Method for producing 3-hydroxypropionic acid

Abstract

The present invention relates to a method for preparing 3-HP and / or improving the ability to produce 3-HP by culturing cells at a high concentration and adjusting the concentration of initial glycerol and the concentration and addition time of additional glycerol while producing and culturing the high-concentration cell culture fluid by fed batch culture, and can produce high-concentration 3HP from high-concentration glycerol.

Description

Technical Field

[0001] Cross-references to related applications

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0015266, filed on January 31, 2024, and the entire contents disclosed in the corresponding Korean patent application are incorporated herein by reference.

[0003] This application relates to methods for preparing 3-hydroxypropionic acid (hereinafter also referred to as "3-HP") at high concentrations and high production capacity and / or methods for improving the production capacity of 3-HP, and more specifically, to methods for preparing 3-HP and / or improving the production capacity of 3-HP, said methods being able to produce high concentrations of 3-HP at high production capacity from high concentrations of glycerol while stably maintaining the activity of cells with 3-HP production capacity. Background Technology

[0004] 3-Hydroxypropionic acid (hereinafter also referred to as "3-HP") is a platform compound that can be converted into a variety of chemicals such as acrylic acid, methyl acrylate, and acrylamide. Since being selected as one of the 12 value-added biochemicals by the U.S. Department of Energy (DOE) in 2004, it has been actively studied in academia and industry.

[0005] The production of 3-HP is mainly achieved through two methods: chemical and biological. However, most of these methods produce petrochemical products. Chemical methods are not environmentally friendly because the initial materials are expensive and toxic substances are generated during the production process. There are also concerns about the depletion of petroleum resources. Therefore, when 3-HP is produced using biomass, the side effects of petrochemical products can be reduced, and it can be used as an alternative to petroleum resources that are on the verge of depletion.

[0006] Therefore, research has been conducted on the biotechnology production of 3-HP, but due to low yield and production capacity, continued research is still needed on strain development and production process development, despite the possibility of producing 3-HP. Summary of the Invention

[0007] Technical issues

[0008] Therefore, the inventors have developed an efficient fermentation technology for the commercialization of 3-HP that can increase the production concentration of 3-HP by achieving complete conversion of the maximum amount of glycerol.

[0009] One embodiment of this application provides a method for preparing 3-hydroxypropionic acid (3-HP) and / or a method for improving the production capacity of 3-HP, comprising: high-concentration cell culture of a strain with the production capacity of 3-hydroxypropionic acid (3-HP) in a culture medium; and production (conversion) of 3-HP by transferring the culture medium of the above steps to a production medium containing a substrate, wherein the production is carried out by a fed-batch process, and wherein the production medium contains glycerol, and additional glycerol is added to the production medium during production.

[0010] Technical solution

[0011] This application provides a method for preparing 3-HP and / or a method for improving the production capacity of 3-HP, comprising: culturing a strain with the production capacity of 3-hydroxypropionic acid (3-HP) at high concentration in a culture medium, and then transferring the culture medium to a production medium containing a substrate to produce (convert) 3-HP, wherein the method converts 3-HP at high concentration and high production capacity under substrate inhibition concentration and maximum production capacity conditions, based on initial conditions having substrate inhibition concentration and maximum production conditions, and additional glycerol supply and supply time conditions for slow consumption of glycerol, by consuming all glycerol via a fed-batch process.

[0012] More specifically, the method,

[0013] Methods relating to the preparation of 3-HP and / or methods for improving the production capacity of 3-HP, comprising the following steps:

[0014] (1) High-concentration cell culture of strains capable of producing 3-hydroxypropionic acid (3-HP) in a culture medium; and

[0015] (2) 3-HP is produced (converted) by transferring the culture medium from step (1) to a production medium containing the substrate.

[0016] Furthermore, the method is characterized in that: the production of 3-HP in step (2) is carried out through a feed-in batching process, and

[0017] The production medium contains glycerol, and additional glycerol is added to the production medium during production.

[0018] The invention will be described in more detail below.

[0019] In step (1) of the method for preparing 3-HP and / or for improving the production capacity of 3-HP provided in this specification, cells with the production capacity of 3-hydroxypropionic acid are cultured at high concentrations in a growth medium. After high-concentration culture, the resulting culture can be used directly in step (2) to produce 3-HP without any separate cell recovery step. Specifically, the culture obtained in step (1) can be transferred to a production medium without undergoing an additional cell recovery step.

[0020] In step (1), the culture medium may not contain glycerol as a carbon source.

[0021] In this specification, 3-hydroxypropionic acid producing cells (hereinafter, they may be used interchangeably with "cells capable of producing 3-hydroxypropionic acid") may be selected from microorganisms that can produce 3-HP from a carbon source (e.g., glycerol) in a production culture medium, such as Escherichia (Escherichia coli). The microorganisms comprised of, but not limited to, microorganisms such as *Escherichia coli*, *Pseudomonas*, *Enterobacteria*, *Brevibacterium*, *Corynebacterium*, *Klebsiella*, *Citrobacter*, *Clostridium*, *Streptomyces*, *Bacillus*, *Lactobacillus*, *Pseudomonas*, *Saccharomyces*, and *Aspergillus*. In one specific embodiment, the 3-hydroxypropionic acid producing cells may be *Escherichia coli*.

[0022] In one embodiment, the 3-hydroxypropionic acid producing cell may contain a gene encoding at least one (e.g., one or both) selected from glycerol dehydratase and aldehyde dehydrogenase. In one embodiment, the 3-HP producing cell may also additionally contain a gene (gdrAB) encoding glycerol dehydratase reactivase (GdrAB). In one embodiment, the 3-HP producing cell may also be a cell additionally capable of biosynthesizing vitamin B12.

[0023] Glyceryl dehydratase can be encoded by the dhaB gene (GenBank accession number U30903.1), but is not limited to it. The dhaB gene can be an enzyme derived from Klebsiella pneumoniae, but is not limited to it. Genes encoding glyceryl dehydratase can include genes encoding dhaB1, dhaB2, and / or dhaB3. The glyceryl dehydratase protein and the gene encoding it can contain mutations in the gene and / or amino acid sequence within the range that maintain the enzymatic activity of breaking down glycerol into 3-hydroxypropionaldehyde (3-HPA) and water (H2O).

[0024] The gene (aldH) encoding aldehyde dehydrogenase (ALDH) can be, for example, the aldH gene (GenBank accession number U00096.3; EaldH) derived from *Escherichia coli* or the *Escherichia coli* K12MG1655 cell line, the puuC gene derived from *Klebsiella pneumoniae*, and / or the KGSADH gene derived from *Azospirillum brasilense*, but is not limited thereto. The aldehyde dehydrogenase protein and the gene encoding it can include mutations in the gene and / or amino acid sequence that maintain the activity of producing 3-HP from 3-HPA.

[0025] 3-HP production cells may contain genes encoding at least one, at least two, or all three proteins selected from glycerol dehydratase, aldehyde dehydrogenase, and glycerol dehydratase reactivator, or recombinant vectors containing said genes.

[0026] Recombinant vectors can be used to express genes encoding at least one, at least two, or all three proteins selected from glycerol dehydratase, aldehyde dehydrogenase, and glycerol dehydratase reactivator in cells by means known in the art, by replacing the promoter and regulatory sites within the target range.

[0027] High-concentration culture can be performed without limitation using methods known in the art to ensure large-scale production of 3-HP cells, and in one embodiment, the culture can be performed by fed-batch culture.

[0028] In one embodiment, fed-batch culture can be performed using the pH-stat method, the DO-stat method, the continuous feeding method, or a combination thereof. In one embodiment, when performing fed-batch culture using the pH-stat method, glucose can be added at a concentration of 1 g / L to 5 g / L, but is not limited thereto. In one embodiment, when performing fed-batch culture using a continuous feeding method, glucose can be added at a rate of 7 g / L / hour to 21 g / L / hour, but is not limited thereto.

[0029] According to one embodiment, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphate, and sulfate to the microbial culture medium in an appropriate manner during cultivation. In one embodiment, during high-concentration cultivation, the pH can be maintained at 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, but is not limited thereto.

[0030] In one embodiment, during high-concentration culture, the carbon source can be used without limitation by selecting monosaccharides, disaccharides, and / or polysaccharides within the range intended for the purpose of high-concentration culture. For example, the carbon source can be at least one, two or more, three or more, four or more, five or more, ten or more, or all eleven of glucose, fructose, galactose, mannose, arabinose, xylose, ribose, sucrose, maltose, lactose, and cellobiose. The culture medium used during high-concentration culture may not contain glycerol as a carbon source. In this way, since the culture medium used during high-concentration culture does not contain glycerol, 3-HP production does not occur during the high-concentration culture step.

[0031] In one implementation, the cell concentration after high-concentration culture is based on OD. 600 The value can be 10 or greater, 30 or greater, 50 or greater, 70 or greater, 100 or greater, or 110 or greater, and for example, when cultured for 20 hours, OD 600 Values ​​can be 10 to 500, 10 to 400, 10 to 300, 10 to 250, 10 to 200, 10 to 150, 30 to 500, 30 to 400, 30 to 300, 30 to 250, 30 to 200, 30 to 150, 50 to 500, 50 to 400, 50 to 300, 50 to 250, 50 to 200, 50 to 150, 70 to 500. 70 to 400, 70 to 300, 70 to 250, 70 to 200, 70 to 150, 100 to 500, 100 to 400, 100 to 300, 100 to 250, 100 to 200, 100 to 150, 110 to 500, 110 to 400, 110 to 300, 110 to 250, 110 to 200, or 110 to 150, but not limited to these.

[0032] In one implementation, based on the cell dry weight (g) per 1 L of culture medium, the cell concentration after high-concentration culture can be 10 g / L to 100 g / L, 10 g / L to 80 g / L, 10 g / L to 70 g / L, 10 g / L to 60 g / L, 10 g / L to 55 g / L, 20 g / L to 100 g / L, 20 g / L to 80 g / L, 20 g / L to 70 g / L, 20 g / L to 60 g / L, 20 g / L to 55 g / L, 30 g / L to 100 g / L, 30 g / L to 80 g / L, 30 g / L to 70 g / L, 30 g / L to 60 g / L, 30 g / L to 55 g / L, 40 g / L to 100 g / L, 40 g / L to 80 g / L, 40 g / L to 7 ...100 g / L, 40 g / L to 80 g / L, 40 g / L to 70 g / L, 40 g / L to 100 g / L, 40 g / L to 80 g / L, 40 g / L to 70 g / L, 40 g / L to 100 g / L, 40 g / L to g / L to 60 g / L, 40 g / L to 55 g / L, 45 g / L to 100 g / L, 45 g / L to 80 g / L, 45 g / L to 70 g / L, 45 g / L to 60 g / L, or 45 g / L to 55 g / L, such as 50 g / L, but not limited to these.

[0033] According to one embodiment, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphate, and sulfate to the microbial culture medium in an appropriate manner during cultivation. In one embodiment, during high-concentration cultivation, the pH can be maintained at 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, but is not limited thereto.

[0034] The temperature of the culture medium can be 20°C to 45°C, 25°C to 40°C, or 30°C to 37°C, and for example, it can be 37°C.

[0035] In this invention, for high-concentration cell culture, 3-HP can produce cells at 20 or less, or 2 or more per hour OD. 600 It is generated at a concentration of optical density (OD) measured at 600 nm.

[0036] In one implementation, the cell concentration after high-concentration culture can be 10 or greater, 50 or greater, 100 or greater, 150 or greater, 200 or greater, 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 OD. 600 However, this is not the only one. Specifically, cell concentration is expressed in terms of OD... 600 The count can be between 100 and 200.

[0037] According to one embodiment, in addition to a carbon source, the culture medium may contain a nitrogen source and trace element components. Available nitrogen sources may include peptone, yeast extract, meat broth, malt extract, corn steep liquor, soybean flour, and urea; or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources may be used alone or as a mixture, but are not limited to this. Available phosphorus sources may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or their corresponding sodium-containing salts, but are not limited to this. Furthermore, the culture medium may contain metal salts required for growth, such as magnesium sulfate or ferric sulfate, but are not limited to this. In addition, essential growth substances such as amino acids and vitamins may be included. Furthermore, precursors suitable for the culture medium may be used. The culture medium or individual components may be added to the culture medium during the culture process using appropriate methods, either in a fed-batch or continuous manner, but are not limited to this.

[0038] Step (2) is a step of producing (converting) 3-HP by transferring the culture medium of step (1) to a production medium containing the substrate, and is a step carried out by a fed-batch process, wherein the production medium then contains glycerol, and additional glycerol is added to the production medium by the fed-batch culture process.

[0039] The substrate can be glucose and / or glycerol, but is not limited to these.

[0040] Specifically, step (2) is a step of producing 3-HP by seeding cells with the ability to produce 3-hydroxypropionic acid (3-HP) into a 3-hydroxypropionic acid production medium and transferring them to a production medium, and producing 3-HP by a fed-batch process under conditions of initial glycerol concentration and additional glycerol addition time and rate.

[0041] 3-hydroxypropionic acid production medium can be used without restriction for the purpose of enabling cells to produce 3-HP without causing proliferation (cell division, growth, or growth and development) of 3-HP producing cells.

[0042] In one embodiment, the carbon source of the production medium may be glycerol, but is not limited thereto. In one embodiment, the production medium may also contain vitamin B12. In one embodiment, the production medium may not contain glucose during cell inoculation.

[0043] In this invention, the problem of production inhibition caused by high concentration of glycerol is overcome by distributing glycerol and adding glycerol as a carbon source, and by consuming the maximum amount of glycerol to convert high concentration 3-HP.

[0044] Therefore, the present invention is characterized by controlling the glycerol concentration (initial glycerol concentration) in the production culture medium at the start of the culture in step (2). Specifically, the initial glycerol concentration can be 100 g / L to 150 g / L, 100 g / L to 145 g / L, 100 g / L to 140 g / L, 100 g / L to 135 g / L, 100 g / L to 130 g / L, 100 g / L to 125 g / L, 100 g / L to 120 g / L, 110 g / L to 150 g / L, 110 g / L to 145 g / L, 110 g / L to 140 g / L, 110 g / L to 135 g / L, 110 g / L to 130 g / L, 110 g / L to 125 g / L, 110 g / L to 120 g / L, 115 g / L to 150 g / L, 115 g / L to 145 g / L, 115 g / L to 140 g / L. 115 g / L to 135 g / L, 115 g / L to 130 g / L, 115 g / L to 125 g / L, 115 g / L to 120 g / L, 120 g / L to 150 g / L, 120 g / L to 145 g / L, 120 g / L to 140 g / L, 120 g / L to 135 g / L, 120 g / L to 130 g / L, 125 g / L to 150 g / L, 125 g / L to 145 g / L, 125 g / L to 135 g / L, 125 g / L to 130 g / L, 130 g / L to 150 g / L, 130 g / L to 145 g / L, 130 g / L to 145 g / L, 130 g / L to 145 g / L, 130 g / L to 145 g / L, 130 g / L to 135 g / L, 125 g / L to 130 ... The concentrations of glycerol are preferably within the ranges of 120 g / L, 125 g / L, 135 g / L to 140 g / L, 130 g / L to 135 g / L, 135 g / L to 145 g / L, 140 g / L to 150 g / L, 140 g / L to 145 g / L, or 145 g / L to 150 g / L, for example, 120 g / L, 125 g / L, 135 g / L, or 145 g / L. This is preferred when the initial glycerol concentration is within the above ranges because 3-HP is produced at a high concentration and there is no residual glycerol.

[0045] Furthermore, in step (2), the characteristic is the addition of additional glycerol to the production medium during 3-HP production. In one embodiment, the concentration of glycerol additionally added to the production medium, based on the total volume of the production medium, can be the following amounts: 70 g / L to 140 g / L, 70 g / L to 120 g / L, 70 g / L to 110 g / L, 80 g / L to 140 g / L, 80 g / L to 120 g / L, 80 g / L to 110 g / L, 90 g / L to 140 g / L, 90 g / L to 120 g / L, or 90 g / L to 110 g / L, for example 100 g / L, but not limited thereto.

[0046] In one embodiment, the additional supply of glycerol may be added at a time when the strain capable of producing 3-HP can produce 3-HP under optimal conditions after the start of production, and for example, it may be added at a uniform rate after 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 5 hours, 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 2 to 5 hours, 3 to 10 hours, 3 to 8 hours, 3 to 6 hours, or 3 to 5 hours, for example, 4 hours, but is not limited thereto.

[0047] The additional glycerin can be supplied for 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 3 to 10 hours, 3 to 8 hours, 3 to 6 hours, 5 to 10 hours, 5 to 8 hours, or 5 to 6 hours, but is not limited to these.

[0048] In one embodiment, the medium may be a synthetic medium or a semi-synthetic medium, but is not limited thereto.

[0049] After high-concentration culture of cells with 3-hydroxypropionic acid (3-HP) production capacity in step (1), cells for inoculation can be prepared in the form of cell culture medium without a separate cell recovery process, but is not limited thereto.

[0050] The seed concentration (cell concentration during seeding) of high-concentration cultured cells can be appropriately adjusted or determined by those skilled in the art within the range required for the production of 3-HP. In one embodiment, the seed concentration (based on dry cell weight (DCW) / culture medium volume (L)) can be 1 g / L to 20 g / L, 1 g / L to 16 g / L, 1 g / L to 12 g / L, 1 g / L to 9 g / L, 2 g / L to 20 g / L, 2 g / L to 16 g / L, 2 g / L to 12 g / L, 2 g / L to 9 g / L, 4 g / L to 20 g / L, 4 g / L to 16 g / L, 4 g / L to 12 g / L, or 4 g / L to 9 g / L, but is not limited thereto.

[0051] In the production of 3-HP, proliferation of the seeded cells may not occur. In one embodiment, 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 seeded cells, for example, 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%, but is not limited thereto.

[0052] In one embodiment, DO (dissolved oxygen) can be adjusted by selecting at least one of stirring rate control, air supply control, and pressure control during cultivation. For example, to increase dissolved oxygen in the culture medium or culture, (1) at least one of stirring rate, air supply, and pressure can be increased, and (2) to decrease dissolved oxygen, at least one of stirring rate, air supply, and pressure can be decreased to maintain dissolved oxygen in the culture medium or culture within a certain range, but is not limited thereto.

[0053] According to one embodiment, in order to maintain the pH constant within the above range, a compound selected from calcium hydroxide, magnesium hydroxide, ammonium hydroxide, sodium hydroxide, and potassium hydroxide can be added to the microbial culture medium in an appropriate manner during cultivation. Furthermore, according to one embodiment, conventional temperature control methods can be used to maintain the temperature of the culture medium.

[0054] The incubation period can continue until a useful substance (e.g., 3-HP) is obtained at the desired yield, and for example, it can be 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, and for example, it can be 24 hours, but is not limited thereto.

[0055] The 3-HP yield provided in this specification for the methods of preparing 3-HP and / or for improving the production capacity of 3-HP can be, for example, 80% or greater, 85% or greater, 90% or greater, 93% or greater, or 95% or greater, but is not limited thereto. The 3-HP yield can be calculated as the amount of 3-HP produced in the culture medium (culture) in the step of producing 3-HP relative to the amount (moles) of glycerol used in the culture medium (production medium), and in one embodiment, it can be calculated using the following Equation 1.

[0056] [Equation 1]

[0057]

[0058] The 3-HP production capacity of the method for preparing 3-HP and / or the method for improving the production capacity of 3-HP provided in this invention can be 6.0 g / L / hour or greater, 6.5 g / L / hour or greater, 6.6 g / L / hour or greater, 6.0 g / L / hour to 60 g / L / hour, 6.0 g / L / hour to 60 g / L / hour, 6.0 g / L / hour to 20 g / L / hour, 6.0 g / L / hour to 10 g / L / hour, 6.5 g / L / hour to 60 g / L / hour, 6.5 g / L / hour to 40 g / L / hour, 6.5 g / L / hour to 20 g / L / hour, 6.6 g / L / hour to 60 g / L / hour, 6.6 g / L / hour to 40 g / L / hour, 6.6 g / L / hour to 2 ... g / L / hour, or 6.6 g / L / hour to 10 g / L / hour, such as 6.68 g / L / hour, but not limited thereto. The 3-HP production capacity can be calculated by dividing the final 3-HP production concentration in the 3-HP production step by the total 3-HP production time, and in one embodiment, it can be calculated by the following Equation 2.

[0059] [Equation 2]

[0060]

[0061] Based on the 3-HP content (g) per 1 L of culture medium during 20 to 30 hours of cultivation (3-HP production) (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours), the 3-HP production rate of the methods for preparing 3-HP and / or for improving the production capacity of 3-HP provided in this specification can be, for example, 100 g / L or greater, 110 g / L or greater, 120 g / L or greater, 130 g / L or greater, 140 g / L or greater, 145 g / L or greater, 100 g / L to 1000 g / L, 110 g / L to 1000 g / L, 120 g / L to 1000 g / L, 130 g / L to 1000 g / L, 140 g / L to 1000 g / L, or 145 g / L. g / L to 1000 g / L (the upper limit can be selected from 130 g / L to 1000 g / L without particular limitation, and for example, it can be 1000 g / L, 500 g / L, 400 g / L, 300 g / L, 200 g / L, 160 g / L or 147 g / L, but is not limited thereto).

[0062] The methods for preparing 3-HP and / or for improving the production capacity of 3-HP provided in this specification can consume most of the glycerol added for 3-HP production. The remaining glycerol after production can be generated in the total culture medium at a concentration of 2 g / L or less, 1.8 g / L or less, 1.5 g / L or less, 1.3 g / L or less, or 1.0 g / L or less (then the lower limit of the byproduct concentration can be selected from 0 g / L to 0.000001 g / L, but is not limited thereto), or it can be generated at a concentration of 0 g / L (byproducts are not generated at a detectable concentration).

[0063] The methods for preparing 3-HP and / or for improving the production capacity of 3-HP provided in this specification can produce byproducts resulting from the production of 3-HP in low quantities. These byproducts may be at least one selected from acetic acid, orotic acid, propionic acid, succinic acid, formic acid, uracil acid, and citric acid.

[0064] The high-concentration and high-yield 3-HP preparation method provided in this invention can exhibit a 3-HP production capacity (g / L / hour) that is 1.05 times or higher or 1.1 times or higher than that of batch fermentation methods, and for example, it can be 1.05 times to 10 times, 1.05 times to 5 times, 1.05 times to 2 times, 1.05 times to 1.7 times, 1.05 times to 1.5 times, 1.05 times to 1.2 times, 1.1 times to 10 times, 1.1 times to 5 times, 1.1 times to 2 times, 1.1 times to 1.7 times, 1.1 times to 1.5 times, or 1.1 times to 1.3 times higher, and for example, it can be about 1.13 times higher.

[0065] Other implementations provide cultures of 3-hydroxypropionic acid-producing cells with high 3-hydroxypropionic acid content and low byproduct content.

[0066] Based on the total culture, the culture may contain 100 g / L or greater, 110 g / L or greater, 120 g / L or greater, 130 g / L or greater, 140 g / L or greater, 145 g / L or greater, 100 g / L to 1000 g / L, 110 g / L to 1000 g / L, 120 g / L to 1000 g / L, 130 g / L to 1000 g / L, 140 g / L to 1000 g / L, or 145 g / L to 1000 g / L (the upper limit may be selected from 130 g / L to 1000 g / L without particular limitation, and for example, it may be 1000 g / L, 500 g / L, 400 g / L, 300 g / L, 200 g / L, 160 g / L or 147 g / L, but is not limited thereto). As one specific implementation, the culture may contain byproducts and 3-hydroxypropionic acid at a concentration of 100 g / L or greater.

[0067] In addition, based on the total culture volume, the culture may contain acetic acid in the following amounts: 0 g / L to 1 g / L, 0 g / L to 0.5 g / L, 0 g / L to 0.3 g / L, 0.01 g / L to 1 g / L, 0.01 g / L to 0.5 g / L, or 0.01 g / L to 0.3 g / L, for example, 0.3 g / L, but not limited thereto.

[0068] Based on the total culture volume, the culture may contain orotic acid in the following amounts: 0 g / L to 0.5 g / L, 0 g / L to 0.3 g / L, 0 g / L to 0.2 g / L, 0 g / L to 0.15 g / L, 0.01 g / L to 0.5 g / L, 0.01 g / L to 0.3 g / L, 0.01 g / L to 0.2 g / L, or 0.01 g / L to 0.15 g / L, for example, 0.14 g / L, but not limited thereto.

[0069] Based on the total culture volume, the culture may contain propionic acid in the following amounts: 0 g / L to 0.5 g / L, 0 g / L to 0.3 g / L, 0 g / L to 0.2 g / L, 0.01 g / L to 0.5 g / L, 0.01 g / L to 0.3 g / L, or 0.01 g / L to 0.2 g / L, for example, 0.19 g / L, but not limited thereto.

[0070] Based on the total culture volume, the culture may contain succinic acid in the following concentrations: 0 g / L to 1 g / L, 0 g / L to 0.5 g / L, 0 g / L to 0.3 g / L, 0 g / L to 0.25 g / L, 0.01 g / L to 1 g / L, 0.01 g / L to 0.5 g / L, 0.01 g / L to 0.3 g / L, or 0 g / L to 0.3 g / L, for example, 0.2 g / L, but not limited thereto.

[0071] Based on the total culture volume, the culture may contain formic acid in the following amounts: 0 g / L to 0.5 g / L, 0 g / L to 0.3 g / L, 0 g / L to 0.2 g / L, 0 g / L to 0.15 g / L, 0.01 g / L to 0.5 g / L, 0.01 g / L to 0.3 g / L, 0.01 g / L to 0.2 g / L, or 0.01 g / L to 0.15 g / L, for example, 0.14 g / L, but not limited thereto.

[0072] Based on the total culture volume, the culture may contain uracil acid in the following concentrations: 0 g / L to 0.1 g / L, 0 g / L to 0.05 g / L, 0 g / L to 0.03 g / L, 0.01 g / L to 0.1 g / L, 0.01 g / L to 0.05 g / L, or 0.01 g / L to 0.03 g / L, for example, 0.02 g / L, but not limited thereto.

[0073] Based on the total culture volume, the culture may contain citric acid in the following amounts: 0 g / L to 0.1 g / L, 0 g / L to 0.05 g / L, 0 g / L to 0.02 g / L, or 0 g / L to 0.01 g / L, but is not limited thereto.

[0074] In one specific embodiment, the culture can be obtained by the methods described above for preparing 3-HP and / or for improving the production capacity of 3-HP, and byproducts containing the above-described content range may be advantageous in terms of 3-HP preparation and production capacity improvement.

[0075] Specifically, in the culture, byproducts can be included in the total culture with high 3-HP content produced during 3-HP production, and "orotic acid" is an intermediate in the pyrimidine biosynthesis pathway of microorganisms and can be produced when 3-HP is produced by aerobic microorganisms.

[0076] The culture may contain byproducts (e.g., orotic acid), but it may be characterized by low byproduct content, and for example, a culture produced by the methods for preparing 3-HP and / or for improving the production capacity of 3-HP provided in this specification and / or a culture with high 3-HP content may have a lower content of byproducts (e.g., orotic acid) compared to a culture produced by methods for preparing 3-HP and / or for improving the production capacity of 3-HP in different ways and / or a culture containing 3-HP with different compositions.

[0077] As one specific implementation, the culture may contain the above-mentioned amounts of byproducts and 100 g / L or less, 200 g / L or less, or 300 g / L or less of 3-hydroxypropionic acid.

[0078] In one specific implementation, the culture can be obtained by the aforementioned methods for preparing 3-HP and / or by methods for improving the production capacity of 3-HP, but is not limited thereto.

[0079] In one specific implementation, the culture can be obtained by the aforementioned methods for preparing 3-HP and / or by methods for improving the production capacity of 3-HP, but is not limited thereto.

[0080] The culture can be used in the production of 3-hydroxypropionic acid.

[0081] Therefore, other embodiments provide compositions comprising the culture for the production of 3-hydroxypropionic acid.

[0082] Other embodiments provide methods for producing 3-hydroxypropionic acid, including separating, recovering, and / or purifying 3-hydroxypropionic acid from compositions used for producing 3-hydroxypropionic acid.

[0083] Beneficial effects

[0084] The method for producing 3-HP and / or the method for improving the production capacity of 3-HP provided in this invention can produce 3-HP at high concentration and high capacity by applying methods for setting the feed batching process and the initial concentration and time of glycerol, as well as the concentration and time of additional additions, and the rate conditions during 3-HP production. By producing 3-HP in this high-concentration and high-capacity manner, the investment cost for the same production volume can be reduced. In particular, not only the cost of raw materials can be reduced, but also the operating costs for separation and purification can be reduced, thereby reducing the overall production cost. Attached Figure Description

[0085] Figure 1 A schematic diagram illustrating the technical features of the present invention compared to the prior art.

[0086] Figure 2 The graph shows the results of measuring the production of 3-HP during the two-step culture under the conditions of the control group (Comparative Example 1) and Example 1.

[0087] Figure 3 A graph showing the results of measuring the production yield (g / L) of 3-HP during 3-HP production, depending on the initial glycerol concentration.

[0088] Figure 4 A graph showing the results of measuring the production yield (g / L) of 3-HP during production, depending on the concentration of initial glycerol and additionally added glycerol. Detailed Implementation

[0089] The invention will be described in more detail below through embodiments. However, the following embodiments are intended to illustrate the content of the invention only, and the scope of the invention is not limited to the following embodiments.

[0090] In this invention, unless otherwise stated, all temperatures are based on Celsius, and unless otherwise specified, nucleic acid sequences are based on... -to- Directional writing.

[0091] Refer to Example 1. Preparation of 3-HP producing strain

[0092] The 3-HP production strain for use in the preparation 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 following comparative examples and embodiments.

[0093] Comparative Example 1. Preparation of 3-HP via fed-batch culture during a two-step cell culture process for 3-HP production.

[0094] Comparative Example 1-1. High-concentration cell culture (first step culture)

[0095] The 3-HP production strain prepared in Example 1 was cultured at high concentrations in a 5 L fermenter (working volume 2 L) using a fed-batch culture method.

[0096] Specifically, 20 g / L glucose was added separately to MR medium (6.67 g KH2PO4 and 4 g (NH4)2HPO4 per L). 0.8 g of citric acid and 5 mL of trace metal solution; in this paper, the trace metal solution is 5 mL of 5 M HCl per 1 L. 10 g, CaCl2 2 g 2.2 g 0.5 g 1 g 0.1 g and 0.02 g) was added to the solution and used as a cell culture medium, and the temperature was maintained at 35°C. The pH was maintained at 6.95 using ammonia.

[0097] High-concentration cell culture was performed using fed-batch culture (specifically, pH-stat or DO-stat feeding). When the glucose concentration in the culture medium reached 0 g / L, a glucose solution with a concentration of 700 g / L was supplied at an average rate of 17.5 mL / hour per 1 L of culture medium. This ensured that all added sugars were consumed by the microorganisms and the glucose concentration was maintained at 0 g / L. The optical density (OD) was measured using a UV spectrometer to determine the cell concentration, and the OD was recorded. 600 The value remained within the range of 6 to 20. At 24 hours after the start of incubation, OD... 600 It reached approximately 150 (cell dry weight 50 g / L).

[0098] After high-concentration cell culture is completed, the cell culture medium is used as is in subsequent steps without the need for a separate recycling process.

[0099] Comparative Examples 1-2. Production using 3-HP with high concentrations of cultured cells (second-step culture)

[0100] A culture medium for the production of 3-HP was prepared by adding 120 g / L glycerol and 10 μM vitamin B12 to a 10 mM phosphate buffer that does not contain glucose. The cell culture medium prepared in Comparative Example 1-1 was inoculated into the 3-HP production medium at a cell inoculation density of 10 g / L (based on cell dry weight), and the 3-HP production step was carried out in a 5 L fermenter (working volume 2 L).

[0101] As part of the 3-HP production conditions, to maintain a DO of 5%, the stirring speed of the culture medium was set to 300 rpm, and aeration was sequentially adjusted at a supply of 1 vvm to create aerobic conditions. Other culture conditions were maintained at a temperature of 35°C and a pH of 6.8 using Ca(OH)2 (comparative example).

[0102] Example 1. Preparation of 3-HP via a fed-batch process and control of initial and additionally added glycerol during two-step cell culture for 3-HP production.

[0103] By applying a fed-batch process for producing 3-HP using a two-step preparation method similar to that of Comparative Example 1, which involves producing a high-concentration cell culture medium and then inoculating it into a production medium, but by allocating the amount of glycerol added to overcome the inhibition of production due to the high concentration of glycerol and consuming the maximum amount of glycerol to convert to high-concentration 3-HP, an increase in the production of 3-HP was determined.

[0104] Specifically, a culture medium for producing 3-HP is prepared such that the initial glycerol concentration can be 120 g / L, and the 3-HP production step is carried out under the following conditions: 4 hours after the start of culture, glycerol corresponding to 100 g / L is additionally supplied to the culture medium for producing 3-HP in a constant amount for 5 to 6 hours (Example).

[0105] The concentration of 3-HP produced according to the methods used in the comparative examples and embodiments above was determined by HPLC analysis, and the results are shown in... Figure 2 middle.

[0106] like Figure 2 As shown, when 3-HP is produced by the fed-batch process according to the present invention (Example), a high concentration of 3-HP is produced at 147 g / L, compared to 130 g / L (Comparative Example) in the case where 3-HP is produced by a batch process. This is achieved by overcoming the inhibition of initial 3-HP production that depends on glycerol concentration in order to maintain cell viability at its maximum. Furthermore, it is produced at a capacity of 6.68 g / L / hour, higher than the Comparative Example's 6.55 g / L / hour, based on the corresponding production volume as measured by Equation 2 below. Most of the added glycerol is consumed (the remaining glycerol concentration is 1 g / L or lower), thereby minimizing the separation and purification load.

[0107] [Equation 2]

[0108]

[0109] Example 2. Comparison of 3-HP production rates depending on initial glycerol concentration during 3-HP production.

[0110] While producing 3-HP using the method of Example 1, the production amount (g / L) of 3-HP during the production process, depending on the initial glycerol concentration, was measured.

[0111] Specifically, high-concentration cell culture and 3-HP production culture were performed using the method of Example 1, but the initial glycerol concentration in the two-step 3-HP production medium was adjusted to 125 g / L, 135 g / L, 145 g / L, 160 g / L, 180 g / L, and 190 g / L, respectively, and the results of 3-HP production by the corresponding methods are shown in [the table below]. Figure 3 middle.

[0112] like Figure 3 As shown, based on an initial glycerol concentration of 125 g / L in the culture medium, the production capacity of 3-HP was inhibited as the glycerol concentration increased.

[0113] At a glycerol concentration of 190 g / L above 180 g / L, not all glycerol was consumed, and 3-HP production remained at the level observed at the initial glycerol concentration of 120 g / L.

[0114] In addition, slight differences were observed at glycerol levels of 125 g / L, 135 g / L, and 145 g / L, but all showed similar levels of 3-HP production capacity.

[0115] Example 3. Exploration of optimal fed-batch culture conditions for 3-HP production

[0116] Since similar 3-HP production capacity was observed in Example 2 when the initial glycerol concentrations were 125 g / L, 135 g / L, and 145 g / L, the following experiments were conducted to determine the 3-HP production effect depending on the additional glycerol concentration supplied, based on initial glycerol concentration conditions below or above the stated concentration range and the feed batching process.

[0117] Specifically, 3-HP was produced according to Example 1 above, but under (i) initial low glycerol concentration conditions (glycerol concentration: 70 g / L) and subsequently under conditions where 80 g / L of glycerol was added 6 hours after the start of the production culture, and (ii) initial high glycerol concentration conditions (glycerol concentration: 160 g / L) and subsequently under conditions where 60 g / L of glycerol was added 4 hours after the start of the production culture, 3-HP was produced via a fed-batch process, and the results are shown in... Figure 4 middle.

[0118] like Figure 4As shown, the production concentration of 3-HP was determined to be limited to 90 g / L, and therefore it was no longer produced under initial low glycerol conditions and additional glycerol supply conditions.

[0119] Furthermore, it was determined that 140 g / L of 3-HP, which was higher than 120 g / L (the production concentration under batch conditions (comparative example), was produced under initial high-concentration glycerol conditions and additional supply conditions, but it was determined that approximately 16 g / L of glycerol was not converted.

[0120] Based on these results, it can be seen that when the initial glycerol concentration is 120 g / L as in Example 1, and an additional 100 g / L of glycerol is supplied during the production culture, 147 g / L of 3-HP can be optimally produced because all the glycerol is consumed.

Claims

1. A method for producing 3-hydroxypropionic acid, comprising the following steps: (1) High-concentration cell culture of strains capable of producing 3-hydroxypropionic acid (3-HP) in a culture medium; and (2) 3-HP is produced by transferring the culture medium from step (1) to a production medium containing the substrate. The production of 3-HP in step (2) is carried out through a feed-in batching process, and The production medium contains glycerol, and additional glycerol is added to the production medium during the production of 3-HP.

2. The method of claim 1, wherein the cell having 3-HP production capacity contains a gene encoding at least one protein selected from glycerol dehydratase and aldehyde dehydrogenase.

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

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

5. The method according to claim 1, wherein after the culture in step (1), the culture medium itself is transferred to the production medium without undergoing additional steps including cell recovery.

6. The method according to claim 1, wherein at the start of the culture in step (2), the concentration of glycerol in the production culture medium is 100 g / L to 150 g / L.

7. The method of claim 1, wherein the additional glycerol is added in an amount corresponding to 70 g / L to 140 g / L, based on the total volume of the production culture medium.

8. The method of claim 1, wherein the addition of the additional glycerol is carried out for 5 to 6 hours.

9. The method according to claim 1, wherein the 3-HP production capacity of the method is 6 g / L / hour or greater.

10. The method of claim 1, wherein a production rate of 100 g / L to 1000 g / L of 3-HP is observed during a culture period of 20 to 30 hours.

11. The method of claim 1, wherein the concentration of residual glycerol in the total culture medium after production is 2 g / L or lower.

12. A cell culture for producing 3-hydroxypropionic acid, characterized in that... Prepared by the method for production according to any one of claims 1 to 11.

13. A composition for preparing 3-hydroxypropionic acid, comprising the culture according to claim 12.

14. The cell culture for producing 3-hydroxypropionic acid according to claim 12, wherein the culture comprises 3-hydroxypropionic acid and orotic acid at a concentration of 0.01 g / L to 0.5 g / L as a byproduct.

15. The cell culture for producing 3-hydroxypropionic acid according to claim 14, wherein the cell culture comprises at least one byproduct selected from 0.01 g / L to 1 g / L acetic acid, 0.01 g / L to 0.5 g / L propionic acid, 0.01 g / L to 1 g / L succinic acid, 0.01 g / L to 0.5 g / L formic acid, 0.01 g / L to 0.1 g / L uracil acid, and 0.01 g / L to 0.1 g / L citric acid.

16. A cell culture for producing 3-hydroxypropionic acid according to claim 14 or claim 15, wherein the content of 3-hydroxypropionic acid in the culture is 100 g / L or greater.

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