Medium composition for herbicidin production comprising amino acid

Abstract

The present invention relates to a medium composition for herbicidin production comprising one or more amino acids among isoleucine, glutamine, proline, and leucine; a composition for herbicidin production; a method for producing herbicidin, the method comprising a step of culturing a Streptomyces scopuliridis strain in a medium containing one or more amino acids among isoleucine, glutamine, proline, and leucine; a method for increasing herbicidin production; or a use of the composition for herbicidin production.

Description

Medium composition for producing herbicidin containing amino acids

[0001] The present invention relates to a medium composition for producing herbicidin, comprising at least one amino acid selected from the group consisting of isoleucine, glutamine, proline, and leucine; a composition for producing herbicidin; a method for producing herbicidin, comprising the step of culturing a Streptomyces scopuliridis strain in a medium comprising at least one amino acid selected from the group consisting of isoleucine, glutamine, proline, and leucine; or a method for increasing herbicidin production.

[0002]

[0003] As global population growth and food demand increase, increasing crop productivity within limited arable land is a critical challenge for ensuring a stable food supply. Against this backdrop, weed infestation in agricultural fields can lead to reduced crop yields and quality due to competition with crops for water and nutrients. While yield reductions due to weeds vary depending on the crop, they have been reported to range from 30% to 100%. Consequently, weed control plays a significant role in increasing crop productivity. In particular, the rapid decline in the rural population, coupled with labor shortages and an aging population, has made the use of herbicides essential for reducing agricultural production costs.

[0004] However, due to the severe environmental pollution caused by long-term use of synthetic organic herbicides, the need for eco-friendly herbicides has recently emerged. Accordingly, active research is being conducted on eco-friendly herbicides and their manufacturing methods (KR 10-2011-0006507 A). Among these, herbicidal ingredients produced by microorganisms are attracting attention as key ingredients in the development of new herbicides due to their environmental friendliness and superior herbicidal activity.

[0005] Meanwhile, herbicidin, a metabolite produced by strains of the genus Streptomyces, is known to possess various biological activities, including herbicidal activity. While herbicidin exhibits valuable biological activities, including excellent herbicidal activity, the technology for biosynthesizing herbicidin at high yields remains insufficient. Therefore, there is a growing need for high-efficiency, mass-production methods through microbial fermentation to manufacture eco-friendly herbicides containing herbicidin.

[0006]

[0007] The problem to be solved by the present invention is to provide a medium composition for producing herbicidin, comprising one or more amino acids selected from isoleucine, glutamine, proline, and leucine; a method for producing herbicidin, comprising a step of culturing a Streptomyces scopuliridis strain in a medium selected from isoleucine, glutamine, proline, and leucine; a method for increasing herbicidin production; and a use of the composition for producing herbicidin.

[0008]

[0009] One object of the present invention is to provide a medium composition for producing herbicidin, comprising at least one amino acid selected from the group consisting of isoleucine, glutamine, proline and leucine.

[0010] Another object of the present invention is to provide a composition for producing herbicidin, comprising a Streptomyces scopuliridis strain or a culture thereof and at least one amino acid selected from the group consisting of isoleucine, glutamine, proline and leucine.

[0011] Another object of the present invention is to provide a method for producing herbicidin, comprising the step of culturing a Streptomyces scopuliridis strain in a medium containing at least one amino acid selected from isoleucine, glutamine, proline, and leucine.

[0012] Another object of the present invention is to provide a method for increasing herbicidin production, comprising the step of culturing a Streptomyces scopuliridis strain in a medium containing one or more amino acids selected from isoleucine, glutamine, proline and leucine.

[0013]

[0014] The medium composition of the present invention can significantly increase the biosynthesis efficiency of a herbicidin compound using a Streptomyces genus strain, compared to a conventional medium composition, by including at least one amino acid selected from isoleucine, glutamine, proline, and leucine.

[0015]

[0016] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification in their entirety by reference to more clearly explain the level of the technical field to which the present invention belongs and the contents of the present invention.

[0017]

[0018] One aspect of the present invention provides a medium composition for producing herbicidin, comprising an amino acid.

[0019]

[0020] The term "herbicidin" in the present invention refers to a metabolite derived from soil actinomycetes of the genus Streptomyces, which has been reported to exhibit herbicidal activity, anti-algal activity, and antifungal activity. Herbicidin is an adenosine-derived nucleoside antibiotic and has a specific tricyclic core structure. The herbicidin may include herbicidin A, herbicidin B, herbicidin C, herbicidin F, herbicidin H, or at least one selected therefrom.

[0021] For example, the herbicidin may be herbicidin A. In the present invention, the term "herbicidin A" is a compound represented by the following chemical formula 1, and is the most widely known component among the herbicidins, and is known to have excellent herbicidal and antibacterial properties.

[0022]

[0023]

[0024]

[0025] Herbicidin is emerging as an environmentally friendly herbicide with powerful weed control. However, research on a system capable of producing it at high yields remains limited. The present invention holds significant technical significance in demonstrating that the biosynthetic efficiency of herbicidin compounds can be significantly increased using Streptomyces strains through the addition of specific amino acids.

[0026]

[0027] A more specific aspect of the present invention provides a medium composition for producing herbicidin, comprising one or more amino acids selected from isoleucine, glutamine, leucine and proline.

[0028]

[0029] Specifically, the medium composition may further comprise a strain for producing herbicidin.

[0030] In the present invention, the term "microorganism" or "strain" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may also include microorganisms that have a specific mechanism weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and microorganisms that include genetic modification for the production of herbicidin. For example, the strain for producing herbicidin may be a microorganism that naturally has the ability to produce herbicidin, or a microorganism that has the ability to produce herbicidin strengthened or granted to a parent strain that does not have the ability to produce herbicidin through natural or artificial genetic modification, but is not limited thereto.

[0031] In one embodiment, the strain for producing herbicidin may be a strain of the genus Streptomyces (Streptomyces sp.). Specifically, the strain for producing herbicidin of the present invention may be, but is not limited to, Streptomyces scopuliridis.

[0032] For example, the Streptomyces scopuliridis strain may be the Streptomyces scopuliridis strain KR-001 deposited with the accession number KCTC 12156BP, and as another example, it may be the Streptomyces scopuliridis N29 strain deposited with the accession number KCTC 13057BP, which uses the KR-001 strain as a parent strain, but is not limited thereto.

[0033]

[0034] In the present invention, the term "isoleucine" is a type of nonpolar amino acid and has a molecular formula of C6H 13It is NO2. The above isoleucine is represented by the following chemical formula 2. Isoleucine is also used interchangeably with the names isoleucine, isoleucine, and isoleucine, and is abbreviated as Ile, I, etc. L-isoleucine exists in various proteins. D-isoleucine is a component of the antibiotic bacitracin, but does not exist in proteins. Other stereoisomers include L- and D-alloisoleucine, and L-isoleucine is an essential amino acid.

[0035]

[0036]

[0037]

[0038] In the present invention, the term "glutamine" is a type of polar neutral amino acid and is the monoamide of glutamic acid. Glutamine is abbreviated as Gln, Q, etc. The molecular formula of glutamine is C5H 10 It is represented by the chemical formula 3 below, N2O3. Glutamine is a needle-shaped crystal. In the body, glutamine serves as a storage agent for amino acids, is involved in the production of purine nuclei in nucleic acids, and performs detoxification functions.

[0039]

[0040]

[0041]

[0042] In the present invention, the term "leucine" is a type of non-polar amino acid, also called leucine, α-aminoisocaproic acid, and abbreviated as Leu, L, etc. The molecular formula of the leucine is C6H 13 NO2, represented by the following chemical formula 4. Leucine is widely distributed in nature in its free form. It is a precursor to various aromatic compounds and is also used as a food additive, nutritional enhancer, and flavor enhancer.

[0043]

[0044]

[0045]

[0046] In the present invention, the term "proline" refers to a type of polar amino acid, abbreviated as Pro, P, etc. The molecular formula of proline is C5H9NO 2. , and is represented by the following chemical formula 5. Proline is a columnar crystal with a slightly sweet taste and hygroscopicity. Proline is produced from glutamic acid in the body and is used as a food additive and as a nutritional enhancer.

[0047]

[0048]

[0049]

[0050] In a specific embodiment of the present invention, when a strain for producing herbicidin was cultured in a medium supplemented with one or more amino acids of isoleucine, glutamine, proline, and leucine to biosynthesize herbicidin, it was confirmed that the amount of herbicidin produced was significantly increased compared to a medium not supplemented with the amino acids. In the case of a medium supplemented solely with amino acids other than isoleucine, glutamine, proline, and leucine, the amount of herbicidin produced actually decreased or herbicidin was not produced, confirming that this increase in the amount of herbicidin biosynthesis was a specific effect of the amino acids.

[0051] Therefore, the medium composition of the present invention can be a medium composition for high production of herbicidin in which the production of herbicidin is significantly increased.

[0052] For example, the medium composition may have an increased herbicidin productivity of about 10% or more, specifically about 11% or more, about 12% or more, about 13% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 80% or more, compared to a medium composition that does not include one or more amino acids of isoleucine, glutamine, proline, and leucine, but is not limited thereto as long as the herbicidin productivity has a positive increase compared to before the addition of one or more amino acids of isoleucine, glutamine, proline, and leucine.

[0053] The term "about" above includes, but is not limited to, a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range equal to or similar to the numerical value following the term "about."

[0054]

[0055] In the present invention, the term "medium" means a material mixed with nutrients as the main ingredient necessary for culturing microorganisms for producing herbicidin, and supplies nutrients and growth factors, including water essential for survival and growth.

[0056]

[0057] Specifically, the amino acid may be included in a concentration (%) of 0.001 to 5.0 relative to the entire medium composition, and specifically, about 0.05 to 5 concentration (%), about 0.05 to 4 concentration (%), about 0.05 to 3 concentration (%), about 0.05 to 2 concentration (%), about 0.05 to 1.5 concentration (%), about 0.05 to 1 concentration (%), about 0.08 to 2 concentration (%), about 0.08 to 1.5 concentration (%), about 0.08 to 1 concentration (%), about 0.1 to 1.5 concentration (%), about 0.1 to 1.2 concentration (%), about 0.1 to 1 concentration (%), about 0.1 to 0.9 concentration (%), about 0.2 to 0.8 concentration (%), about 0.1 to 0.5 concentration (%), about 0.3 to It may include, but is not limited to, 0.7 concentration(%), about 0.2 to 0.6 concentration(%), about 0.4 to 0.8 concentration(%), about 0.3 to 0.9 concentration(%), about 0.1 to 0.7 concentration(%), about 0.4 to 0.9 concentration(%), or about 0.1 to 0.5 concentration(%).

[0058]

[0059] In one embodiment, the isoleucine may be included in a concentration (%) of 0.1 to 1.0 relative to the entire medium composition, specifically, a concentration (%) of 0.1 to 0.7 relative to the entire medium composition, more specifically, a concentration (%) of 0.1 to 0.5 relative to the entire medium composition, or a concentration (%) of 0.17 to 0.45 relative to the entire medium composition, even more specifically, a concentration (%) of 0.18 to 0.42 relative to the entire medium composition, and for example, a concentration (%) of 0.2 to 0.4 relative to the entire medium composition. In an embodiment of the present invention, when isoleucine is included in the medium composition in the same concentration (%) range as described above, it was confirmed that the increase in the production of herbicidin was greatly increased around the upper and lower limits of the range, suggesting that high production of herbicidin may be particularly possible when isoleucine is included in a specific concentration range.

[0060] In one embodiment, the glutamine may be included in a concentration (%) of 0.1 to 1.0 relative to the entire medium composition, specifically, a concentration (%) of 0.1 to 0.7% relative to the entire medium composition, more specifically, a concentration (%) of 0.2 to 0.8% relative to the entire medium composition, or a concentration (%) of 0.3 to 0.75% relative to the entire medium composition, even more specifically, a concentration (%) of 0.3 to 0.7% relative to the entire medium composition, and for example, a concentration (%) of 0.4 to 0.6. In an embodiment of the present invention, when glutamine is included in the medium composition in the same concentration (%) range as described above, it was confirmed that the increase in the production of herbicidin was greatly increased around the upper and lower limits of the range, suggesting that high production of herbicidin may be particularly possible when glutamine is included in a specific concentration range.

[0061] In one embodiment, the leucine may be included in a concentration (%) of 0.1 to 1.0 relative to the entire medium composition, specifically, in a concentration (%) of 0.2 to 1.0 relative to the entire medium composition, and for example, in a concentration (%) of 0.4 to 0.8 relative to the entire medium composition. In an embodiment of the present invention, it was confirmed that the herbicidin production was excellent when glutamine was included in the medium composition in the same concentration (%) range as described above.

[0062] In one embodiment, the proline may be included in a concentration (%) of 0.1 to 1.0 relative to the total medium composition, specifically in a concentration (%) of 0.2 to 1.0% relative to the total medium composition, more specifically in a concentration (%) of 0.2 to 0.7% relative to the total medium composition, 0.3 to 0.8% relative to the total medium composition, or 0.4 to 0.9% relative to the total medium composition.

[0063]

[0064] In this way, the present invention has another major technical significance in that it has elucidated that the efficiency of herbicidin compound biosynthesis using a Streptomyces genus strain can be maximized by adding a specific amino acid to a medium at a specific concentration range.

[0065]

[0066] The medium of the present invention may contain only one kind of amino acid among isoleucine, glutamine, leucine and proline, and the inventors of the present invention confirmed in an example that even when only one kind of isoleucine, glutamine, leucine and proline is contained in the medium composition, the production of herbicidin is greatly increased compared to the control group.

[0067] The medium of the present invention may contain a combination of two or more amino acids selected from isoleucine, glutamine, leucine, and proline.

[0068] In one embodiment, the medium of the present invention may comprise a) isoleucine and glutamine, b) isoleucine and leucine, c) isoleucine and proline, d) glutamine and leucine, e) glutamine and proline, or f) leucine and proline. In an embodiment of the present invention, it was confirmed that the production of herbicidin was excellently increased even when two or more types of amino acids among isoleucine, glutamine, leucine, and proline were mixed and added.

[0069] In another embodiment, the medium of the present invention may comprise a combination of three or more kinds of amino acids, or four kinds of amino acids, of isoleucine, glutamine, leucine and proline.

[0070] Even when the medium composition of the present invention includes a mixed sample of two or more amino acids as described above, the concentration (%) of each amino acid may be included as described above. In addition, the mixed sample of amino acids may be included at a concentration (%) of 0.001 to 5.0 relative to the entire medium composition, but is not limited thereto.

[0071]

[0072] In one embodiment, the medium composition of the present invention may additionally include, but is not limited to, a substance necessary for producing herbicidin.

[0073] The medium and other culture conditions used for culturing the Streptomyces microorganism of the present invention may be any medium that already contains one or more amino acids among isoleucine, glutamine, proline, and leucine, or any medium used for culturing a conventional microorganism that may further contain one or more amino acids among isoleucine, glutamine, proline, and leucine, without any particular limitation.

[0074] The medium of the present invention may be a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus, inorganic compounds, amino acids and / or vitamins, etc., with temperature, pH, etc. controlled, but is not limited thereto.

[0075] In the present invention, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto. For example, the carbon source may include glucose and potato starch.

[0076] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto. For example, the nitrogen source may include soybean powder.

[0077] The above-mentioned personnel may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and the like. In addition, amino acids other than isoleucine, glutamine, proline, and leucine, vitamins, and / or suitable precursors may be included. For example, the medium of the present invention may include trace amounts of ZnSO4-7H2O (Zinc sulfate heptahydrate), nicotinic acid, or a combination thereof.

[0078]

[0079] Another aspect of the present invention provides a composition for producing herbicidin, comprising a strain for producing herbicidin or a culture thereof; and one or more amino acids selected from isoleucine, glutamine, proline, and leucine.

[0080] At this time, the definitions of herbicidin, herbicidin producing strain, isoleucine, glutamine, proline, and leucine are the same as described above.

[0081]

[0082] Another aspect of the present invention provides a method for producing herbicidin, comprising the step of culturing a strain for producing herbicidin in a medium containing one or more amino acids selected from isoleucine, glutamine, proline, and leucine. In this case, the strain for producing herbicidin may be a strain of the genus Streptomyces, and more specifically, a strain of Streptomyces scopuliridis.

[0083] Specifically, the method for producing herbicidin of the present invention may additionally include, for example, a step of preparing a microorganism of the genus Streptomyces of the present invention, a step of preparing a medium for producing herbicidin for culturing the microorganism, or a combination thereof (in any order), prior to the culturing step.

[0084] In addition, the method for producing herbicidin of the present invention may further include, prior to the culturing step, a step of inoculating a Streptomyces scopuliridis strain into a medium containing one or more amino acids among isoleucine, glutamine, proline, and leucine.

[0085]

[0086] In the present invention, the term "cultivation" refers to growing the Streptomyces strain of the present invention under appropriately controlled environmental conditions. In the present invention, as long as a medium containing one or more amino acids of isoleucine, glutamine, proline, and leucine is used, the culturing process can be performed according to an appropriate medium and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0087] The Streptomyces strain of the present invention can be cultured under aerobic conditions in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids other than isoleucine, glutamine, proline, and leucine, and / or vitamins, while controlling temperature, pH, etc. These components or precursors can be added to the medium in batch or continuous manner. However, the present invention is not limited thereto.

[0088] Specifically, during the above culturing, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the culturing, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain anaerobic and microaerobic states, nitrogen, hydrogen, or carbon dioxide gas can be injected without injecting gas, but is not limited thereto.

[0089]

[0090] In the above culture, the culture temperature may be 20 to 35°C, specifically 25 to 30°C, but is not limited thereto.

[0091] The above culturing may be performed for a culture period of about 10 hours to 20 days, about 1 day to 10 days, about 3 days to 9 days, about 4 days to 8 days, or about 5 days to 7 days, but is not limited thereto.

[0092] Herbicidin produced by the above culture may be secreted into the medium or remain within the microorganism.

[0093]

[0094] Specifically, the method for producing herbicidin of the present invention may further include a step of recovering herbicidin A from the medium or strain. The recovering step may be additionally included after the culturing step.

[0095] The above recovery may be performed by collecting the target herbicidin using a suitable method known in the art according to the culture method of the microorganism of the present invention, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out method), extraction, cell disruption, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and the target herbicidin can be recovered from the medium or microorganism using a suitable method known in the art.

[0096] Additionally, the method for producing herbicidin of the present invention may additionally include a purification step. The purification may be performed using any suitable method known in the art. In one example, when the method for producing herbicidin of the present invention includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially (or sequentially) regardless of the order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

[0097]

[0098] Another aspect of the present invention provides a method for increasing herbicidin production, comprising the step of culturing a Streptomyces spp. strain in a medium containing one or more amino acids selected from isoleucine, glutamine, proline and leucine.

[0099] At this time, the definitions of Streptomyces spp. strain, isoleucine, glutamine, proline, leucine, medium, culture and herbicidin are the same as described above.

[0100]

[0101] Another aspect of the present invention provides a use for producing herbicidin, comprising a composition comprising one or more amino acids selected from isoleucine, glutamine, leucine and proline.

[0102] Specifically, the composition may further comprise a strain for producing herbicidin or a culture thereof.

[0103] At this time, the definitions of the strain for producing herbicidin, isoleucine, glutamine, proline, leucine, culture, and herbicidin are the same as described above.

[0104]

[0105] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited by these examples.

[0106]

[0107] Example 1. Optimization of culture conditions for mass production of microbial herbicides.

[0108] First, we sought to explore amino acid components suitable as effective ingredients of the medium for high production of herbicidin.

[0109] Specifically, Streptomyces scopuliridis N29 mutant was inoculated into a basic medium containing glucose, potato starch, and soybean flour as main components, and 1.5 mM ZnSO4-7H2O (Zinc sulfate heptahydrate) and 0.5 mM nicotinic acid as trace components, and used as a control group. An experimental group was prepared by adding 0.6% concentration of each of the 17 amino acids to the basic medium, and the same amount of N29 mutant was inoculated and cultured for 6 days.

[0110]

[0111] On the fifth and sixth days of cultivation, the culture medium was collected, mixed 1:1 with methanol, centrifuged, and HPLC analysis was performed to investigate the herbicidin content. The results of the analysis of herbicidin production in the control group and the experimental group cultured with the addition of each of the 17 amino acids are shown in Table 1.

[0112]

[0113] Herbicidin 5DAT (g / L) 6DAT (g / L) Production compared to control group (%)*Control 1.76 ± 0.02 1.837 ± 0.04 100 Alanine 0.56 ± 0.02 0.308 ± 0.06 17 Arginine 0.06 ± 0.01 0.023 ± 0.001 Asparagine 0.00 ± 0.000 ± 0.000 Glutamic acid 0.02 ± 0.000 016 ± 0.001 Glutamine 2.11 ± 0.06 2.441 ± 0.07 133 Glycine 0.00 ± 0.000 046 ± 0.032 Histidine 0.04 ± 0.01 0.027 ± 0.011 Isoleucine 2.29 ± 0.042.621 ± 0.04142Leucine2.03 ± 0.002.314 ± 0.02126Lysine1.76 ± 0.011.728 ± 0.0094Methionine0.61 ± 0.010.725 ± 0.0139Phenylalanine1.29 ± 0.021.449 ± 0.0679Proline1.88 ± 0.042.187 ± 0.01119Serine0.45 ± 0.130.522 ± 0.1428Threonine1.40 ± 0.301.194 ± 0.0365Tryptophan1.51 ± 0.011.809 ± 0.0098Valine1.33 ± 0.071.412 ± 0.1977

[0114] * Based on 6DAT production volume

[0115] As can be seen in Table 1 above, for most of the 17 amino acids, the production of herbicidin decreased or was impossible compared to the basic medium. However, in the experimental groups cultured for 6 days with the addition of glutamine, isoleucine, leucine, or proline, the production of herbicidin increased by 33%, 42%, 26%, and 19%, respectively, compared to the control group. In particular, isoleucine, which had the highest herbicidin production compared to the control group, increased its production by approximately 42% compared to the control group.

[0116] From the above results, the possibility of utilizing the medium composition containing glutamine, isoleucine, leucine and proline as active ingredients for efficient production and mass production of herbicidin was confirmed.

[0117]

[0118] Example 2. Analysis of herbicidin production according to the concentration of added glutamine, isoleucine, and leucine.

[0119]

[0120] Next, in Example 1, the three amino acids that showed the best increase in herbicidin production were evaluated to optimize the addition concentration by evaluating the production amount according to the concentration.

[0121]

[0122] Glutamine, isoleucine, and leucine, three amino acids that showed a significant increase in herbicidin production compared to the control group in Table 1, were added to the medium at concentrations of 0.2 to 1.0% (0.2% intervals) and cultured for 6 days. The results of the analysis of herbicidin production are shown in Table 2 below.

[0123]

[0124] Herbicidin 5DAT (g / L) 6DAT (g / L) Production compared to control (%)*Glutamine 0.0% 1.50 ± 0.08 1.76 ± 0.10 100Glutamine 0.2% 0.75 ± 0.05 0.86 ± 0.01 47Glutamine 0.4% 1.95 ± 0.06 2.19 ± 0.09 125Glutamine 0.6% 1.62 ± 0.06 1.98 ± 0.22 113Glutamine 0.8% 1.55 ± 0.03 1.75 ± 0.06 99Glutamine 1.0% 1.47 ± 0.15 1.66 ± 0.05 94Isoleucine 0.0% 1.50 ± 0.08 1.76 ± 0.10100Isoleucine 0.2%1.89 ± 0.032.34 ± 0.02133Isoleucine 0.4%1.79 ± 0.062.36 ± 0.07135Isoleucine 0.6%1.78 ± 0.052.26 ± 0.01129Isoleucine 0.8%1.66 ± 0.082.22 ± 0.02126Isoleucine 1.0%1.53 ± 0.032.17 ± 0.04124Leucine 0.0%1.50 ± 0.081.76 ± 0.10100Leucine 0.2%1.65 ± 0.041.96 ± 0.04112Leucine 0.4%1.63 ± 0.052.10 ± 0.02119Leucine 0.6%1.60 ± 0.102.14 ± 0.04122Leucine 0.8%1.60 ± 0.052.08 ± 0.06119Leucine 1.0%1.44 ± 0.252.01 ± 0.45114

[0125] * HPLC based on 6DAT production volume

[0126] As shown in Table 2 above, the analysis results confirmed that the production of herbicidin increased significantly, especially in the experimental groups that added 0.2% to 0.4% isoleucine. In addition, in the case of glutamine, the production of herbicidin increased most significantly in the experimental groups that added 0.4% and 0.6%. These results suggest that high production of herbicidin may be possible, especially in a specific concentration range when included as an effective ingredient in the medium, depending on the amino acid type.

[0127]

[0128] Based on the results shown in Table 2 above, in order to more precisely determine the optimal addition concentration of isoleucine, which has the highest contribution to increasing herbicidin production, experimental groups added at concentrations of 0.05% to 0.4% (0.05% intervals) were cultured at 28 degrees and 150 rpm for 6 days and analyzed by HPLC. The results are shown in Table 3 below.

[0129]

[0130] Herbicidin 5DAT (g / L) 6DAT (g / L) Production compared to control (%)*Isoleucine 0.0% 1.32 ± 0.02 1.35 ± 0.03 100Isoleucine 0.05% 1.34 ± 0.00 1.42 ± 0.06 105Isoleucine 0.10% 1.56 ± 0.06 1.68 ± 0.05 125Isoleucine 0.15% 1.56 ± 0.04 1.66 ± 0.06 123Isoleucine 0.2% 1.68 ± 0.02 1.79 ± 0.03 133Isoleucine 0.25% 1.64 ± 0.03 1.81 ± 0.05 134Isoleucine 0.30%1.64 ± 0.051.88 ± 0.08139Isoleucine 0.35%1.59 ± 0.101.78 ± 0.11132Isoleucine 0.40%1.63 ± 0.001.84 ± 0.00136

[0131] * Based on 6DAT production volume

[0132] As a result, as can be seen in Table 3 above, when the medium composition included 0.2 to 0.4% of isoleucine, the production of herbicidin significantly increased by more than 30% compared to the control group. In particular, in the experimental group where 0.3% of isoleucine was added, it was confirmed that the production of herbicidin significantly increased by more than 39% compared to the control group.

[0133]

[0134] Example 3. Analysis of herbicidin production according to the addition of mixed amino acids.

[0135]

[0136] Based on the results shown in Examples 1 and 2 above, we aimed to confirm whether an increase in herbicidin production also occurred when a mixture of amino acids was added, targeting the four amino acids that contributed most to an increase in herbicidin production.

[0137] Specifically, an experimental group was prepared in which the remaining three amino acids, glutamine, leucine, and proline, were each added at a concentration of 0.2% to the main components, glucose, potato starch, and soybean flour, and isoleucine was added at a concentration of 0.2%. The amount of herbicidin produced was analyzed by HPLC after culturing for 6 days at 28 degrees and 150 rpm, and the results are shown in Table 4 below.

[0138]

[0139] Herbicidin5DAT (g / L)6DAT (g / L)Production compared to control (%)*Control1.23 ± 0.061.25 ± 0.06100Isoleucine 0.2%, Leu 0.2%1.42 ± 0.051.23 ± 0.06116Isoleucine 0.2%, Pro 0.2%1.44 ± 0.011.49 ± 0.02117Isoleucine 0.3%, Leu 0.2%1.43 ± 0.111.15 ± 0.14116Isoleucine 0.3%, Pro 0.2%1.46 ± 0.001.48 ± 0.02119Isoleucine 0.4%, Leu 0.2%1.27 ± 0.000.93 ± 0.01104Isoleucine 0.4%, Pro 0.2%1.46 ± 0.041.40 ± 0.09119

[0140] * Based on 5DAT production volume

[0141] As a result, as can be seen in Table 4 above, even when amino acids were mixed and used, an increase in herbicidin production occurred, and in particular, an excellent production increase of approximately 19% was confirmed compared to the control group in the experimental group where 0.3% and 0.4% isoleucine were added to 0.2% proline. These results suggest that a medium composition including a combination of two or more amino acids selected from isoleucine, glutamine, proline, and leucine also has an excellent effect on herbicidin production.

[0142]

[0143] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A medium composition for producing herbicidin, comprising at least one amino acid selected from the group consisting of isoleucine, glutamine, proline and leucine.

2. A medium composition for producing herbicidin, in claim 1, further comprising a Streptomyces scopuliridis strain.

3. A medium composition for producing herbicidin, wherein the amino acid is included in a concentration (%) of 0.05 to 1.0 relative to the total medium composition in paragraph 1.

4. A medium composition for producing herbicidin, wherein the amino acid is included in a concentration (%) of 0.1 to 1.0 relative to the total medium composition in paragraph 1.

5. A medium composition for producing herbicidin, wherein in paragraph 1, the isoleucine is included in a concentration (%) of 0.1 to 0.5 relative to the total medium composition.

6. A medium composition for producing herbicidin, wherein in paragraph 1, the isoleucine is included in a concentration (%) of 0.18 to 0.42 relative to the total medium composition.

7. A medium composition for producing herbicidin, wherein in paragraph 1, the glutamine is included in a concentration (%) of 0.3 to 0.7 relative to the total medium composition.

8. A medium composition for producing herbicidin, in the first paragraph, wherein the herbicidin is at least one selected from herbicidin A, herbicidin B, herbicidin C, herbicidin F, and herbicidin H.

9. A medium composition for producing herbicidin, wherein the herbicidin in paragraph 1 is herbicidin A.

10. Streptomyces scopuliridis strain or culture thereof; and A composition for producing herbicidin, comprising one or more amino acids selected from isoleucine, glutamine, proline and leucine.

11. A method for producing herbicidin, comprising the step of culturing a Streptomyces scopuliridis strain in a medium containing one or more amino acids of isoleucine, glutamine, proline and leucine.

12. A method for producing herbicidin, further comprising the step of inoculating a Streptomyces scopuliridis strain into a medium containing one or more amino acids among isoleucine, glutamine, proline, and leucine, in accordance with claim 11.

13. A method for producing herbicidin, further comprising a step of recovering herbicidin from the medium or strain in claim 11.

14. A method for producing herbicidin in claim 11, wherein the culturing is performed for a culturing period of 5 to 7 days.

15. A method for increasing herbicidin production, comprising the step of culturing a Streptomyces scopuliridis strain in a medium containing one or more amino acids of isoleucine, glutamine, proline and leucine.

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