O-acetylhomoserine production process using microbial fermentation

Abstract

The present application relates to a method for producing O-acetylhomoserine, which comprises a main culture step of culturing an O-acetylhomoserine-producing strain in a medium to produce O-acetylhomoserine, and the medium is provided with a potassium salt. According to the present application, in the method for culturing to produce O-acetylhomoserine, potassium ions are further supplied to the medium in the form of a potassium salt, thereby improving O-acetylhomoserine productivity (g / L / hr).

Description

[Technical Field]

[0001] The present application relates to process technology for producing O-acetylhomoserine using microbial fermentation. [Background technology]

[0002] In the prior art, when an O-acetylhomoserine product is produced using a Corynebacterium strain with improved O-acetylhomoserine production ability, there is a problem that even when an improved strain is used, the production volume is limited due to the use of a fermenter with limited resources to produce a fermentation broth containing O-acetylhomoserine. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] US Patent Application Publication No. 2017 / 0101655 Summary of the Invention [Problem to be solved by the invention]

[0004] One object of the present application is to improve productivity by adding a potassium salt during the process of producing O-acetylhomoserine using microbial fermentation. [Means for solving the problem]

[0005] This will be explained in more detail as follows: Meanwhile, each description and embodiment disclosed in this application also applies to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions described below are not considered to limit the category of this application.

[0006] The present application relates to a process for producing O-acetylhomoserine using microbial fermentation, and is a technology for improving O-acetylhomoserine productivity by adding potassium hydroxide to a culture medium during the fermentation process for producing O-acetylhomoserine.

[0007] FIG. 1 is a flow chart showing a method for producing O-acetylhomoserine according to the present application.

[0008] Referring to Figure 1, the method for producing O-acetylhomoserine according to the present invention includes a main culture step in which an O-acetylhomoserine-producing strain is cultured in a medium to produce O-acetylhomoserine, and the medium is provided with a potassium salt. Each step will be described in detail below.

[0009] The main culturing step is a step of preparing a fermentation broth containing O-acetylhomoserine using an O-acetylhomoserine-producing strain.

[0010] In this application, the term "fermented product" refers to the result of enzymatic or metabolic decomposition of organic substances using microorganisms. For example, the fermented product may include a culture obtained by culturing a microorganism in a culture medium, or a concentrate, dried product, or lyophilized product of the culture obtained by removing the strain from the culture. In this case, the fermented liquid may include the entire fermented product containing amino acids, or may be a fermented product containing amino acids from which impurities have been removed.

[0011] The "O-acetylhomoserine-producing strain" used in the main culture step includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and may be microorganisms in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains genetic modifications for the production of a target protein or amino acid.

[0012] The O-acetylhomoserine-producing strain of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce O-acetylhomoserine, or a microorganism in which the ability to produce O-acetylhomoserine has been imparted to a parent strain that does not have the ability to produce O-acetylhomoserine. Specifically, the microorganism that produces O-acetylhomoserine or a target product in the present application, or the microorganism that has the ability to produce O-acetylhomoserine or a target product, may be a microorganism in which some of the genes in the biosynthetic pathway of the target protein or target product have been enhanced or weakened, or some of the genes in the degradation pathway of the target protein or target product have been enhanced or weakened. "Enhanced" or "increased" O-acetylhomoserine-producing ability of the microorganism of the present application means that the O-acetylhomoserine-producing ability of the microorganism of the present application is improved compared to other microorganisms other than the microorganism of the present application, the parent strain, or an unmodified microorganism. For example, the O-acetylhomoserine-producing ability of the microorganism of the present application may be improved by about 1% or more, 10% or more, 100% or more, 200% or more, 500% or more, 1000% or more, 1100% or more, 1200% or more, or 1300% or more compared to that of another microorganism, or may be improved by about 1.01-fold or more, 2-fold or more, 5-fold or more, 10-fold or more, 11-fold or more, 12-fold or more, or 13-fold or more, but is not limited thereto. The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range that is equal to or similar to the numerical value following the term "about," but is not limited thereto.

[0013] The microorganism used in the main culture step may be at least one selected from the group consisting of yeast Candida famata, ascomycetes Eremothecium ashbyii and Ashbyagossypii, bacteria Bacillus subtilis, and Corynebacterium sp.

[0014] When the microorganism used in the main culture step is a Corynebacterium microorganism, the microorganism may be, for example, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, or the like. The bacterial strain may be, but is not limited to, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium crenatum, or Corynebacterium flavescens, and more specifically, Corynebacterium glutamicum.

[0015] Microorganisms of the genus Corynebacterium, particularly Corynebacterium glutamicum, are Gram-positive microorganisms that are widely used for the production of L-amino acids and other useful substances. To produce these L-amino acids and other useful substances, various studies have been conducted to develop highly efficient production microorganisms and fermentation process technologies. For example, target-specific approaches such as increasing the expression of genes encoding enzymes involved in the biosynthesis of L-tryptophan, L-valine, L-isoleucine, L-leucine, L-histidine, O-acetylhomoserine, or L-threonine, or deleting genes unnecessary for biosynthesis, have been primarily used. In the present application, a fermentation broth containing amino acids can be prepared using a strain of the genus Corynebacterium.

[0016] According to one embodiment of the present application, the O-acetylhomoserine-producing strain may be an O-acetylhomoserine-producing Escherichia microorganism in which endogenous citrate synthase protein activity has been attenuated or inactivated, thereby improving the ability to produce O-acetylhomoserine compared to the microorganism before the citrate synthase protein activity was attenuated or inactivated. In particular, KCCM11146P-A167, a representative citrate synthase activity-attenuated strain that has been confirmed to exhibit excellent O-acetylhomoserine production ability (deposited with the Korea Center for Microorganisms (KCCM), an international depository institution under the Budapest Treaty, on November 22, 2013, and assigned accession number KCCM11483P), can be used as the O-acetylhomoserine-producing strain of the present application. When the above-described microorganism capable of producing O-acetylhomoserine is used, O-acetylhomoserine can be produced in a more environmentally friendly manner and at a higher yield than chemical synthesis. Furthermore, the produced O-acetylhomoserine can be used as a precursor for the synthesis of methionine and acetic acid by O-acetylhomoserine sulfhydrylase, thereby enabling highly efficient bioconversion to L-methionine. The converted L-methionine can be widely used not only in animal feed or animal feed additives but also in the production of human foods or food additives.

[0017] In the main culturing step, the O-acetylhomoserine-producing strain is cultured under appropriate culture conditions known in the art. Such a culture process can be easily adjusted by those skilled in the art depending on the strain selected. Specifically, the culture may be performed in a batch, continuous, or fed-batch manner, but is not limited thereto.

[0018] As shown in FIG. 1, potassium salt is provided in the main culture stage. The potassium salt added in the main culture stage reduces the amount of potassium (K +) concentration. In some cases, when potassium concentration in the medium is adjusted by adding other forms of potassium salts other than potassium phosphate, it is possible to adjust the potassium concentration alone without affecting the phosphorus (P) concentration. Potassium is known to be essential for enzyme activity related to protein synthesis during microbial growth. Adjusting the potassium concentration through the addition of KOH can improve the efficiency of O-acetylhomoserine production by O-acetylhomoserine-producing strains. Furthermore, because potassium is involved in the membrane potential of microorganisms, maintaining the potassium concentration within a certain range makes it possible to maintain the potential difference. Maintaining the potential difference also affects the osmotic pressure of the microorganism, ultimately affecting the efficiency of O-acetylhomoserine production.

[0019] The potassium salt used in the main culture step may be at least one selected from the group consisting of potassium hydroxide (KOH), potassium acetate (KAc), potassium chloride (KCl), potassium sulfate (KSO), and potassium carbonate (KCO).

[0020] Potassium salt can be added to the medium so that the potassium ion concentration is 0.3 g / L to 1.5 g / L. The concentration of potassium salt to be added can be determined taking into account the change in O-acetylhomoserine production efficiency due to the potassium ion concentration. It has been confirmed that when the potassium ion concentration in the medium is 0.3 g / L to 1.5 g / L, the addition of potassium is effective in increasing enzyme activity and maintaining the membrane potential difference, and the O-acetylhomoserine production efficiency is most excellent due to the increase in potassium concentration. The potassium ion concentration may be 0.3 g / L to 1.0 g / L, 0.3 g / L to 0.5 g / L, 0.5 g / L to 1.5 g / L, 0.5 g / L to 1.0 g / L, or 1.0 g / L to 1.5 g / L, depending on the situation.

[0021] When KOH is used as the potassium salt to achieve the aforementioned potassium salt concentration, KOH can be added to the medium to achieve a concentration of 0.4 g / L to 2.2 g / L. In some cases, KOH can be added to the medium to achieve a concentration of 0.4 g / L to 1.5 g / L, 0.4 g / L to 1.0 g / L, 1.0 g / L to 2.2 g / L, 1.0 g / L to 1.5 g / L, or 1.5 g / L to 2.2 g / L.

[0022] When potassium acetate (KAc) is used as the potassium salt, potassium acetate can be added to the medium to a concentration of 2.6 g / L to 3.9 g / L. In some cases, potassium acetate can be added to the medium to a concentration of 2.6 g / L to 3.5 g / L, 2.6 g / L to 3.0 g / L, 3.0 g / L to 3.9 g / L, 3.0 g / L to 3.5 g / L, or 3.5 g / L to 3.9 g / L.

[0023] When potassium chloride (KCl) is used as the potassium salt, potassium chloride can be added to the medium to a concentration of 2.0 g / L to 3.0 g / L. In some cases, potassium chloride can be added to the medium to a concentration of 2.0 g / L to 2.5 g / L or 2.5 g / L to 3.0 g / L.

[0024] When potassium sulfate (K2SO4) is used as the potassium salt, potassium sulfate can be added to the medium to a concentration of 4.6 g / L to 6.9 g / L. In some cases, potassium sulfate can be added so that the concentration in the medium is 4.6g / L to 6.5g / L, 4.6g / L to 6.0g / L, 4.6g / L to 5.5g / L, 4.6g / L to 5.0g / L, 5.0g / L to 6.9g / L, 5.0g / L to 6.5g / L, 5.0g / L to 6.0g / L, 5.0g / L to 5.5g / L, 5.5g / L to 6.9g / L, 5.5g / L to 6.5g / L, 5.5g / L to 6.0g / L, 6.0g / L to 6.9g / L, 6.0g / L to 6.5g / L, or 6.5g / L to 6.9g / L.

[0025] When potassium carbonate (K2CO3) is used as the potassium salt, potassium carbonate can be added to the medium to a concentration of 3.7 g / L to 5.5 g / L. In some cases, potassium carbonate can be added to the medium to a concentration of 3.7 g / L to 5.0 g / L, 3.7 g / L to 4.5 g / L, 3.7 g / L to 4.0 g / L, 4.0 g / L to 5.5 g / L, 4.0 g / L to 5.0 g / L, 4.0 g / L to 4.5 g / L, 4.5 g / L to 5.5 g / L, 4.5 g / L to 5.0 g / L, or 5.0 g / L to 5.5 g / L.

[0026] The potassium salt may be added before the initiation of the main culture or during the main culture, i.e., the potassium salt may be added to the medium in advance before the initiation of the main culture, or may be added to the medium at an appropriate time during the main culture.

[0027] During the main culture stage, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. At this time, oxygen or an oxygen-containing gas, such as air, can be continuously injected into the culture to maintain an aerobic state. The temperature of the culture can be maintained between 20°C and 45°C. The main culture can be continued until the desired maximum production of O-acetylhomoserine is achieved. For this purpose, the main culture can be carried out for 10 to 70 hours. O-acetylhomoserine may be excreted in the culture medium or contained within the cells.

[0028] After the main culturing step is completed, a step of separating O-acetylhomoserine from the fermentation broth and producing the separated O-acetylhomoserine as a product may be further carried out.

[0029] For example, the fermentation broth prepared after the main culture step can be concentrated to separate the O-acetylhomoserine wet crystals produced during the concentration process. In this process, water in the fermentation broth is removed to precipitate O-acetylhomoserine in crystalline form. Concentration can be carried out in a variety of ways. Concentration can be carried out in a conventional concentrator (e.g., a paddle dryer, a slurry drying facility, a vacuum concentrator, a forced circulation concentrator, a thin film concentrator, or a rotary concentrator) as appropriate for those skilled in the art.

[0030] In addition, to separate the O-acetylhomoserine wet crystals precipitated by concentration from the mother liquor, a solid-liquid separator such as a vacuum membrane filter, a pressure membrane filter, or a centrifugal separator may be used, but is not limited thereto.

[0031] The mother liquor remaining after the O-acetylhomoserine wet crystallization can be further used for concentration. This allows for recycling of O-acetylhomoserine that does not form wet crystals or O-acetylhomoserine that precipitates as crystals below a certain size and remains in the mother liquor without being separated in the O-acetylhomoserine wet crystallization step. Furthermore, after recycling the mother liquor, a further step such as heating the fermentation liquor may be carried out, if necessary, to further dissolve O-acetylhomoserine precipitated in the form of fine crystals in the fermentation liquor.

[0032] Next, a step of preparing O-acetylhomoserine mixed granules may be carried out by mixing the separated O-acetylhomoserine wet crystals with seeds. The seeds used in this step are also called seed crystals or seed crystals and refer to substances used as catalysts for the crystallization or granulation of liquids. Specifically, the seeds in the present application may be, but are not limited to, amino acid crystals, for example, crystals of O-acetylhomoserine of the same species as the O-acetylhomoserine contained in the fermentation concentrate to be granulated. When the seeds come into contact with the fermentation broth, solid components present in the fermentation broth bind to the seeds, causing aggregation and forming granules. The seeds used in this step may have an average particle size of 150 to 300 μm. Specifically, seeds having an average particle size of 150 to 250 μm, 200 to 300 μm, or 200 to 250 μm may be used, but are not limited thereto. The particle size of the seeds used ultimately affects the productivity of the granule production according to the present application, and can be appropriately selected by those skilled in the art taking into account the desired moisture content, etc.

[0033] In this step, the O-acetylhomoserine mixed granules can be produced using a mixer-type granulator. The mixer-type granulator may be one in which seeds are fed into the mixer-type granulator at a constant rate through a feeder while the previously obtained amino acid wet crystals are simultaneously fed to obtain granules. Here, "granules" refer to macroscopic particles, which are larger permanent aggregates formed by gathering small particles such as powder, and may have an average particle size of 50 μm to 5 mm, 75 μm to 4 mm, or 100 μm to 3 mm.

[0034] Next, the O-acetylhomoserine mixed granules can be dried to obtain an amino acid product. In the drying step, the previously obtained O-acetylhomoserine mixed granules are dried, but the drying method is not limited. After drying, the O-acetylhomoserine product can be obtained. In this application, the term "O-acetylhomoserine product" refers to the amino acid substance contained in the fermentation broth being commercialized in various dosage forms. For example, the O-acetylhomoserine product refers to an amino acid-containing mixture in the form of granules. However, if necessary, the dosage form of the O-acetylhomoserine product can be varied as long as it does not deviate from the concept of the present invention. Furthermore, as described above, subsequent processes can be further performed to realize various dosage forms of the O-acetylhomoserine product. The O-acetylhomoserine product described above can be used as an additive for animal feed, etc., and its uses are not limited.

[0035] Meanwhile, prior to the main culture step, a flask culture step of the O-acetylhomoserine-producing strain and a seed culture step may be performed. The flask culture and seed culture are processes for sufficiently growing microorganisms in a seed culture medium to increase the number of microorganisms required for fermentation, and may be performed by a method commonly used in the art. By performing the above steps, an appropriate number of O-acetylhomoserine-producing strains can be immediately introduced into the main culture, thereby omitting the growth step for increasing the number of O-acetylhomoserine-producing strains. This improves the overall efficiency of the O-acetylhomoserine production process.

[0036] According to one embodiment of the present application, a medium for preparing O-acetylhomoserine through microbial fermentation is provided.

[0037] The medium may be a composition that provides an environment suitable for the metabolism of the O-acetylhomoserine-producing strain for the production of O-acetylhomoserine. The term "medium" refers to a mixture of nutrients, primarily nutrients required for culturing the microorganism, and provides nutrients and growth factors, including water, which are essential for survival and growth.

[0038] The medium of the present application comprises a carbon source, a nitrogen source, a phosphorus source and a potassium salt.

[0039] Carbon sources may include carbohydrates such as glucose, fructose, sucrose, maltose, mannitol, and sorbitol; sugar alcohols, alcohols such as glycerol, pyruvic acid, lactic acid, and citric acid; organic acids, and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) may also be used. A variety of other carbon sources may be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0040] Usable nitrogen sources include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, and defatted soybean cake or its decomposition products. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0041] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner, but are not limited thereto.

[0042] As described above, the potassium salt may be present in the medium at different concentrations depending on the type of potassium salt, and the technical significance of this has been described above.

[0043] Also included may be metal salts such as magnesium sulfate or iron sulfate, and essential growth substances such as vitamins, which are necessary for the growth of the microorganisms.

[0044] The raw materials may be added to the culture in a suitable manner during the cultivation process, either batchwise or continuously. Therefore, in this application, the medium refers not only to the composition added at the start of the main cultivation but also to the entire material added during the cultivation of the O-acetylhomoserine-producing strain. [Effects of the Invention]

[0045] According to the present application, in the method for culturing to produce O-acetylhomoserine, potassium ions in the form of potassium salts are further supplied to the medium, thereby improving O-acetylhomoserine productivity (g / L / hr). [Brief explanation of the drawings]

[0046] [Figure 1] 1 is a flowchart showing a method for producing O-acetylhomoserine according to the present application. DETAILED DESCRIPTION OF THE INVENTION

[0047] The present application will be described in more detail through experimental examples below. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily performed by those of ordinary skill in the technical field of the present application or a similar technical field.

[0048] Experimental Example The method for producing O-acetylhomoserine according to one embodiment of the present application and the culture medium used therein have been described in detail above. Below, the advantageous effects mentioned in the present application will be described in detail through experimental results of examples and comparative examples.

[0049] Experimental Example 1: Comparison of O-acetylhomoserine productivity (g / L / hr) depending on the concentration of potassium hydroxide (KOH) added during main cultivation According to one embodiment of the present invention, the culture was centrifuged at low speed to remove the biomass, and the resulting supernatant was separated and analyzed by ion exchange chromatography.

[0050] [Table 1]

[0051] In Experimental Example 1, the main culture was performed with different KOH concentrations, but all other parameters, such as culture temperature, pH, and total equivalent amount added, were the same. At a KOH concentration of 0.4 g / L (potassium ion concentration of 0.3 g / L), productivity increased by 4.8% compared to when no KOH was added (potassium ion concentration of 0.0 g / L). At a KOH concentration of 0.7 g / L (potassium ion concentration of 0.5 g / L), productivity increased by 6.4% compared to when no KOH was added. At a KOH concentration of 1.1 g / L (potassium ion concentration of 0.8 g / L), productivity increased by 9.3% compared to when no KOH was added. At a KOH concentration of 1.5 g / L (potassium ion concentration of 1.0 g / L), productivity increased by 8.3% compared to when no KOH was added. It was also confirmed that productivity increased when the potassium ion concentration was 1.5 g / L compared to when no KOH was added. However, it was confirmed that productivity decreased when the potassium ion concentration was 1.8 g / L. Therefore, it was confirmed that O-acetylhomoserine productivity was high when the potassium ion concentration was 0.3 g / L to 1.5 g / L.

[0052] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. a main culture step in which the O-acetylhomoserine-producing strain is cultured on a medium to produce O-acetylhomoserine; The method for producing O-acetylhomoserine, wherein the medium is provided with a potassium salt.

2. The potassium salts include potassium hydroxide (KOH), potassium acetate (KAc), potassium chloride (KCl), potassium sulfate (K ２ SO ４ ) and potassium carbonate (K ２ CO ３ 2. The method for producing O-acetylhomoserine according to claim 1, wherein the O-acetylhomoserine is at least one selected from the group consisting of:

3. 3. The method for producing O-acetylhomoserine according to claim 2, wherein the potassium salt is added to the medium so that the potassium ion concentration is 0.3 g / L to 1.5 g / L.

4. 2. The method for producing O-acetylhomoserine according to claim 1, wherein the step of flask culturing the O-acetylhomoserine-producing strain and then seed culturing the strain are carried out before the main culturing step.

5. 2. The method for producing O-acetylhomoserine according to claim 1, further comprising the step of concentrating the fermentation broth containing O-acetylhomoserine and separating the O-acetylhomoserine wet crystals after the main culturing step.

6. comprising a carbon source, a nitrogen source, a phosphorus source, and a potassium salt; A medium capable of producing O-acetylhomoserine by culturing an O-acetylhomoserine-producing strain.

7. The potassium salts include potassium hydroxide (KOH), potassium acetate (KAc), potassium chloride (KCl), potassium sulfate (K ２ SO ４ ) and potassium carbonate (K ２ CO ３ 7. The medium according to claim 6, wherein the medium is at least one selected from the group consisting of:

8. 8. The medium according to claim 7, wherein the potassium salt is added to the medium so that the potassium ion concentration is 0.3 g / L to 1.5 g / L.

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