Method for producing 3-hydroxypropionic acid

Abstract

The present invention relates to a two-step method for producing 3-HP, comprising: a first step of culturing cells at high density; and a second step of producing 3-HP by using the high-density cultured cells as a catalyst, wherein, in the first step, the cell production rate is controlled through high-density cell culturing and, in the second step, DO and / or culture temperature and pH are adjusted to produce 3-HP and / or improve the productivity of 3-HP, and thus high concentrations of 3-HP can be produced from high-concentrations of glycerol while cell activity is stably maintained.

Description

Method for producing 3-hydroxypropionic acid

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority to Republic of Korea Patent Application No. 10-2024-0009485, filed January 22, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present application relates to a method for producing 3-hydroxypropionic acid (hereinafter also referred to as “3-HP”) and / or a method for improving the productivity of 3-HP, and more specifically, to a method for producing 3-HP and / or improving the productivity of 3-HP, which enables the production of a high concentration of 3-HP from a high concentration of glycerol while stably maintaining the activity of cells having 3-HP production ability.

[0004]

[0005] 3-Hydroxypropionic acid is a platform compound that can be converted into various chemicals, including acrylic acid, methyl acrylate, and acrylamide. Since being selected as one of the top 12 value-added biochemicals by the U.S. Department of Energy (DOE) in 2004, it has been actively researched in both academia and industry.

[0006] 3-HP is produced largely through two methods: chemical and biological. However, the chemical method is criticized as being unfriendly due to the high cost of initial materials and the generation of toxic substances during the production process. Therefore, the environmentally friendly bioprocess is gaining attention.

[0007] Glucose and glycerol are mainly used as substrates for 3-HP biosynthesis using microorganisms, but due to low yield and productivity, continuous strain development and production process development research are still necessary despite the potential of 3-HP.

[0008]

[0009] Accordingly, the inventors of the present invention developed a fermentation technology capable of increasing the concentration of 3-HP production while stably maintaining cell activity for commercialization of 3-HP.

[0010] An example of the present application provides a method for producing 3-hydroxypropionic acid (3-HP) and / or a method for improving the productivity of 3-HP, including a step of mass producing 3-hydroxypropionic acid (3-HP) while controlling the cell production rate by culturing cells having 3-HP production ability at a high concentration; and a step of producing 3-HP under optimal dissolved oxygen (hereinafter also referred to as “DO”) and optimal cell stabilization and production conditions.

[0011] The present application provides a method for producing 3-hydroxypropionic acid (3-HP) and / or a method for improving the productivity of 3-HP, including a step of mass producing 3-hydroxypropionic acid (3-HP) while controlling the cell production rate by culturing cells capable of producing 3-HP at a high concentration; and a step of producing 3-HP under optimal dissolved oxygen levels and optimal cell stabilization and production conditions.

[0012] More specifically, the method comprises:

[0013] (1) Cells having 3-hydroxypropionic acid (3-HP) production ability were cultured in a growth medium (growth medium) until the cell concentration reached OD 600 A high-concentration cell culture step in which cell culture is performed until the number reaches 100 to 200; and

[0014] (2) a step of producing 3-HP by transferring the culture medium of the above step (1) to a production medium containing a substrate while maintaining DO (dissolved oxygen) at 1 to 20%,

[0015] The above step (2) may be performed under the following conditions:

[0016] (i) a temperature condition of 30 to 40°C (specifically, 35°C); and / or

[0017] (ii) pH conditions of 6 to 8 (specifically, pH 6.8).

[0018]

[0019] Hereinafter, the present invention will be described in more detail.

[0020]

[0021] In the step (1) of the method for producing 3-HP and / or the method for improving the productivity of 3-HP provided herein, cells having 3-hydroxypropionic acid production ability are cultured at a high concentration in a culture medium (growth medium). After the high-concentration culture, the cell culture solution is used as is in step (2) to produce 3-HP without a separate cell recovery process. Specifically, the method can transfer the culture solution itself to the production medium without going through an additional step including a cell recovery step after the culturing step of step (1).

[0022] In the above step (1), the culture medium (growth medium) may not contain glycerol as a carbon source.

[0023] In the present specification, the 3-hydroxypropionic acid producing cell (hereinafter, interchangeably used with the equivalent meaning of 'cell having 3-hydroxypropionic acid producing ability') refers to a microorganism capable of producing 3-HP from a carbon source (e.g., glycerol) in a production medium, for example, a strain of the genus Escherichia (E. coli, etc.), the genus Pseudomonas, the genus Enterobacteria, the genus Brevibacterium, the genus Corynebacterium, the genus Klebsiella, the genus Citrobacter, the genus Clostridium, the genus Streptomyces, the genus Bacillus, the genus Lactobacillus, the genus Pseudomonas, It may be selected from microorganisms of the genus Saccharomyces and the genus Aspergillus, but is not limited thereto. In one specific example, the 3-hydroxypropionic acid producing cell may be Escherichia coli.

[0024] In one example, the 3-hydroxypropionic acid producing cell may comprise a gene encoding one or more (e.g., one or both) proteins selected from the group consisting of glycerol dehydratase and aldehyde dehydrogenase. In one example, the 3-HP producing cell may further comprise a gene (gdrAB) encoding glycerol dehydratase reactivase (GdrAB). In one example, the 3-HP producing cell may further be a cell capable of biosynthesizing vitamin B12.

[0025] The above glycerol dehydratase may be encoded by, but is not limited to, the dhaB (GenBank accession no. U30903.1) gene. The dhaB gene may be an enzyme derived from, but is not limited to, Klebsiella pneumonia. The gene encoding the glycerol dehydratase may include a gene encoding dhaB1, dhaB2, and / or dhaB3. The glycerol dehydratase protein and the gene encoding the same may include mutations in the gene and / or amino acid sequence within a range that maintains an enzymatic activity that decomposes glycerol into 3-hydroxypropanal (3-HPal) and water (H2O).

[0026] The gene (aldH) encoding the above aldehyde dehydrogenase (ALDH) may be, for example, an aldH (GenBank Accession no. U00096.3; EaldH) gene derived from Escherichia coli or an E. coli K12 MG1655 cell line, a puuC gene derived from Klebsiella pneumoniae, and / or a KGSADH gene derived from Azospirillum brasilense, but is not limited thereto. The above aldehyde dehydrogenase protein and the gene encoding the same may include mutations in the gene and / or amino acid sequence within a range that maintains the activity for producing 3-HP from 3-HPal.

[0027] The above 3-HP producing cell may comprise a gene encoding one or more, two or more, or all three proteins selected from the group consisting of glycerol dehydratase, aldehyde dehydrogenase, and glycerol dehydratase reactivase, or a recombinant vector comprising the gene.

[0028] The above recombinant vector can be used by replacing the promoter and regulatory region by a method known in the art within the scope of the purpose of expressing in a cell a gene encoding one or more, two or more, or all three proteins selected from the group consisting of glycerol dehydratase, aldehyde dehydrogenase, and glycerol dehydratase reactivase.

[0029] The above high-density culture can be performed without limitation using any method known in the art for the purpose of securing a large quantity of 3-HP producing cells, and in one example, the culture can be performed as a fed-batch culture.

[0030] In one example, the fed-batch culture may be performed using a pH-stat method, a DO-stat feeding method, a continuous feeding method, or a combination thereof. In one embodiment, when the fed-batch culture is performed using a pH-stat method, glucose may be added at a concentration of 1 to 5 g / L.

[0031] In one embodiment, when the fed-batch culture is performed in a constant-rate culture mode, glucose can be added at a rate of 7 to 21 g / L / h.

[0032] In one example, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the microbial culture medium in an appropriate manner during cultivation. In one example, the pH during the high-concentration cultivation can be maintained at, but is not limited to, 5 to 7.5, 5 to 7, 5.5 to 7.5, 5.5 to 7, 6 to 7.5, or 6.5 to 6.

[0033] The temperature of the culture medium may be 20°C to 45°C, 25°C to 40°C or 30°C to 37°C, for example 37°C.

[0034] For high-density cell culture, the above 3-HP producing cells were cultured at OD per hour 600 (Absorbance measured at 600 nm (OD)) may be produced (proliferated) at a concentration of 5 to 23, 5 to 22, 5 to 17, 5 to 16, 5 to 10, 5 to 8, 6 to 23, 6 to 22, 6 to 17, 6 to 16, 6 to 10, 6 to 8, 10 to 23, 10 to 22, 10 to 17, 10 to 16, 14 to 23, 14 to 22, 14 to 17, 14 to 16, 17 to 23, 17 to 22, 20 to 23, or 20 to 22, for example, 7, 15, or 21, but is not limited thereto, and the 3-HP manufacturing method and / or 3-HP productivity improvement In terms of 3-HP production in the method, it is advantageous that 3-HP producing cells are produced (proliferated) within the above range.

[0035] In one example, the cell concentration after the high-concentration culture is OD 600 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, 10 to 500, 10 to 400, 10 to 300, 10 to 250, 50 to 500, 50 to 400, 50 to 300, 50 to 250, 100 to 500, 100 to 400, 100 to 300, 100 to 250, 150 to 500, 150 to 400, 150 to 300, 150 to 250, 200 to 500, 200 to 400, 200 to 300, or 200 to 250, but is not limited thereto. Specifically, the cell concentration is OD 600 It can be 100 to 200 days.

[0036] In one example, the carbon source included in the culture medium (growth medium) for the high-density cultivation may be selected without limitation from monosaccharides, disaccharides, and / or polysaccharides within the target range for the high-density cultivation. For example, the carbon source may be one or more, two or more, three or more, four or more, five or more, ten or more, or a combination of all eleven types selected from the group consisting of glucose, fructose, galactose, mannose, arabinose, xylose, ribose, sucrose, maltose, lactose, and cellobiose. The growth medium may not include glycerol as a carbon source.

[0037] The above culture medium (growth medium) contains a carbon source of 1 to 50 g / L, 1 to 40 g / L, 1 to 30 g / L, 1 to 25 g / L, 1 to 23 g / L, 10 to 50 g / L, 10 to 40 g / L, 10 to 30 g / L, 10 to 25 g / L, 10 to 23 g / L, 15 to 50 g / L, 15 to 40 g / L, 15 to 30 g / L, 15 to 25 g / L, 15 to 23 g / L, 17 to 50 g / L, 17 to 40 g / L, 17 to 30 g / L, 17 to 25 g / L, or 17 to 23 g / L, based on the volume of the culture medium (growth medium), for example, 20 May include, but is not limited to, g / L.

[0038] OD per hour of the above 3-HP producing cells 600 To produce a range of (absorbance (OD) measured at 600 nm), the carbon source can be additionally supplied at a constant rate.

[0039] In one example, in the high-concentration cell culture (step (1) above), the concentration of the culture medium (growth medium) is 4 to 25 g / L, 4 to 23 g / L, 4 to 21 g / L, 4 to 17 g / L, 4 to 15 g / L, 4 to 13 g / L, 4 to 9 g / L, 4 to 7 g / L, 7 to 25 g / L, 7 to 23 g / L, 7 to 21 g / L, 7 to 17 g / L, 7 to 15 g / L, 7 to 13 g / L, 7 to 9 g / L, 10 to 25 g / L, 10 to 23 g / L, 10 to 21 g / L, 10 to 17 g / L, 10 to 15 g / L, 13 to 25 g / L, The carbon source may be additionally supplied into the medium in a content of 13 to 23 g / L, 13 to 21 g / L, 13 to 17 g / L, 13 to 15 g / L, 17 to 25 g / L, 17 to 23 g / L, 17 to 21 g / L, 20 to 25 g / L, 20 to 23 g / L, or 20 to 21 g / L, for example, 7, 14, or 21 g / L (concentration; weight of carbon source added (g) / volume of medium (L)), and adding the carbon source in the content range is advantageous in terms of improving the 3-HP production and / or 3-HP productivity.

[0040] The additionally supplied carbon source may be additionally supplied at a time when the concentration of the carbon source in the culture medium (growth medium) becomes 0 g / L, and specifically, may be supplied after 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, or 15 hours from the start of cell culture in the culture medium (growth medium), but is not limited thereto.

[0041] In one embodiment according to the present invention, a glucose solution having a concentration of 700 g / L was added to the medium as a carbon source for high-density cell culture, and the carbon source solution was supplied to the medium at 10 to 30 mL / h per 1 L of the medium so that all of the added sugar in the culture medium was consumed while maintaining the glucose concentration at 0 g / L.

[0042] For example, the medium may include, along with the carbon source described above, a nitrogen source and trace element components. Nitrogen sources that may be used include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may also be used individually or as a mixture, but are not limited thereto. Phosphorus sources that may be used include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, the medium may contain, but are not limited to, metal salts such as magnesium sulfate or iron sulfate required for growth. In addition, essential growth substances such as amino acids and vitamins may be included. Appropriate precursors may also be used in the medium. The above medium or individual components may be added to the culture medium in a batch or continuous manner in an appropriate manner during the culture process, but are not limited thereto.

[0043] The above step (2) is a step of producing 3-HP by transferring the culture solution of the above step (1) to a production medium containing a substrate while maintaining DO (dissolved oxygen) at 1 to 20%, and the above step (2) is a step performed under the following conditions.

[0044] (i) a temperature condition of 30 to 40°C (specifically, 35°C); and / or

[0045] (ii) pH conditions of 6 to 8 (specifically, pH 6.8).

[0046] Specifically, the step (2) is a step of producing 3-HP by inoculating cells having 3-hydroxypropionic acid (3-HP) production ability into a 3-hydroxypropionic acid production medium and moving them to the production medium, and maintaining the dissolved oxygen level at a constant level and also maintaining the temperature and / or pH at a constant level for cell stability.

[0047] The above 3-hydroxypropionic acid culture medium (growth medium) can be used without limitation in the scope of the purpose of enabling the cells to produce 3-HP without causing proliferation (cell division, growth, or outgrowth) of the 3-HP producing cells.

[0048] In one example, the carbon source of the culture medium (growth medium) may be, but is not limited to, glycerol. In one example, the production medium may additionally contain vitamin B12. In one embodiment, the production medium may not contain glucose at the time of cell inoculation.

[0049] In one example, the medium may be, but is not limited to, a synthetic medium or a semisynthetic medium.

[0050] The above inoculation cells may be prepared in the form of a cell culture solution without a separate cell recovery process after culturing cells having 3-hydroxypropionic acid (3-HP) production ability at high concentration in the above step (1), but are not limited thereto.

[0051] The inoculation concentration (cell concentration at the time of inoculation) of the above-described high-concentration cultured cells can be appropriately adjusted or determined by a person skilled in the art within the range of the purpose of 3-HP production. In one example, the inoculation concentration (based on dry cell weight (DCW) / medium volume (L)) may be, but is not limited to, 1 to 20 g / L, 1 to 16 g / L, 1 to 12 g / L, 1 to 9 g / L, 2 to 20 g / L, 2 to 16 g / L, 2 to 12 g / L, 2 to 9 g / L, 4 to 20 g / L, 4 to 16 g / L, 4 to 12 g / L, or 4 to 9 g / L.

[0052] The step of producing the above 3-hydroxypropionic acid can be used by selecting without limitation any culture method known in the art within the scope of the purpose of producing 3-HP, and in one example, the culture step can be performed by fermentation.

[0053] In the above 3-HP production step, proliferation of the inoculated cells may not occur. In one example, the number of cells at the end of the production step may be 150% or less, 130% or less, 100% or less, 90% or less, or 80% or less of the number of inoculated cells, such as, but not limited to, 50 to 150%, 50 to 130%, 50 to 100%, 50 to 90%, 50 to 80%, 70 to 150%, 70 to 130%, 70 to 100%, 70 to 90%, or 70 to 80%.

[0054] In one embodiment, step (2) of the method may be maintaining a state in which the dissolved oxygen (DO) is 1 to 20%, 1 to 15%, 1 to 12%, 1 to 10%, 1 to 8%, 1 to 6%, 1 to 5%, 3 to 20%, 3 to 15%, 3 to 12%, 3 to 10%, 3 to 8%, 3 to 6%, 3 to 5%, 4 to 20%, 4 to 15%, 4 to 12%, 4 to 10%, 4 to 8%, 4 to 6%, 4 to 5%, 5 to 20%, 5 to 15%, 5 to 12%, 5 to 10%, 5 to 8%, or 5 to 6%, for example, 5%, but is not limited thereto. It is not.

[0055] In this specification, the “change value of dissolved oxygen” may mean a change value of dissolved oxygen in the process of cultivating and / or producing a specific substance, and specifically, may be a difference between the maximum and minimum values ​​of dissolved oxygen in the process of cultivating and / or producing a specific substance, for example, if the minimum value of dissolved oxygen in the process of cultivating and / or producing a specific substance is 4% and the maximum value is 6%, the change value of dissolved oxygen may be expressed as 2%.

[0056] The change value of the dissolved oxygen amount in step (2) of the above method may be, but is not limited to, 10% or less (less than), 7% or less (less than), 5% or less (less than), 3% or less (less than), 2% or less (less than), 1% or less (less than), 0 to 10%, 0 to 7%, 0 to 5%, 0 to 3%, 0 to 2%, or 0 to 1%.

[0057] Maintaining the above DO (dissolved oxygen) at a constant level (e.g., DO of 4 to 6%) may be performed by at least one selected from the group consisting of adjusting the stirring speed during cultivation, adjusting the air supply amount, and adjusting the pressure, but is not limited thereto. For example, when the dissolved oxygen level in the medium or culture is lower than the above range, (1) at least one of the stirring speed, the air supply amount, and the pressure is increased, and (2) when the dissolved oxygen level in the medium or culture is higher than the above range, at least one of the stirring speed, the air supply amount, and the pressure is decreased, thereby maintaining the dissolved oxygen level in the medium or culture within a predetermined range, but is not limited thereto. Specifically, in an embodiment according to the present invention, in order to maintain DO of 5%, the stirring speed and the air supply amount were sequentially adjusted during cultivation to form an aerobic condition, thereby producing 3-HP.

[0058] In one example, to maintain the DO conditions and ensure accurate oxygen delivery, a process for removing bubbles from the surface of the culture medium may be further included. For example, this bubble removal process may utilize an antifoaming agent. The antifoaming agent may be one that does not affect cell stability, and may be added in an amount that does not generate bubbles even under the highest stirring speed and air supply conditions, which can be appropriately selected by those skilled in the art. For example, bubble formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester.

[0059] In one example, the method for producing 3-HP by maintaining (regulating) the dissolved oxygen content may be one in which the 3-HP production increases by 30% or more, 50% or more, 70% or more, 30 to 1000%, 30 to 500%, 30 to 300%, 30 to 100%, 50 to 1000%, 50 to 500%, 50 to 300%, 50 to 100%, 70 to 1000%, 70 to 500%, 70 to 300%, or 70 to 100% compared to a control group that does not include a dissolved oxygen content maintenance condition, but is not limited thereto.

[0060] In one example, in the step (2), for cell stability,

[0061] (i) a temperature condition of 30 to 40°C (specifically, 35°C); and / or

[0062] (ii) It can be performed under pH conditions of 6 to 8 (specifically, pH 6.8).

[0063] More specifically, the above step (2)

[0064] (i) Performed under temperature conditions of 37℃ to 40℃ or pH conditions of 7 to 8,

[0065] (ii) It can be performed under temperature conditions of 30℃ or higher and less than 37℃ or pH conditions of 6 or higher and less than 7.

[0066] More specifically, the above step (2)

[0067] (i) temperature conditions of 35℃; and / or

[0068] (ii) It can be performed under pH conditions of 6.8.

[0069] For example, to maintain a constant pH within the aforementioned range, a compound selected from the group consisting of calcium hydroxide, magnesium hydroxide, ammonia hydroxide, sodium hydroxide, and potassium hydroxide may be appropriately added to the microbial culture during cultivation. Additionally, for example, a conventional temperature maintenance method may be used to maintain the temperature of the culture.

[0070] The incubation period may continue until the desired yield of the useful substance (e.g., 3-HP) is obtained, for example, 3 to 60 hours, 3 to 48 hours, 3 to 36 hours, 3 to 28 hours, 6 to 60 hours, 6 to 48 hours, 6 to 36 hours, 6 to 28 hours, 12 to 60 hours, 12 to 48 hours, 12 to 36 hours, 12 to 28 hours, 20 to 60 hours, 20 to 48 hours, 20 to 36 hours or 20 to 28 hours, for example, but not limited to, 24 hours.

[0071] The 3-HP yield of the method for producing 3-HP and / or the method for improving the productivity of 3-HP provided herein may be, for example, 80% or more, 85% or more, 90% or more, 93% or more, or 95% or more, but is not limited thereto. The 3-HP yield may be calculated as the 3-HP production amount in the medium (culture) relative to the amount of glycerol used (moles) in the medium (production medium) in the step of producing 3-HP, and in one example, may be calculated as in the following mathematical formula 1.

[0072] [Mathematical Formula 1]

[0073] 3-HP production yield (yield, %) = [(final 3-HP (g)) / {(Glycerol (g) before cultivation) - (Glycerol (g) remaining after cultivation)}] * 100

[0074] The 3-HP productivity, which represents the 3-HP production amount (production concentration) according to the 3-HP production time of the 3-HP manufacturing method and / or the 3-HP productivity improvement method provided in the present specification, can be calculated as in the following mathematical formula 2. Specifically, the 3-HP production amount may be the 3-HP production concentration measured by an HPLC measurement method or the like.

[0075] [Equation 2]

[0076] 3-HP productivity = (final 3-HP production concentration) / (total 3-HP production time)

[0077] The 3-HP productivity of the 3-HP manufacturing method and / or the 3-HP productivity improvement method provided by the present invention may be 4.0 g / L / h or more, 4.5 g / L / h or more, 4.0 to 60 g / L / h, 4.0 to 40 g / L / h, 4.0 to 20 g / L / h, 4.0 to 10 g / L / h, 5.0 to 60 g / L / h, 5.0 to 40 g / L / h, 5.0 to 20 g / L / h, 5.0 to 10 g / L / h, 6.0 to 60 g / L / h, 6.0 to 40 g / L / h, 6.0 to 20 g / L / h, or 6.0 to 10 g / L / h, but is not limited thereto.

[0078] Another example provides a culture of 3-hydroxypropionic acid producing cells having a high 3-hydroxypropionic acid content and a low by-product content. Specifically, the present invention provides a culture of 3-hydroxypropionic acid producing cells characterized by being produced by the above 3-HP production method and / or the method for improving the productivity of 3-HP.

[0079] The above culture may contain 3-hydroxypropionic acid, based on the entire culture, at 60 g / L or more, 62 g / L or more, 65 g / L or more, 67 g / L or more, 70 g / L or more, 72 g / L or more, 75 g / L or more, 77 g / L or more, 78 g / L or more, 79 g / L or more, 80 g / L or more, 81 g / L or more, 82 g / L or more, 83 g / L or more, 84 g / L or more, 85 g / L or more, 86 g / L or more, 87 g / L or more, 88 g / L or more, 89 g / L or more, or 90 g / L or more (the upper limit may be selected without any particular limitation from 85 to 1000 g / L, for example, 1000 g / L, 500 g / L, or (It may be 200g / L, but is not limited thereto).

[0080] The culture may include at least one selected from the group consisting of acetic acid, orotic acid, propionic acid, succinic acid, formic acid, uracil acid, and citric acid, and may include acetic acid in an amount of 0 to 1 g / L, 0 to 0.5 g / L, 0 to 0.3 g / L, 0.01 to 1 g / L, 0.01 to 0.5 g / L, or 0.01 to 0.3 g / L, for example, 0.3 g / L, based on the entire culture having a high 3-hydroxypropionic acid (3-HP) content, but is not limited thereto.

[0081] The culture may contain, but is not limited to, a content of orotic acid of 0 to 0.5 g / L, 0 to 0.3 g / L, 0 to 0.2 g / L, 0 to 0.15 g / L, 0.01 to 0.5 g / L, 0.01 to 0.3 g / L, 0.01 to 0.2 g / L, or 0.01 to 0.15 g / L, for example, 0.14 g / L, based on the total culture having a high 3-hydroxypropionic acid (3-HP) content.

[0082] The culture may contain, but is not limited to, a total culture having a high 3-hydroxypropionic acid (3-HP) content, of propionic acid in a content of 0 to 0.5 g / L, 0 to 0.3 g / L, 0 to 0.2 g / L, 0.01 to 0.5 g / L, 0.01 to 0.3 g / L, or 0.01 to 0.2 g / L, for example, 0.19 g / L.

[0083] The culture may contain, but is not limited to, a content of 0 to 1 g / L, 0 to 0.5 g / L, 0 to 0.3 g / L, 0 to 0.25 g / L, 0.01 to 1 g / L, 0.01 to 0.5 g / L, 0.01 to 0.3 g / L, or 0 to 0.3 g / L, for example, 0.2 g / L, of succinic acid based on the total culture having a high 3-hydroxypropionic acid (3-HP) content.

[0084] The culture may contain, based on the total culture having a high 3-hydroxypropionic acid (3-HP) content, a content of formic acid of 0 to 0.5 g / L, 0 to 0.3 g / L, 0 to 0.2 g / L, 0 to 0.15 g / L, 0.01 to 0.5 g / L, 0.01 to 0.3 g / L, 0.01 to 0.2 g / L, or 0.01 to 0.15 g / L, for example, 0.14 g / L, but is not limited thereto.

[0085] The culture may contain uracil acid in a content of 0 to 0.1 g / L, 0 to 0.05 g / L, 0 to 0.03 g / L, 0.01 to 0.1 g / L, 0.01 to 0.05 g / L, or 0.01 to 0.03 g / L, for example, 0.02 g / L, based on the total culture having a high 3-hydroxypropionic acid (3-HP) content, but is not limited thereto.

[0086] The above culture may contain citric acid in a content of 0 to 0.1 g / L, 0 to 0.05 g / L, 0 to 0.02 g / L, or 0 to 0.01 g / L, based on the total culture having a high 3-hydroxypropionic acid (3-HP) content, but is not limited thereto.

[0087] In one specific example, the culture may be obtained by the 3-HP production method and / or the 3-HP productivity improvement method described above, and it may be advantageous to include by-products in the above content range in terms of 3-HP production and productivity improvement.

[0088] In particular, by-products in the above culture may be included in the whole culture having a high 3-HP content produced during 3-HP production, and "orotic acid" may be produced by aerobic microorganisms during 3-HP production as an intermediate in the pyrimidine biosynthetic pathway of microorganisms.

[0089] The culture may include the by-product (e.g., orotic acid), but may be characterized by a low content of the by-product, for example, a culture produced by the method for producing 3-HP and / or the method for improving the productivity of 3-HP provided herein and / or a culture having a high content of the by-product may have a lower content of the by-product (e.g., orotic acid) compared to a culture produced by a method for producing 3-HP in another way and / or a method for improving the productivity of 3-HP and / or a culture containing 3-HP of another composition.

[0090] The above culture can be used for the production of 3-hydroxypropionic acid.

[0091] Accordingly, another example provides a composition for producing 3-hydroxypropionic acid comprising the above culture.

[0092] Another example provides a method for producing 3-hydroxypropionic acid, comprising the step of isolating, recovering, and / or purifying 3-hydroxypropionic acid from the composition for producing 3-hydroxypropionic acid.

[0093] In another aspect, the present invention provides an OD per hour 600 A method for producing a cell having 3-hydroxypropionic acid (3-HP) production ability, comprising a step of producing a cell having 3-hydroxypropionic acid production ability at a concentration of 5 to 23, 5 to 22, 5 to 17, 5 to 16, 5 to 10, 5 to 8, 6 to 23, 6 to 22, 6 to 17, 6 to 16, 6 to 10, 6 to 8, 10 to 23, 10 to 22, 10 to 17, 10 to 16, 14 to 23, 14 to 22, 14 to 17, 14 to 16, 17 to 23, 17 to 22, 20 to 23, or 20 to 22, for example, 7, 15, or 21. A method for high-density cell culture is provided.

[0094] The 3-HP production method and / or productivity improvement method provided by the present invention can produce 3-HP at high concentration and high productivity by applying a method of controlling the cell production rate by controlling the sugar supply rate under high-concentration cell culture conditions and a method of setting conversion conditions in the 3-HP production step. In this way, by producing 3-HP at high concentration and high productivity, it is possible to reduce investment costs compared to the same production volume, and it is expected that the effect of reducing not only the raw material cost but also the separation and purification operating cost can be reduced.

[0095] Figure 1 is a graph showing the results of measuring the production amount of 3-HP during two-stage cultivation under the conditions of Example 1 and Example 2.

[0096] Figures 2a and 2b are graphs showing the results of measuring the production amount (g / L) of 3-HP during two-stage cultivation under the conditions of Example 2-1 and Example 2-2, respectively.

[0097] Figures 3a and 3b are graphs showing the results of measuring the production amount (g / L) of 3-HP according to the temperature of the culture medium during 3-HP production.

[0098] Figures 4a and 4b are graphs showing the results of measuring the production amount (g / L) of 3-HP according to the pH of the culture solution during 3-HP production.

[0099] Figure 5 is a graph showing the results of measuring the production amount (g / L) of 3-HP by adjusting the temperature and pH conditions of the culture solution during 3-HP production to (i) pH 7.25 and 37°C, (ii) pH 7.25 and 35°C, (iii) pH 6.85 and 37°C, and (iv) pH 6.8 and 35°C.

[0100] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended only to illustrate the content of the present invention, and the scope of the present invention is not limited by the following examples.

[0101] Unless otherwise specified in this specification, all temperatures are in degrees Celsius.

[0102]

[0103] Reference Example 1. Production of 3-HP producing strain

[0104] A 3-HP producing strain for use in the manufacturing method according to the present invention was prepared according to the method disclosed in Korean Patent Application No. 10-2021-0151641 and used in the examples below.

[0105]

[0106] Example 1. Production of 3-HP using adaptation conditions and optimal conditions (2-stage culture + OD 600 (Adjust the value)

[0107] Example 1-1. High-density cell culture (stage 1 culture)

[0108] The 3-HP producing strain manufactured in the above Reference Example 1 was subjected to high-density cell culture using a 5 L fermenter (working volume 2 L) using a fed-batch culture method.

[0109] Specifically, MR medium (per 1 L KH2PO4 6.67 g, (NH4)2HPO4 4 g, MgSO4·7H2O 0.8 g, citric acid 0.8 g, and trace metal solution 5 mL; here, the trace metal solution is 5 mL 5 M HCl, 10 g FeSO4·7H2O, 2 g CaCl2, 2.2 g ZnSO4·7H2O, 0.5 g MnSO4·4H2O, 1 g CuSO4·5H2O, 0.1 g (NH4)6Mo7O2·4H2O, and 0.02 g Na2B4O2·10H2O) with 20 g / L glucose was separately added and used as a cell culture medium, and the temperature was maintained at 35 degrees Celsius. The pH was maintained at 6.95 using ammonia water.

[0110] The high-density cell culture above was performed using fed-batch culture, specifically, the pH-stat feeding method or the DO-stat feeding method. In order to maintain the glucose concentration at 0 g / L while the microorganisms consumed all the added sugars in the culture medium, a glucose solution with a concentration of 700 g / L per 1 L of the medium was supplied at an average of 17.5 mL / h when the glucose concentration in the culture medium became 0 g / L. The cell concentration was measured by measuring the optical density (OD) using a UV-Spectrometer, and the OD per hour was measured. 600 The values ​​were set to 6 to 21. OD 24 hours after the start of culture 600 It showed about 150 (dry cell weight 50 g / L).

[0111] After completing the high-concentration cell culture above, the cell culture solution was used as is in the subsequent steps without a separate recovery process.

[0112]

[0113] Example 1-2. 3-HP production using high-density cultured cells (two-stage culture)

[0114] The medium for 3-HP production was prepared by adding 190 g / L of glycerol and 10 μM of vitamin B12 to 10 mM phosphate buffer containing no glucose. The cell culture solution prepared in Example 1-1 was inoculated into the medium for 3-HP production at a cell inoculum amount of 10 g / L (based on dry cell weight), and the 3-HP production step was performed in a 5 L fermenter (working volume 2 L).

[0115] As 3-HP production conditions, DO was maintained at 5%, the stirring speed of the culture medium was performed at 300 rpm so that the DO change value was less than 2, and aeration was supplied at 1 vvm to sequentially control aerobic conditions. Other culture conditions were maintained at 37°C and pH at 7.0 using Ca(OH)2.

[0116] The 3-HP production amount (production concentration) (g / L) produced during the process was measured through HPLC analysis, and the results are shown in Fig. 1 (Example 1).

[0117]

[0118] Example 2. Conditions for controlling air supply to activate the electron transport chain (two-stage cultivation + DO control)

[0119] Example 2-1. High-density cell culture (stage 1 culture)

[0120] High-concentration cell culture was performed in substantially the same manner as in Example 1-1 above, and after completing the high-concentration cell culture, the cell culture solution was used as is in the subsequent step without a separate recovery process.

[0121]

[0122] Example 2-2. 3-HP production using high-density cultured cells (two-stage culture)

[0123] To overcome the production inhibition caused by applying stable conditions rather than optimal conditions for producing high-concentration 3-HP, the following method was used to produce high-concentration 3-HP.

[0124] Specifically, the medium for 3-HP production was prepared by adding 190 g / L of glycerol and 10 μM of vitamin B12 to 10 mM phosphate buffer containing no glucose. The cell culture solution prepared in Example 2-1 was inoculated into the medium for 3-HP production at a cell inoculum amount of 10 g / L (based on dry cell weight), and the 3-HP production step was performed in a 5 L fermenter (working volume 2 L).

[0125] As 3-HP production conditions, DO was maintained at 5%, the stirring speed of the culture solution was performed at 300 rpm so that the DO change value was less than 2, and aeration was supplied at 1 vvm to sequentially control aerobic conditions.

[0126] In addition, the cell stabilization process included a step of applying temperature and pH that considered cell stability rather than the initial production conditions, and the production conditions after applying the adaptation conditions and initial optimal conditions included a 3-HP production step that applied long-term stability conditions.

[0127] Specifically, in order to produce high-concentration 3-HP by maintaining high-concentration production conditions from the beginning, the 3-HP production step was performed by maintaining pH 6.8 and temperature conditions of 35℃ (Example 2-1). In the beginning, the optimal conditions were started, and in order to produce high-concentration 3-HP by switching to high-concentration production conditions from the middle, the temperature conditions of pH 7.0 and 37℃ were maintained for about 5 hours from the start of fermentation, and thereafter, 3-HP was produced by maintaining high-concentration production conditions of pH 6.8 and 35℃ (Example 2-2). The 3-HP production amount (production concentration) (g / L) produced according to each process was measured through HPLC analysis, and the measurement results of each process are shown in Figures 2a and 2b. In addition, based on the measured 3-HP production amount, the 3-HP production yield was measured by the following mathematical formula 1, and the 3-HP productivity was measured by the following mathematical formula 2:

[0128] [Mathematical Formula 1]

[0129] 3-HP production yield (yield, %) = [(final 3-HP (g)) / {(Glycerol (g) before cultivation) - (Glycerol (g) remaining after cultivation)}] * 100

[0130] [Equation 2]

[0131] 3-HP productivity = (final 3-HP production concentration (g / L)) / (total 3-HP production time)

[0132] As a result, both methods of Example 2-1 and Example 2-2 showed a 3-HP production concentration of 131 g / L, a production yield of 95%, and a productivity of 6.55 g / L / h. Since the 3-HP production concentration patterns of the two methods were substantially the same, the results of the two methods were expressed together in Example 2 and shown in Fig. 1, and the composition of the culture including 3-hydroxypropionic acid cultured under pH 6.8 and 35°C temperature conditions was measured through HPLC analysis, and the composition information is shown in Table 1 below.

[0133] Composition concentration (g / L) 3-Hydroxypropionic acid 131.1 Glycerol 0.1 Acetic acid 0.3 Orotic acid 0.14 Propionic acid 0.19 Succinic acid 0.2 Formic acid 0.1 Uracile acid 0.02 Citric acid -

[0134]

[0135] Example 3. Comparison of 3-HP production according to culture temperature during 3-HP production

[0136] While producing 3-HP using the method of Example 1 above, the production amount (g / L) of 3-HP according to the culture temperature during 3-HP production was measured.

[0137] Specifically, after the first stage of culture using the method of Example 1-1, a culture solution containing a strain for producing 3-HP (the strain produced in Reference Example 1) was inoculated under the same conditions as the second stage of culture in Example 1-2, and culture was performed, and the 3-HP production amount was measured using substantially the same method.

[0138] However, the culture temperature in the second stage culture was adjusted to 34, 35, 36 and 37, 38, 38.5 and 39℃, respectively, and the results of measuring 3-HP production are shown in Figures 3a and 3b.

[0139] As can be seen in Fig. 3a, the production rate decreased as the culture temperature decreased below 37°C, but the enzyme stability of the cells was maintained, so all glycerol was consumed and 3-HP was produced at the same yield.

[0140] In addition, as can be confirmed in Fig. 3b, based on the culture temperature of 37℃, the higher the temperature, the better the initial productivity, but the stability of the cells decreased, resulting in a decrease in cell activity and a lower production concentration of 3-HP.

[0141]

[0142] Example 4. Comparison of 3-HP production according to culture pH during 3-HP production

[0143] While producing 3-HP using the method of Example 1 above, the production amount of 3-HP (g / L) according to the pH of the culture solution during 3-HP production was measured.

[0144] Specifically, after the first stage of culture using the method of Example 1-1, a culture solution containing a strain for producing 3-HP (the strain produced in Reference Example 1) was inoculated under the same conditions as the second stage of culture in Example 1-2, and culture was performed, and the 3-HP production amount was measured using substantially the same method.

[0145] However, the culture pH in the second stage culture was adjusted to 6.5, 6.85, 7.0, 7.25, and 7.5, respectively, and the results of measuring 3-HP production are shown in Figures 4a and 4b.

[0146] As can be seen in Fig. 4a, when the culture pH was 7.5, good initial productivity was observed, but due to a decrease in cell activity, cell stability decreased after 10 hours of culture.

[0147] Additionally, as can be confirmed in Fig. 4b, at pH 6.5, cell stability decreased in the later stage of culture and 3-HP production stopped, but at pH 6.85, although the initial productivity was somewhat slower than at 7.0, a constant productivity was observed, indicating cell stability. In other words, it was shown that stable pH conditions were between 6.85 and 7.25.

[0148]

[0149] Example 5. Comparison of 3-HP production according to cell production per hour at high-concentration cell culture stage during 3-HP production

[0150] While culturing high-concentration cells and producing 3-HP using the method of Example 1 above, the production amount (g / L) of 3-HP according to the cell production per hour during high-concentration cell culture was measured.

[0151] In the first stage of culture using the method of the above Example 1-1, the cell production rate per hour is determined by the supply rate of the glucose solution added to the culture medium, and thus, high-concentration cell culture solutions with different cell production rates per hour by varying the glucose supply rate were inoculated under the same conditions as the second stage of culture of the above Example 1-2 and cultured, and the cell concentration per hour (cell production rate) and 3-HP production rate (g / L) were measured using substantially the same method as in Examples 1-1 and 1-2.

[0152] Specifically, the first stage culture was performed by adjusting the glucose supply rate to 5, 10, 20, 30, or 40 mL / L / h, and the pH and temperature conditions in the second stage culture were set to pH 6.8 and 35°C, respectively, and the experiment was conducted in a 145 g / L glycerol medium, and the results of measuring 3-HP production are shown in Table 2.

[0153] Glucose supply rate (mL / L / h)Cell production per hour (OD600 / h)3-HP production concentration (g / L)546210710420151093021101402583

[0154] As a result of measuring 3-HP production, in the first stage of culture, under the condition of glucose supply rate of 20, i.e., cell production per hour is OD 600 In the case of 15, the 3-HP production concentration was the best.

[0155]

[0156] Example 6. Exploration of high-concentration 3-HP production conditions

[0157] In order to produce a higher concentration of 3-HP than the conditions producing the highest concentration of 3-HP under the optimal conditions (temperature, pH, glycerol concentration) explored in Examples 3 and 4 above, the effects of the conditions of 35°C and pH 6.85, which showed the same production although the initial production rate was somewhat slower, on 3-HP production were confirmed, taking cell stability into consideration.

[0158] Specifically, after the first stage of culture using the method of Example 1-1, a culture solution containing a strain for producing 3-HP (the strain produced in Reference Example 1) was inoculated under the same conditions as the second stage of culture in Example 1-2, and culture was performed, and the 3-HP production amount was measured using substantially the same method.

[0159] However, the culture pH and temperature conditions were adjusted to (i) pH 7.25 and 37°C, (ii) pH 7.25 and 35°C, (iii) pH 6.85 and 37°C, and (iv) pH 6.8 and 35°C, respectively, and the experiment was conducted in a 190 g / L glycerol medium for high-concentration 3-HP production, and the results of measuring 3-HP production under each condition are shown in Fig. 5.

[0160] As can be seen in Fig. 5, when comparing the cases where the pH was maintained at 7.25 and only the culture temperature was adjusted to 37°C and 35°C, when the temperature was lowered to 35°C and 3-HP was produced, 125 g / L was produced, the same as when the temperature was 37°C.

[0161] When comparing the cases where the culture temperature was maintained at 37℃ but only the pH was adjusted to 7.25 and 6.85, when the pH was lowered to 6.85, 127.5 g / L of 3-HP was produced, which was higher than the 3-HP production (125 g / L) when the pH was 7.25.

[0162] In addition, when comparing the 3-HP production under different culture temperature and pH conditions, the 3-HP production under the culture temperature of 37°C and pH 7.25 was 125 g / L, while the 3-HP production under the culture temperature of 35°C and pH 6.85, where both temperature and pH were low, was 131 g / L.

Claims

1. A method for producing 3-hydroxypropionic acid (3-HP), comprising the following steps: (1) Cells with 3-hydroxypropionic acid (3-HP) production ability were cultured in a culture medium until the cell concentration reached OD 600 A high-concentration cell culture step in which cell culture is performed until the number reaches 100 to 200; and (2) a step of producing 3-HP by transferring the culture medium of the above step (1) to a production medium containing a substrate while maintaining DO (dissolved oxygen) at 1 to 20%, The above step (2) may be performed under the following conditions: (i) temperature conditions of 30 to 40°C; and / or (ii) pH conditions of 6 to 8.

2. A method according to claim 1, wherein the cell having the 3-HP production ability comprises a gene encoding at least one protein selected from the group consisting of glycerol dehydratase and aldehyde dehydrogenase.

3. A method according to claim 1, wherein the culture medium of step (1) does not contain glycerol as a carbon source.

4. A method according to claim 3, wherein the carbon source is glucose.

5. A method in which, in the first paragraph, a carbon source having a content of 4 to 25 g / L (concentration; weight of carbon source added (g) / volume of medium (L)) per hour based on the volume of culture medium is additionally supplied into the culture medium in step (1).

6. A method in which, in the first paragraph, after the culturing step of step (1), the culture solution itself is transferred to the production medium without going through an additional step including a cell recovery step.

7. A method in which, in the first paragraph, the change value of the dissolved oxygen amount in step (2) is 0 to 5%.

8. A method in which, in the first paragraph, the amount of dissolved oxygen in step (2) is maintained by controlling the stirring speed and the amount of air supplied.

9. In the first paragraph, the step (2) (i) Performed under temperature conditions of 37℃ to 40℃ or pH conditions of 7 to 8, (ii) A method performed under a temperature condition of 30℃ or higher and less than 37℃ or a pH condition of 6 or higher and less than 7.

10. A method for producing 3-hydroxypropionic acid, having a productivity of 3-hydroxypropionic acid of 6 g / L / h or more, according to any one of claims 1 to 9.

11. A culture of 3-hydroxypropionic acid producing cells, characterized in that it is produced by a production method according to any one of claims 1 to 9.

12. A composition for producing 3-hydroxypropionic acid, comprising the culture of item 11. A culture of 3-hydroxypropionic acid producing cells, wherein the culture contains 0.01 to 0.5 g / L of 3-hydroxypropionic acid and orotic acid as a by-product.

14. A culture of 3-hydroxypropionic acid producing cells, wherein the production cell culture in claim 13 further comprises at least one by-product selected from the group consisting of 0.01 to 1 g / L of acetic acid, 0.01 to 0.5 g / L of propionic acid, 0.01 to 1 g / L of succinic acid, 0.01 to 0.5 g / L of formic acid, 0.01 to 0.1 g / L of uracilic acid, and 0.01 to 0.1 g / L of citric acid.

15. OD per hour 600 A method for high-density culturing of cells having 3-hydroxypropionic acid production ability, comprising a step of producing cells having 3-hydroxypropionic acid (3-HP) production ability at a concentration of 5 to 23.

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