 3-dehydroshikimic acid, 3-dehydroquinic acid, protocatechuic acid, and chorismic acid; and salts thereof.

[0137] In some embodiments, the target substance is at least one selected from the group consisting of amino acids, aromatic compounds, organic acids, hydrocarbons, salts thereof, alkanols, and alcohols.

[0138] Furthermore, in certain embodiments, the target substance is preferably an amino acid or a derivative thereof, or a salt thereof. Specifically, the amino acid includes valine, leucine, isoleucine, glutamine, aspartic acid, glutamic acid, arginine, alanine, proline, cysteine, lysine (lysine), threonine, asparagine, phenylalanine, serine, methionine, glycine, tyrosine, histidine, tryptophan, cystine, and theanine. The amino acid may be in any of the L-, D-, and DL-forms (racemic).

[0139] Furthermore, the amino acid derivatives are specifically metabolic products derived from L-amino acids in the metabolic system of genetically modified microorganisms.

[0140] Furthermore, in certain embodiments, the target substance is L-aspartic acid or a downstream metabolite derived therefrom, including amino acids and amino acid derivatives such as L-threonine, L-lysine, L-asparagine, L-homoserine, 3-hydroxypropionic acid, nicotine adenine dinucleotide (NAD), L-homocysteine, L-methionine, β-alanine, and pantothenic acid.

[0141] In another embodiment, the target substance is at least one selected from the group consisting of citric acid, cis-aconitic acid, D-isocitric acid, α-ketoglutaric acid, succinyl CoA, succinic acid, a further downstream metabolite derived from any one of these metabolites, and a salt thereof.

[0142] In yet another embodiment, the target substance is at least one selected from the group consisting of oxaloacetic acid, L-malic acid, fumaric acid, a metabolite derived from any one of these metabolites, and a salt thereof. These metabolites can be efficiently produced by using bacteria (e.g., coryneform bacteria, Escherichia bacteria, etc.) that drive the reductive TCA cycle or an incomplete reductive TCA pathway when cultured or reacted under reducing conditions that substantially prevent growth.

[0143] In yet another embodiment, the target substance is at least one selected from the group consisting of oxaloacetate, fumarate, malate, succinate, downstream metabolites via the biosynthetic pathway of at least one of these compounds, and salts thereof. In a preferred embodiment, the target substance is at least one selected from the group consisting of aspartate, metabolites derived from aspartate, and salts thereof. In another preferred embodiment, the target substance is at least one selected from the group consisting of aspartate, beta-alanine, asparagine, and salts thereof.

[0144] In some embodiments, aspartate as a target substance or intermediate metabolite can be biosynthesized from fumarate by an enzyme activity specified by EC 4.3.1.1 (AspA, for example, an enzyme encoded by the aspA gene in bacteria such as Corynebacterium glutamicum or Escherichia coli). In some embodiments, aspartate as a target substance or intermediate metabolite can be biosynthesized from oxaloacetate by an enzyme activity specified by EC 1.4.1.21 (AspDH, for example, an enzyme encoded by the nadX gene in bacteria such as Corynebacterium glutamicum). In some embodiments, aspartate as a target substance or intermediate metabolite can be biosynthesized from oxaloacetate by an enzyme activity specified by EC 2.6.1.1 (AspAT, for example, an enzyme encoded by the aspC or aspB gene in bacteria such as Escherichia coli). In certain embodiments, aspartate as a target substance or intermediate metabolite can be biosynthesized by an enzymatic activity specified by at least one of EC 4.3.1.1 and EC 1.4.1.21.

[0145] Additionally, in some embodiments, beta-alanine as a target substance or intermediate metabolite can be biosynthesized from aspartate by the enzyme activity specified by EC 4.1.1.11 (aspartate 1-decarboxylase, e.g., the enzyme encoded by the gene panD in bacteria such as Corynebacterium glutamicum or Escherichia coli).

[0146] Furthermore, in some embodiments, asparagine as a target substance or intermediate metabolite can be biosynthesized from aspartic acid by an enzyme activity specified by EC 6.3.5.4 (asparagine synthase, e.g., the enzyme encoded by the gene asnB in bacteria such as Corynebacterium glutamicum or Escherichia coli).

[0147] In yet another embodiment, the target substance is at least one selected from the group consisting of metabolites in the TCA cycle, the reductive TCA cycle, or the incomplete reductive TCA pathway (e.g., oxaloacetate, fumarate, malate, succinate, succinyl-CoA, α-ketoglutarate, D-isocitrate, cis-aconitate, citric acid, acetyl-CoA), downstream metabolites via the biosynthetic pathway of any one of these metabolites, and salts thereof. In yet another embodiment, the target substance is at least one selected from the group consisting of metabolites in the glycolysis pathway, the reductive pentose phosphate pathway, the acetyl-CoA pathway, the anaplerotic pathway, or the glyoxylate cycle, metabolites via the biosynthetic pathway of any one of these metabolites, and salts thereof.

[0148] In the present invention, the proteins or enzyme proteins that realize the various enzymatic activities described above may be (l) encoded by genes inherently possessed by the microorganism, or (m) encoded by genes exogenously introduced into the microorganism by genetic engineering technology, and it should be understood that various embodiments employing any combination of the specific enzymatic activities or enzymes described above in accordance with the form of (l) or (m) are explicitly described in this specification.

[0149] <Regarding Step (r)> The method according to the present invention may include "(r) recovering the target substance" after step (q).

[0150] The term "recovering the target substance" in step (r) naturally includes extraction and / or purification of the target substance by various extraction and / or purification methods after step (q), but also includes recovery of the target substance by collecting the culture or reaction product itself containing the microbial cells and the culture medium or reaction medium (X), or the microbial cells (solid fraction) containing the target substance or the supernatant (liquid fraction) of the culture or reaction product.

[0151] In embodiments in which a target substance is extracted and / or purified, an appropriate extraction / purification technique may be employed depending on desired conditions, such as purity. While not particularly limited, the target substance can be separated, purified, and recovered by using, alone or in combination, various crystallization methods, various filtration techniques such as ultrafiltration, various chromatography techniques such as ion exchange chromatography, affinity chromatography, hydrophobic chromatography, and reverse phase chromatography, concentration methods, dialysis, activated carbon adsorption, etc. Various extraction / purification techniques for these substances are known, and any of these may be utilized as appropriate.

[0152] Additionally, the method of the present invention may optionally include further steps such as washing, drying, crushing, powdering or granulating, and / or packaging the target material.

[0153] <Step (p)> Furthermore, the method according to the present invention may optionally further comprise, prior to step (q), the step of: (p) culturing and growing a microorganism, and obtaining the grown microorganism or a culture containing the same, which is not necessarily an essential element. In this case, the grown microorganism or a culture containing the same obtained in step (p) is subjected to step (q).

[0154] Step (p) is a so-called pre-culture step, and can be arbitrarily adopted for the purpose of realizing efficient target substance production in step (q) by growing microbial cells. In step (p), any operation and conditions for culturing and growing the microorganisms can be adopted as long as they allow the growth of microbial cells, and known methods can also be appropriately adopted. In addition, the operations and conditions described for step (q) above can be adopted, but when using microorganisms that do not grow under anaerobic or microaerobic conditions but grow under aerobic conditions, the culture and growth of the microorganisms in step (p) will necessarily be carried out under aerobic conditions. On the other hand, when using microorganisms that do not grow under aerobic conditions but grow under anaerobic or microaerobic conditions, the culture and growth of the microorganisms in step (p) will necessarily be carried out under anaerobic or microaerobic conditions. However, the operation and conditions for culturing and growing the microorganisms in step (p) are not limited as long as the microorganisms grow.

[0155] Although specific embodiments of the present invention have been described in detail above, it goes without saying that the present invention is not limited to the above-described embodiments. Various modifications, alterations, and combinations of each configuration, element, and feature may be employed without departing from the spirit and scope of the present invention. In the present invention, the terms "comprise," "contain," "have," "hold," and "possess" do not exclude the presence of elements other than those referred to by these terms, unless otherwise specified, and these terms are used interchangeably. Furthermore, when a microorganism, etc., is said to contain an enzyme, it specifically means that a gene (nucleic acid portion) encoding the enzyme is present in an expressible form in the genome (chromosomal DNA) or extrachromosomal nucleic acid molecule (e.g., plasmid) of the microorganism, etc., regardless of whether the microorganism, etc., is wild-type or recombinant. Furthermore, in this application, the term "containing an enzyme" can be interpreted as "containing a gene (nucleic acid) encoding the enzyme," and embodiments based on these interpretations are also explicitly described in the present specification. Furthermore, the contents of each prior art document referred to in this specification are incorporated herein by reference as part of this specification and may constitute part of embodiments of the present invention.

[0156] EXAMPLES The present invention will be explained in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

[0157] The compositions of the various basal media used in the following test examples are shown in Table 4.

[0158]

[0159] Test Examples 1 and 2 Test Example 1: Example using urea-free feed solution In this test example, Corynebacterium glutamicum ATCC13032 (GES524 strain) in which ldh, sdh, and poxB had been inactivated was transformed with a plasmid (pGE333) carrying an aspartate feedback inhibition-resistant phosphoenolpyruvate carboxylase gene to obtain strain GES524 / pGE333 (see Examples in WO2020208842A1). An aspartic acid production reaction was carried out by using the strain GES524 / pGE333 as the aspartic acid-producing microorganism and supplying a urea-free feed solution to the reaction system. The test procedure is described below.

[0160] The above-mentioned strains, which had been frozen and stored in a clean bench, were scraped and spread onto A agar medium (containing 25 μg / L of kanamycin) and cultured overnight in a 33°C incubator. Next, colonies of the strains grown on A agar medium were scraped and inoculated into 10 mL of A medium (containing 25 μg / L of kanamycin) in a test tube, and cultured for 12 hours (33°C). The entire amount of the resulting culture was inoculated into 100 mL of A medium placed in a 500 mL flask, and cultured with shaking at 33°C and 200 rpm for 12 hours.

[0161] The culture obtained by the flask culture described above was added to 3 L of molasses medium (Molasses (Hokkaido Sugar Industry): 400 mL, KH ２ P.O. ４ :3.5g, MgSO ４ ・7H ２ The mixture was added to a mixture of 2.5 g of HO, 5 mL of antifoaming agent CB-442 (NOF Corp.), and the remainder was distilled water, and pre-culture was carried out under the following aerobic culture conditions.

[0162] (Aerobic culture conditions) Agitation: 600 rpm Temperature: 35°C pH: 7.0 Aeration: 2.5 L / min. A 15N aqueous ammonia solution was used as the pH adjusting solution (feed solution).

[0163] After 17 to 18 hours from the start of cultivation, when the pH of the culture solution began to rise, aeration and stirring were stopped temporarily, and the OD of the culture solution was ６１０ When the OD ６１０= approximately 60. Medium components were removed from the resulting culture by centrifugation (Avanti J-26S, manufactured by Beckman Coulter), and the separated bacterial cell fraction was suspended in distilled water to obtain a bacterial cell suspension with a total volume of 1 L. The reaction solution obtained by mixing 90 mL of the bacterial cell suspension with 10 mL of 70% aqueous glucose solution was added to a Bio Jr. 8 (ABLE), and an aspartic acid production reaction was carried out under the following reaction conditions using the pH-adjusted solution (2) (a mixture of 75 vol% 2 M aqueous ammonium bicarbonate solution and 25 vol% 15 N aqueous ammonia solution) shown in Table 7 below.

[0164] (Reaction conditions) Agitation: 330 rpm Temperature: 35°C pH: 7.5 Aeration: 10 mL / min

[0165] After the start of the reaction, the reaction was continued until 24 hours had elapsed, when it was confirmed that the glucose concentration in the reaction solution had fallen to 5 mM or less, and the reaction was stopped at that time. For each sample in Test Examples 1 and 2, the start of the reaction was designated as time 0, and 0.5 mL of the reaction solution was sampled at time 0, 8, 20, and 24 hours (when the reaction was stopped) from the start of the reaction, and the supernatant was recovered by centrifugation, and the concentrations of glucose, various amino acids, and various organic acids contained in the supernatant were measured. The amino acid analysis system Prominence (Shimadzu) was used to measure the various amino acids, and the organic acids were measured by HPLC analysis using a TSKgel OApak column (Tosoh).

[0166] Test Example 2: Example using urea-containing feed solution An aspartic acid production reaction was carried out using the same procedures and conditions as in Test Example 1, except that a 4 M urea aqueous solution was used instead of the pH adjusting solution (2) used in the aspartic acid production reaction in Test Example 1, and the reaction was stopped 23 hours after the start of the reaction, when it was confirmed that the glucose concentration in the reaction solution had become 5 mM or less.

[0167] (Results of Test Examples 1 and 2) Table 5 shows the measured values ​​of glucose and various amino acids and organic acids in the reaction samples at 0, 8, 20, 23, and 24 hours after the start of the reaction for each of Test Examples 1 and 2. Furthermore, Figure 6 shows a graph plotting the measured values ​​of glucose concentration and aspartic acid concentration (titer) in the reaction samples. Furthermore, Table 6 and Figures 7 and 8 show the measured values ​​of aspartic acid concentration (titer) in the reaction samples at 24 hours after the start of the reaction (end of the reaction), as well as the moles of aspartic acid produced per 0.5 mol of glucose taken up until the end of the reaction (mol / 2 mol glucose).

[0168]

[0169]

[0170] As can be seen from the results shown in Table 5 and Figure 6, after the start of the reaction, production of various organic acids such as succinic acid, acetic acid, malic acid, and fumaric acid, as well as various amino acids including aspartic acid, was confirmed as glucose was consumed in both Test Examples 1 and 2. In both Test Examples 1 and 2, since the recombinant Corynebacterium glutamicum with enhanced aspartic acid production ability was used as the microorganism as described above, it was confirmed that the concentration (titer) of aspartic acid had increased considerably 23 or 24 hours after the end of the reaction.

[0171] As a pH adjusting solution, ammonium bicarbonate (NH ４ HCO ３ ) and ammonia (NH ３Compared with Test Example 1, in which a feed solution containing urea was used, Test Example 2, in which a feed solution containing urea was used as the pH-adjusting solution, showed a relatively higher Asp titer (concentration) and also a higher Asp yield per 0.5 mol of glucose (Table 6, Figures 7 and 8). Thus, this Test Example demonstrated that the use of a urea-containing feed solution (pH-adjusting solution) in a target substance production process using a microorganism not only enables efficient production of the target substance (aspartic acid), but also enables the production of a preparation having a high target substance concentration (titer) without the concentration of the target substance (aspartic acid) produced in the reaction solution being diluted by the feed solution.

[0172] As described above, urea in the urea-containing feed solution serves as a nitrogen source in the biosynthesis of aspartic acid and also provides carbonate ions (CO ) that can be utilized in certain metabolic reactions. ３ ２－ ) / bicarbonate ion (HCO ３ － It is believed that these ions are efficiently incorporated into the metabolic reaction, resulting in efficient production of aspartic acid. In addition, as shown in Table 5, in each reaction step of Test Example 1 and Test Example 2, the redox potential of the reaction solution shifted in the negative direction from the start of the reaction to the end of the reaction, ranging from -287 mV to -416 mV in Test Example 1 and from -264 mV to -360 mV in Test Example 2.

[0173] <Test Examples 3 to 6> In the following Test Examples 3 to 6, a recombinant Corynebacterium glutamicum strain in which the aspartic acid-producing ability was improved by similarly satisfying all of the above-mentioned conditions (I) to (IV) in accordance with the genetic modification of the above-mentioned GES524 / pGE333 strain was used as an aspartic acid-producing microorganism, and an aspartic acid production reaction was carried out in which a feed solution of a predetermined composition or a urea-containing feed solution was supplied to the reaction system, and the aspartic acid production efficiency was analyzed and compared.

[0174] Test Example 3: Example using ammonium bicarbonate-containing feed solution In a clean bench, the above-mentioned strain that had been frozen was scraped, spread on an Agar medium, and cultured overnight in a constant temperature bath at 33°C. Next, a colony of the strain grown on the A agar medium was scraped and inoculated into 10 mL of A medium in a test tube, which was then cultured for 12 hours (33°C). The entire amount of the obtained culture solution was inoculated into 100 mL of A medium placed in a 500 mL flask, which was then cultured with shaking at 33°C and 200 rpm for 12 hours.

[0175] Using a 5 L jar (ABLE, BMS-05NP4), the culture obtained by the above-mentioned flask culture was poured into 2 L of glucose medium, and preculture was carried out under the following aerobic culture conditions.

[0176] (Aerobic culture conditions) Agitation: 600 rpm Temperature: 35°C pH: 7.0 Aeration: 1.5 L / min. A 15N aqueous ammonia solution was used as the pH adjusting solution (feed solution).

[0177] After 13 to 14 hours from the start of cultivation, when the glucose concentration of the culture solution became zero and the pH began to rise, aeration and stirring were stopped temporarily, and the OD of the culture solution was measured. ６１０ After measuring the pH, 400 mL of 50% glucose was added to the culture solution, and aspartic acid production reactions were carried out under the following reaction conditions and using various pH-adjusting solutions (the compositions of which are shown in Table 7 below).

[0178] (Reaction conditions) Agitation: 330 rpm Temperature: 35°C pH: 7.5 Aeration: 0.1 L / min

[0179] (Composition of each pH-adjusting solution)

[0180] 19 to 22 hours after the start of the reaction, 150 mL of 50% glucose was added to the reaction solution, and the reaction was continued until the glucose concentration in the reaction solution reached 20 mM or less. Once it was confirmed that the glucose concentration in the reaction solution had reached 20 mM or less, 0.5 mL of the reaction solution was sampled and the supernatant was recovered by centrifugation, and the amounts of glucose consumed and amino acids produced were calculated and measured. Amino acids were measured using the Prominence amino acid analysis system (Shimadzu).

[0181] Test Example 4: Example using a potassium carbonate-containing feed solution The steps from cultivation to the start of the reaction (preculture) were carried out under the same conditions and procedures as in Test Example 2 to obtain a preculture of the above-mentioned strain. Then, an aspartic acid production reaction was carried out under the following conditions and procedures.

[0182] The aspartic acid production reaction was initiated under the reaction conditions shown in Test Example 2, with 250 mL of 70% glucose added to the culture medium and pH-adjusted solutions having various compositions shown in Table 8 below.

[0183] (Composition of each pH-adjusting solution)

[0184] Nine to ten hours after the start of the reaction, 100 mL of 70% glucose was added, and 22 to 24 hours after the start of the reaction, another 100 mL of 70% glucose was added. Since no decrease in the reaction rate was observed, glucose was continued to be added as needed, and the reaction was allowed to continue until 96 hours had elapsed from the start of the reaction. Then, 0.5 mL of the reaction solution was sampled, and the supernatant was recovered by centrifugation. The amounts of glucose consumed and amino acids produced were measured and calculated. Amino acids were measured using the Prominence amino acid analysis system (Shimadzu).

[0185] Test Example 5: Example using urea-containing feed solution A preculture of coryneform bacteria was prepared in the same manner as in Test Example 2, and an aspartic acid production reaction was carried out under the same reaction conditions and by the same procedures as in Test Example 2, except that an 8 M urea aqueous solution was used as the pH adjusting solution (7).

[0186] 22 hours after the start of the reaction, 200 mL of 70% glucose was added to the reaction mixture, and the reaction was continued for 56 hours. After that, 0.5 mL of the reaction mixture was sampled and centrifuged to recover the supernatant. The amounts of glucose consumed and amino acids produced were measured and calculated. Amino acids were measured using the Prominence amino acid analysis system (Shimadzu).

[0187] Test Example 6: Example using pH-adjusted solution containing urea and other components A preculture of a coryneform bacterium was prepared using the same procedure as in Test Example 2, and an aspartic acid production reaction was carried out using the same reaction conditions and procedures as in Test Example 2, except that a pH-adjusted solution (8) having the composition shown in Table 9 below was used.

[0188]

[0189] After 10, 22, and 47 hours from the start of the reaction, 100, 200, and 100 mL of 70% glucose were added to the reaction solution, respectively, and the reaction was continued until 94 hours had elapsed. After that, 0.5 mL of the reaction solution was sampled and the supernatant was recovered by centrifugation. The amounts of glucose consumed and amino acids produced were measured and calculated. Amino acids were measured using the Prominence amino acid analysis system (Shimadzu).

[0190] (Results of Test Examples 3 to 6) Table 10 shows the following information for each reaction solution sample in Test Examples 3 to 6. 1) pH adjusting solution used and its composition, 2) total amount of pH adjusting solution supplied to the reaction solution, 3) glucose concentration in the reaction solution at the end of the reaction, 4) aspartic acid concentration (titer) at the end of the reaction, 5) number of moles of aspartic acid produced per 0.5 mol of glucose incorporated into the reaction by the end of the reaction (molar yield), 6) amount of pH adjusting solution supplied (mL) per total amount of glucose consumed (mmol), 7) CO2 per total amount of glucose consumed (mmol). ３ ２－ or HCO ３ － 8) NH per total glucose consumption (mmol) ３ － Supply amount (mmol)

[0191] In addition, Figures 9, 10, 11, 12 and 13 show graphs in which the values ​​of 4) to 8) above are plotted, respectively.

[0192] The amount (mL) of pH adjusting solution supplied in 6) above is a value obtained by dividing the total amount (mL) of pH adjusting solution supplied to the reaction solution during the reaction by the total amount (mmol) of glucose consumed during the reaction. ３ ２－ or HCO ３ － The amount (mmol) of carbonate ions (CO ) supplied to the reaction solution during the reaction ３ ２－ ) or bicarbonate ion (HCO ３ － The value is calculated by dividing the amount (mmol) of glucose consumed during the reaction by the total amount (mmol) of glucose consumed during the reaction. Note that in the case of a pH adjusting solution containing urea, all of the urea is decomposed into ammonia and carbon dioxide, and all of the carbon dioxide thus produced is converted into carbonate ions (CO ３ ２－ ) or bicarbonate ion (HCO ３ － ) and CO ３ ２－ or HCO ３ － The amount (mmol) of the NH ３ － The amount (mmol) of ammonium ions (NH ４ ＋ ) (mmol) divided by the total amount (mmol) of glucose consumed during the reaction. For the pH adjusting solution containing urea, it is assumed that all urea is decomposed into ammonia and carbon dioxide, and the NH ３ － The amount of feed (mmol) was calculated.

[0193]

[0194] First, as can be seen from Table 10 and FIG. 9, the sample according to Test Example 5, which used the pH adjusting solution (7) containing 8M urea alone, had a low NH ４ HCO ３Compared with the samples according to Test Example 3, which used pH adjusting solutions (1) to (3) containing urea and ammonia, respectively, the total amount of pH adjusting solution supplied to the reaction solution was small (Table 10 and FIG. 11), but the aspartic acid titer (concentration) was higher than that of the samples using pH adjusting solutions (1) and (3), respectively, demonstrating that the configuration of Test Example 5, which used a pH adjusting solution containing urea, can achieve a good aspartic acid titer (concentration). ４ HCO ３ and a pH adjusting solution (2) containing ammonia, and K ２ CO ３ Although the titer values ​​are smaller than those of the samples using the pH adjusting solutions (4) to (6) containing ammonia, respectively, it can be said that the configuration of Test Example 5 can achieve satisfactory titers of the target substance even in the case of actual production.

[0195] Furthermore, in Table 10 and FIG. 10, when attention is paid to the molar yield of aspartic acid per 0.5 mol of glucose incorporated into the reaction, the sample according to Test Example 5, which used the pH-adjusted solution (7) containing 8 M urea alone, had a NH ４ HCO ３ When compared with the samples using pH adjusting solutions (1) to (3) containing ammonia, the values ​​were comparable. ２ CO ３ The results were higher than those of the samples using the pH-adjusting solutions (4) to (6) containing urea and ammonia, respectively. Thus, it was revealed that the configuration of Test Example 5 using a pH-adjusting solution containing urea can achieve good aspartic acid production efficiency while minimizing the total amount of pH-adjusting solution supplied to the reaction solution.

[0196] As described above, the configuration of Test Example 5, which utilizes the pH-adjusted solution (7) containing 8 M urea alone, has been shown to be able to efficiently produce a preparation containing the target substance (aspartic acid) at a high concentration by reducing the amount of feed solution supplied to the reaction solution, thereby preventing dilution of the target substance (aspartic acid) produced in the reaction solution.

[0197] Next, as can be seen from Table 10 and Figures 9, 10 and 11, 8M urea, NH ４ HCO ３ , K. ２ CO ３ The sample of Test Example 6, which utilized a pH-adjusting solution (8) containing a mixture of ammonium and ammonium hydroxide, had a relatively large amount of pH-adjusting solution supplied to the reaction solution, but it exhibited the highest values ​​for both the aspartic acid titer and the asparagine molar yield among the samples of each Test Example. This demonstrates that the configuration of Test Example 6 enables the production of a preparation containing a high concentration of a target substance with excellent aspartic acid production efficiency.

[0198] 11 to 13, the pH-adjusting solutions (7) and (8) containing urea can supply sufficient amounts of carbon dioxide (carbonate ions / bicarbonate ions) and ammonia (ammonium ions), which are potential reaction components, to the metabolic reaction system within the microbial cells, while minimizing the amount of pH-adjusting solution supplied to the reaction solution. As a result, the use of a pH-adjusting solution containing urea can avoid dilution of the target substance due to the pH-adjusting solution being carried into the reaction solution, and can provide sufficient amounts of carbonate ions / bicarbonate ions and ammonia (ammonium ions), which are potential reaction components, for a specific metabolic reaction in the biosynthetic pathway of the target substance. As demonstrated by the results of each test example, it is believed that this will enable the efficient production of highly concentrated preparations of the target substance.

[0199] The present invention has high industrial applicability in the fields of biotechnology, production of substances such as fine chemicals, and the like.

Claims

1. A method for producing a target substance, comprising: (q) culturing or reacting cells of a microorganism having urease activity or a processed product of the cells in a culture medium or reaction medium (X) under anaerobic or microaerobic conditions to produce a target substance; and during step (q), adding a solution (Y) containing urea to the culture medium or reaction medium (X).

2. The method according to claim 1, wherein the target substance is biosynthesized through a metabolic reaction that utilizes the reductive TCA cycle or an incomplete reductive TCA pathway and bicarbonate ions as a reaction substrate.

3. The method of claim 1, wherein the solution (Y) further comprises at least one selected from the group consisting of ammonium bicarbonate, potassium carbonate, and ammonia.

4. The method according to claim 1, wherein in step (q), the pH of the culture medium or reaction medium (X) is monitored and controlled at a predetermined value or within a predetermined range by adding a predetermined amount of solution (Y) to the culture medium or reaction medium (X).

5. The method according to claim 1, wherein the microorganism is a recombinant microorganism that satisfies all of the following conditions (I) to (III): condition (I) fumarate reductase activity, which catalyzes the conversion of fumarate to succinate in the reductive TCA cycle or the incomplete reductive TCA pathway, is reduced or inactivated; condition (II) lactate dehydrogenase activity is reduced or inactivated compared to the wild-type microorganism; and condition (III) the microorganism has a modified phosphoenolpyruvate carboxylase activity that is resistant to feedback inhibition by aspartate in wild-type phosphoenolpyruvate carboxylase activity, or an exogenous phosphoenolpyruvate carboxylase activity that is more resistant to feedback inhibition by aspartate than the wild-type phosphoenolpyruvate carboxylase activity exhibited by the wild-type microorganism.

6. The method according to claim 1, wherein the microorganism is a coryneform bacterium or an Escherichia bacterium.

7. The method of claim 1, wherein the microorganism is Corynebacterium glutamicum or Escherichia coli.

8. The method according to claim 1, wherein the target substance is at least one selected from the group consisting of amino acids, organic acids, and salts thereof.

9. The method according to claim 1, wherein the compound is at least one selected from the group consisting of downstream metabolites biosynthesized via oxaloacetate or fumarate as intermediate metabolites and salts thereof.

10. The method according to claim 1, wherein the target substance is at least one selected from the group consisting of aspartic acid, downstream metabolites biosynthesized via aspartic acid as an intermediate metabolite, and salts thereof.

11. The method of claim 1, wherein the target substance is at least one selected from aspartic acid, beta-alanine, and asparagine, and salts thereof.

12. The method according to claim 1, wherein in step (q), the oxidation-reduction potential of the culture medium or reaction medium (X) is controlled in the range of -200 mV to -550 mV.

13. The method according to claim 1, wherein solution (Y) contains 1 to 12.5 M urea, and in step (q), solution (Y) is added in a proportion of 2 to 1100 (v / v)% relative to the total volume of the culture medium or reaction medium (X).

14. The method according to claim 1, wherein solution (Y) contains 1 to 10 M urea, and solution (Y) is added to culture medium or reaction medium (X) during step (q) so as to satisfy all of the following conditions (a) to (d): (a) amount of solution (Y) added / volume of culture medium or reaction medium (X) = 2 to 1100 (v / v)%; (b) amount of solution (Y) added / total amount of glucose consumed = 0.08 to 0.40 mL / mmol; (c) CO ３ ２－ or HCO ３ － (d) NH ３ Amount of glucose supplied / amount of glucose consumed=2.00 to 3.50 mmol / mmol, provided that CO ３ ２－ or HCO ３ － Supply amount and NH under condition (d) ３ The amount of urea supplied is determined so that all of the urea contained in the solution (Y) supplied to the culture medium or reaction medium (X) is converted into CO ２ and N.H. ３ The CO ２ All of these are CO ３ ２－ or HCO ３ － The theoretical value is calculated assuming that the 15. The method according to claim 1, wherein the target substance is at least one selected from the group consisting of aspartic acid, downstream metabolites biosynthesized via aspartic acid as an intermediate metabolite, and salts thereof, the solution (Y) further contains at least one selected from the group consisting of ammonium bicarbonate, potassium carbonate, and ammonia, and the microorganism is a coryneform bacterium or an Escherichia bacterium.

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