Two-step process for producing 3-hydroxypropionic acid

Abstract

To provide a method for producing 3-hydroxypropionic acid (3-HP) and / or a method for improving 3-HP productivity, more specifically, a two-step process for producing 3-HP comprising a first step for culturing cells to a high density and a second step for producing 3-HP using the cells cultured to a high density as a catalyst.SOLUTION: The invention relates to a method for producing 3-hydroxypropionic acid (3-HP) comprising the steps of: (1) conducting high density culture of a 3-HP-producing strain; and (2) isolating the cells cultured to a high density to inoculate a 3-hydroxypropionic acid production medium to produce 3-HP, and / or a method for improving the productivity of 3-HP. This enables improved productivity and yield of 3-HP.SELECTED DRAWING: None

Description

[Technical field]

[0001] Cross-reference to related application(s) This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0096238 filed on July 31, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a method for producing 3-hydroxypropionic acid (3-HP) and / or a method for improving the productivity of 3-HP, and more specifically, to a two-step production method comprising a first step of culturing cells to a high density and a second step of producing 3-HP using the highly concentrated cultured cells as a catalyst. [Background technology]

[0003] 3-hydroxypropionic acid is a platform compound that can be converted into various chemicals such as acrylic acid, methyl acrylate, and acrylamide. Since being selected as one of the Top 12 value-added bio-chemicals by the US Department of Energy (DOE) in 2004, it has been actively researched in academia and industry.

[0004] 3-HP can be produced mainly through two methods: chemical and biological. However, it has been pointed out that the chemical method is not environmentally friendly due to the high cost of the initial materials and the production of toxic substances during the production process, so environmentally friendly bioprocesses are now attracting attention.

[0005] Glucose and glycerol are the main substrates used for 3-HP biosynthesis using microorganisms, and currently research is being conducted on a one-step fermentation method in which 3-HP production and cell growth occur simultaneously.

[0006] However, in the one-step production, the substrate for 3-HP production is also used for cell growth, resulting in the generation of by-products such as acetate and lactate, which reduces the yield and productivity of 3-HP. Due to these low yields and productivity, 3-HP has not yet been commercialized despite its potential. Summary of the Invention [Problem to be solved by the invention]

[0007] One example of the present application provides a method for producing 3-hydroxypropionic acid (3-HP) and / or a method for improving productivity of 3-HP, the method including: (1) a step of high-concentration cell culture of a 3-hydroxypropionic acid (3-HP)-producing strain; and (2) a step of isolating the high-concentration cultured cells and inoculating them into a 3-hydroxypropionic acid production medium and / or culturing them in a 3-hydroxypropionic acid production medium to produce 3-hydroxypropionic acid.

[0008] The two-step production method can improve the decrease in 3-HP productivity caused by cell growth or by-product generation.

[0009] Another example provides a culture medium of a 3-hydroxypropionic acid producing strain containing a high concentration of 3-hydroxypropionic acid. In one example, the culture medium may be produced by the two-step production method described above. The culture medium may be used for the production of 3-hydroxypropionic acid. [Means for solving the problem]

[0010] An example is: (1) High-density cell culture of a 3-hydroxypropionic acid (3-HP)-producing strain; and (2) isolating the cells cultured at high concentration and inoculating them into a medium for producing 3-hydroxypropionic acid and / or culturing them in a medium for producing 3-hydroxypropionic acid to produce 3-hydroxypropionic acid. The present invention provides a method for producing 3-hydroxypropionic acid (3-HP) and / or a method for improving 3-HP productivity, comprising the steps of: Another example provides a culture medium of a 3-hydroxypropionic acid producing strain containing 3-hydroxypropionic acid at a high concentration. The culture medium can be used for the production of 3-hydroxypropionic acid. The culture medium can be produced by the production method described above.

[0011] The present invention will now be described in more detail.

[0012] Two-stage 3-HP production (1) High-density cultivation of 3-HP-producing strains The methods for producing 3-HP and / or methods for improving 3-HP productivity provided herein include (1) culturing a 3-hydroxypropionic acid-producing strain at high concentration.

[0013] The 3-hydroxypropionic acid producing strain can be selected from among all natural or genetically modified recombinant microorganisms capable of 3-hydroxypropionic acid production. In one example, the microorganism may be selected from the genera Escherichia (e.g., E. coli), Pseudomonas, Enterobacteria, Brevibacterium, Corynebacterium, Klebsiella, Citrobacter, Clostridium, Streptomyces, Bacillus, Lactobacillus, Pseudomonas, Saccharomyces, and Aspergillus, but is not limited thereto. In one specific example, the 3-hydroxypropionic acid producing strain may be E. coli.

[0014] The 3-hydroxypropionic acid producing strain may contain a gene encoding one or more proteins selected from the group consisting of glycerol dehydratase and aldehyde dehydrogenase, or the two proteins. In one example, the 3-HP producing strain may additionally contain a gene (gdrAB) encoding a glycerol dehydratase reactivating enzyme (GdrAB). In one example, the 3-HP producing strain may additionally contain a gene (gdrAB) encoding a vitamin B 12 The bacterial strain may be capable of biosynthesizing

[0015] The glycerol dehydratase may be, but is not limited to, encoded by the dhaB (GenBank accession no. U30903.1) gene. The dhaB gene may be, but is not limited to, an enzyme derived from Klebsiella pneumonia. The gene encoding the glycerol dehydratase may include genes encoding dhaB1, dhaB2 and / or dhaB3. The glycerol dehydratase protein and the gene encoding it convert glycerol into 3-hydroxypropanal (3-HPA) and water (HPA). 2 The gene and / or amino acid sequence may include mutations within the range that maintains the enzyme activity of decomposing the protein into O).

[0016] The gene (aldH) encoding aldehyde dehydrogenase (ALDH) may be, for example, but is not limited to, the aldH (GenBank Accession no. U00096.3; EaldH) gene derived from Escherichia coli or E. coli K12 MG1655 cell strain, the puuC gene derived from Klebsiella pneumoniae, and / or the KGSADH gene derived from Azospirillum brasilense. The aldehyde dehydrogenase protein and the gene encoding it may contain mutations in the gene and / or amino acid sequence within the range that maintains the activity for producing 3-HP from 3-HPA.

[0017] The gene encoding the glycerol dehydratase reactivating enzyme (GdrAB) can be the gdrAB gene from Klebsiella pneumoniae.

[0018] The 3-HP producing strain can comprise a recombinant vector containing genes encoding one or more, two or more, or all three proteins selected from the group consisting of glycerol dehydratase, aldehyde dehydrogenase, and glycerol dehydratase reactivating enzyme.

[0019] The recombinant vector can be used by replacing the promoter and regulatory site by a method known in the art within the desired range for 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 reactivating enzyme.

[0020] The high-concentration culture can be carried out using any method known in the art for the purpose of obtaining a large amount of the 3-HP-producing strain, without limitation. In one example, the culture can be carried out as a fed-batch culture.

[0021] In one embodiment, the fed-batch culture may be performed in a pH-stat mode, a constant-rate (continuous feeding) culture mode, or a combination thereof. In one embodiment, when the fed-batch culture is performed in a pH-stat mode, glucose may be added at a concentration of 1 to 5 g / L, but is not limited thereto. In one embodiment, when the fed-batch culture is performed in a constant-rate culture mode, glucose may be added at a rate of 10 to 20 g / L / h, but is not limited thereto.

[0022] In one example, the pH during the high-density culture 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.

[0023] In one example, the carbon source during high-concentration culture may be selected from monosaccharides, disaccharides, and / or polysaccharides in the desired range for high-concentration culture without any restrictions. 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 selected from the group consisting of glucose, fructose, galactose, mannose, arabinose, xylose, ribose, sucrose, maltose, lactose, and cellobiose. The medium used during high-concentration culture may not contain glycerol as a carbon source. In this way, if the medium used during high-concentration culture does not contain glycerol, 3-HP production may not occur at the high-concentration culture stage.

[0024] In one example, the cell concentration after high density culture was OD 600 Based on the value, the OD value may be 10 or more, 30 or more, 50 or more, 70 or more, 100 or more, or 110 or more. For example, the OD value may be 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, or 20 hours after culturing. 600 Values ​​are 10~500, 10~400, 10~300, 10~250, 10~200, 10~150, 30~500, 30~400, 30~300, 30~250, 30~200, 30~150, 50~500, 50~400, 50~300, 50~250, 50~200, 50~150, 70~500, 70~400, 70~300 , 70-250, 70-200, 70-150, 100-500, 100-400, 100-300, 100-250, 100-200, 100-150, 110-500, 110-400, 110-300, 110-250, 110-200, or 110-150, for example, but not limited to, 120.

[0025] In one example, the cell concentration after high-concentration culture may be, based on the cell dry weight (g) per 1 L of medium, 10 to 100 g / L, 10 to 80 g / L, 10 to 50 g / L, 10 to 40 g / L, 10 to 35 g / L, 15 to 100 g / L, 15 to 80 g / L, 15 to 50 g / L, 15 to 40 g / L, 15 to 35 g / L, 20 to 100 g / L, 20 to 80 g / L, 20 to 50 g / L, 20 to 40 g / L, 20 to 35 g / L, 25 to 100 g / L, 25 to 80 g / L, 25 to 50 g / L, 25 to 40 g / L, or 25 to 35 g / L, but is not limited thereto.

[0026] (2) 3-HP production stage The method for producing 3-HP and / or the method for improving the productivity of 3-HP provided herein include (2) separating the cells cultured at a high concentration and inoculating and / or culturing them in a medium for producing 3-hydroxypropionic acid to produce 3-hydroxypropionic acid.

[0027] Separation of the high-density cultured cells may be performed by one or more, two or more, or all of the steps selected from the group consisting of centrifuging the cells, removing the supernatant, and resuspending the pellet in a buffer. In one example, the buffer may be, but is not limited to, phosphate buffered saline (PBS).

[0028] The medium for producing 3-hydroxypropionic acid can be used without restriction within the intended range as long as no additional growth of the 3-HP producing strain occurs and the strain is capable of producing 3-HP.

[0029] In one example, the carbon source of the 3-hydroxypropionic acid production medium can be, but is not limited to, glycerol. In one example, the production medium contains vitamin B 12 The medium for producing 3-hydroxypropionic acid may be free of glucose as a carbon source.

[0030] In one example, the medium may be, but is not limited to, a synthetic media or a semisynthetic media.

[0031] The inoculation of the high-concentration cultured cells can be appropriately changed by a skilled technician to a cell inoculation concentration within the target range of 3-HP production. In one example, the cell concentration at the time of inoculation (based on dry cell weight (DCW) per 1 L of medium) can be, but is not limited to, 1-20 g / L, 1-16 g / L, 1-12 g / L, 1-9 g / L, 2-20 g / L, 2-16 g / L, 2-12 g / L, 2-9 g / L, 4-20 g / L, 4-16 g / L, 4-12 g / L, or 4-9 g / L.

[0032] The step of producing 3-hydroxypropionic acid may be performed by selecting from among culture methods known in the art without limitation within the scope of the objective of producing 3-HP. In one example, the culture step may be performed by fermentation.

[0033] The 3-HP production phase may be one in which no proliferation of the inoculated cells occurs. In one example, the number of cells at the end of the production phase may be, but is not limited to, 150% or less, 130% or less, 100% or less, 90% or less, 80% or less, 50-150%, 50-130%, 50-100%, 50-90%, 50-80%, 70-150%, 70-130%, 70-100%, 70-90%, or 70-80% of the number of cells inoculated at the start of the production phase.

[0034] The 3-HP yield of the method for producing 3-HP and / or the method for improving 3-HP productivity provided herein may be, for example, but is not limited to, 80% or more, 85% or more, 90% or more, 93% or more, or 95% or more based on 29 hours of culture (3-HP production). The 3-HP yield may be calculated based on the amount of 3-HP produced in the medium relative to the amount of glycerol used (molar number) in the medium in step (2), and in one example, may be calculated according to the following Equation 1.

[0035] [Formula 1] Yield (%) = (Final 3-HP (g)) / {(Glycerol before fermentation (g))-(Glycerol remaining after fermentation (g))}

[0036] The 3-HP productivity (3-HP production amount (g) per hour and per L of medium) of the 3-HP production method and / or method for improving 3-HP productivity provided herein can be, but is not limited to, 2.0 g / L / h or more, 2.5 g / L / h or more, 2.0 to 30 g / L / h, 2.0 to 20 g / L / h, 2.0 to 10 g / L / h, 2.0 to 5 g / L / h, 2.5 to 30 g / L / h, 2.5 to 20 g / L / h, 2.5 to 10 g / L / h, or 2.5 to 5 g / L / h.

[0037] In one embodiment, the productivity of 3-HP produced by the two-step 3-HP production method of the present invention was measured to be 2.94 g / L / h, which is the highest value reported in the literature to date.

[0038] The 3-HP production amount of the 3-HP production method and / or the method for improving 3-HP productivity provided herein is, for example, 40 g / L or more, 41 g / L or more, 42 g / L or more, or 43 g / L or more based on the 3-HP content (g) per 1 L of medium during culture (3-HP production) for 20 to 30 hours (e.g., 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, or 30 hours). , 44g / L or more, 45g / L or more, 46g / L or more, 47g / L or more, 48g / L, 49g / L or more, 50g / L or more, 51g / L or more, 52g / L or more, or 53g / L or more (the upper limit may be selected without any particular limitation within the range of 60 to 1000g / L, for example, 1000g / L, 500g / L, 100g / L, 90g / L, 80g / L, 70g / L, or 60g / L, but is not limited thereto).

[0039] The method for producing 3-HP and / or the method for improving the productivity of 3-HP provided by the present invention may not produce by-products due to 3-HP production. The by-products may be acetate and / or lactate. In one example, the by-products may be produced at a concentration of 1% (w / v) or less, 0.5% (w / v) or less, 0.1% (w / v) or less, 0.01% (w / v) or less, 0.001% (w / v) or less, 0.0001% (w / v), or 0.00001% (w / v) or less in the total culture solution (in this case, the lower limit of the by-product concentration may be selected from 0 to 0.000001 (w / v), but is not limited thereto), or may be produced at a concentration of 0% (w / v) (no by-products are produced at a detectable concentration).

[0040] The two-stage 3-HP production method provided herein may provide a 3-HP yield (%) that is 1.05-fold or more or 1.1-fold or more higher than that of a one-stage fermentation mode in which cell growth and 3-HP production occur simultaneously, for example, 1.05-10-fold, 1.05-5-fold, 1.05-2-fold, 1.05-1.7-fold, 1.05-1.5-fold, 1.05-1.2-fold, 1.1-10-fold, 1.1-5-fold, 1.1-2-fold, 1.1-1.7-fold, 1.1-1.5-fold, or 1.1-1.2-fold higher, for example, about 1.13-fold higher.

[0041] The two-stage 3-HP production method of the present invention may have a 3-HP productivity (g / L / h) that is 1.1 times or more, 1.3 times or more, or 1.5 times or more higher than that of the one-stage fermentation method, for example, 1.1 to 10 times, 1.1 to 5 times, 1.1 to 3 times, 1.1 to 2.5 times, 1.1 to 2 times, 1.3 to 10 times, 1.3 to 5 times, 1.3 to 3 times, 1.3 to 2.5 times, 1.3 to 2 times, 1.5 to 10 times, 1.5 to 5 times, 1.5 to 3 times, 1.5 to 2.5 times, 1.5 to 2 times, 1.9 to 3 times, 1.9 to 2.5 times, or 1.9 to 2 times higher, for example, about 1.96 times higher.

[0042] The two-step method for producing 3-HP provided by the present invention may further include a step of (2) isolating the highly-concentrated cultured cells and inoculating and / or culturing them in a medium for producing 3-hydroxypropionic acid to produce 3-hydroxypropionic acid, followed by isolating, recovering, and / or purifying 3-hydroxypropionic acid from the culture medium.

[0043] Culture medium containing 3-HP Another embodiment of the present application provides a culture medium of a 3-hydroxypropionic acid producing strain containing a high concentration of 3-hydroxypropionic acid. The culture medium may be free of or contain cells (3-hydroxypropionic acid producing strain). The culture medium may be free of glucose.

[0044] The 3-hydroxypropionic acid producing strain was as described above. The culture medium may be a culture medium containing or not containing cells (cells) obtained by inoculating a 3-hydroxypropionic acid producing strain cultured in high-concentration cells into a medium for producing 3-hydroxypropionic acid, and the medium for producing 3-hydroxypropionic acid and / or the culture medium may not contain glucose. The high-concentration cell culture is as described in the above step (1).

[0045] In one example, the culture medium may be obtained by steps (1) and (2) described above.

[0046] The culture medium may have a 3-hydroxypropionic acid concentration of 41 g / L or more, 42 g / L or more, 43 g / L or more, 44 g / L or more, 45 g / L or more, 46 g / L or more, 47 g / L or more, 48 g / L, 49 g / L or more, 50 g / L or more, 51 g / L or more, 52 g / L or more, or 53 g / L or more (the upper limit may be selected from 60 to 1000 g / L without any particular limitation, for example, 1000 g / L, 500 g / L, 100 g / L, 90 g / L, 80 g / L, 70 g / L, or 60 g / L, but is not limited thereto). The culture medium may have a concentration of a by-product selected from the group consisting of acetate and lactic acid of 1% (w / v) or less, 0.5% (w / v) or less, 0.1% (w / v) or less, 0.01% (w / v) or less, 0.001% (w / v), 0.0001% (w / v), or 0.00001% (w / v) or less (in this case, the lower limit of the by-product concentration may be selected from the range of 0 to 0.000001 (w / v), but is not limited thereto), or may be 0% (w / v).

[0047] The culture medium can be used for producing 3-hydroxypropionic acid.

[0048] Another embodiment provides a composition for producing 3-hydroxypropionic acid, comprising the culture medium.

[0049] Another example provides a method for producing 3-hydroxypropionic acid, comprising the step of separating, recovering, and / or purifying 3-hydroxypropionic acid from the 3-hydroxypropionic acid producing composition. Effect of the Invention

[0050] The two-step 3-HP production method of the present invention significantly improves 3-HP productivity and yield compared to the one-step culture, and can be useful for commercialization of 3-HP. [Brief description of the drawings]

[0051] [Figure 1] 1 is a graph showing OD changes over time as a result of high-density cell culture of a 3-HP-producing strain according to an embodiment of the present invention. [Diagram 2] FIG. 1 is a graph showing the results of measuring the concentrations of 3-HP (3HP), glycerol, acetate, and lactate over time after inoculating a 3-HP-producing strain cultured at high concentration according to an embodiment of the present invention into a medium for 3-HP production (0 h). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples.

[0053] Unless otherwise specified herein, all temperatures are in degrees Celsius, and nucleic acid sequences are written from the 5' to the 3' end unless otherwise specified. EXAMPLES

[0054] Example 1.3 - High-density cell culture of HP-producing strains 1-1. Preparation of 3-HP-producing strain

[0055] A recombinant vector was prepared by introducing genes encoding glycerol dehydratase and aldehyde dehydrogenase, which are known to produce 3-hydroxypropionic acid (3-HP) using glycerol as a substrate. The recombinant vector was introduced into E. coli W3110 to produce a 3-HP producing strain.

[0056] Specifically, the gene encoding glycerol dehydratase (dhaB), the gene encoding aldehyde dehydrogenase (aldH), and the gene encoding glycerol dehydratase reactivating enzyme (gdrAB) were cloned into the plasmid pCDF to prepare a recombinant vector for producing 3HP (pCDF_J23101_dhaB_gdrAB_J23100_aldH).

[0057] The pCDFDuetJ23 vector used to construct the recombinant vector is a vector in which the promoter portion of the pCDFDuet-1 vector has been replaced with the J23101 and J23100 promoters. The dhaB (U30903.1; approximately 2.7 kb; including dhaB1, dhaB2, and dhaB3) and gdrAB genes (approximately 2.2 kb; gdrA, gdrB) to be inserted into the vector were amplified from the chromosome of Klebsiella pneumoniae (ATCC 25955) using the primers in Table 1 below. The dhaB123 and gdrA genes are located next to each other on the chromosome of Klebsiella pneumoniae, so they were amplified together, but since gdrB is located in the opposite direction to dhaB123 and gdrA, only gdrB was amplified separately.

[0058] The aldH gene was amplified and isolated from the genome of E. coli K12 MG1655 strain using a promoter pair consisting of the nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:6 in Table 1 below. The nucleic acid sequences of the primers used for amplifying each gene are shown in Table 1 below, and "-F" in the name indicates the forward promoter, and "-R" indicates the reverse promoter.

[0059] [Table 1]

[0060] After amplification of each gene, the dhaB123 and gdrA genes were cloned downstream of the J23101 promoter of the pCDFDuetaJ23 vector using the restriction enzymes EcoRI and HindIII, and the gdrB gene was cloned downstream of the J23101 promoter using the restriction enzymes HindIII and AflII. The aldH gene was cloned downstream of the J23108 promoter using the restriction enzymes KpnI and NdeI. The cloning method for each gene was performed by a method known in the art. The plasmid was electroporated into E. coli W3110 (KCCM 40219) to generate a 3HP producing strain.

[0061] 1-2. High-density cell culture The 3-HP producing strain thus prepared was cultured at high density in a 5L fermenter (working volume 2L) using a fed-batch culture method.

[0062] Specifically, MR medium (KH per 1L) 2 PO 4 6.67g, (NH 4 ) 2 HPO 4 4 g MgSO4·7H 2 0.8 g O, 0.8 g citric acid, and 5 mL of trace metal solution; where the trace metal solution is 5 mL of 5 M HCl, FeSO 4 7H 2 O 10g, CaCl 2 2g ZnSO 4 7H 2 2.2g O, MnSO 4 4H 2 0.5g O, CuSO 4 5H 2 O 1g, (NH 4 ) 6 Mo 7 O 2 4H 2 O 0.1g, and Na 2 B 4 O 210H 2 The cell culture medium was prepared by adding 20g / L glucose and 25mg / L antibiotic (streptomycin) for selection to 0.02g of glucose (O), and the temperature was maintained at 35 degrees Celsius. The pH was maintained at 6.95 using ammonia water, the dissolved oxygen (DO) was maintained at 20% by gradually increasing the stirring speed to 900 rpm, and the aeration was maintained at 1vvm.

[0063] The fed-batch culture was carried out using the pH-stat feeding method, and glucose was added at 3 g / L.

[0064] The cell concentration was measured by measuring the optical density (OD) using a UV-Spectrometer over the course of culture time. Figure 1 shows the OD over time. 600 The graph below shows the change in the values. OD 600 was approximately 120 (30 g / L dry cell weight).

[0065] After the 20-hour high-density cell culture, the cell culture medium was centrifuged at 6,000 rpm and 4° C. for 10 minutes to collect the cells. The collected cells were suspended in phosphate-buffered saline (PBS) and used in the subsequent steps.

[0066] Example 2.3 - HP Production Stage The medium for 3-HP production is glucose-free M9 medium supplemented with 70 g / L glycerol and vitamin B 12 The cell suspension prepared in Example 1-2 was inoculated into the 3-HP production medium at a cell inoculation amount of 5 g / L (based on dry cell weight), and the 3-HP production step was carried out in a 5 L fermenter (working volume 2 L).

[0067] The culture conditions for 3-HP production were maintained at 35°C, agitation speed 300 rpm, aeration at 1 vvm, and Ca(OH) 2The pH was maintained at 7.0 using

[0068] The concentrations of 3-HP, glycerol, acetate, and lactate over time during the 3-HP production process were measured by high pressure liquid chromatography (HPLC), and the results are shown in Table 2 and Figure 2. Table 2 shows the results of measuring the concentrations (g / L) of 3-HP, glycerol, acetate, and lactate over the course of fermentation time after inoculation in the 3-HP production culture step (two-stage culture).

[0069] [Table 2]

[0070] As shown in FIG. 2, 17 hours after the strain was inoculated into the 3-HP production medium, the 3-HP concentration was 50.0 g / L or more, and the productivity was confirmed to be 2.94 g / L / h, which is the highest 3-HP productivity reported in the literature to date.

[0071] 29 hours after inoculation, the final 3-HP concentration was confirmed to be 53.3 g / L, and the yield was approximately 95.7%, demonstrating excellent 3-HP productivity. However, the by-products of 3-HP production, lactic acid and acetate, were hardly produced until 29 hours had passed.

[0072] Comparison Example 1. Comparison with 1-step 3-HP production method In order to compare the 3-HP production ability of the two-step production method using the glucose-free 3-HP production medium provided in the present application with the existing one-step production method, the 3-HP-producing strain prepared in Example 1-1 above was cultured by fed-batch culture, and glucose necessary for cell growth and glycerol, a substrate, were simultaneously added to the culture medium to simultaneously grow cells and produce 3-HP (one-step production method).

[0073] The one-step production process carried out for comparison is described in more detail below. The culture medium used was the MR medium of Example 1-2 supplemented with 20 g / L glucose and 25 mg / L antibiotic (streptomycin) for selection, and the temperature was maintained at 35 degrees Celsius. The pH was maintained at 6.95 using ammonia water, the dissolved oxygen (DO) was maintained at 20% while gradually increasing the stirring speed to 900 rpm, and the aeration was maintained at 1 vvm.

[0074] When the glucose added in the culture medium at the beginning was completely consumed, 3 g / L of glucose was added in a constant-feeding culture method, and 70 g / L of glycerol was added 20 hours after the culture. The subsequent process was the same as in Example 2.

[0075] The yield and productivity of 3-HP produced through the 3-HP production process were measured by high pressure liquid chromatography (HPLC). The results of the 3-HP yield and productivity obtained in this manner are shown in Table 3 below, in comparison with the 3-HP yield and productivity through the two-step 3-HP production process carried out in Examples 1 and 2.

[0076] [Table 3]

[0077] As seen in Table 3, the two-stage 3-HP production method is superior in 3-HP yield and productivity compared to the existing one-stage production method. In addition, unlike the one-stage production method, the two-stage 3-HP production method does not produce by-products such as acetate and lactate, making the two-stage 3-HP production method superior.

Claims

1. (1) high-density cell culture of a 3-hydroxypropionic acid (3-HP) producing strain; and (2) isolating the high-density cultured cells and inoculating them into a medium for producing 3-hydroxypropionic acid; The 3-HP producing strain is an E. coli strain containing genes encoding glycerol dehydratase and aldehyde dehydrogenase proteins, and into which a recombinant vector in which the promoter portion is replaced with the J23101 and J23100 promoters has been introduced, The step (2) is a step in which cell growth does not occur, and is carried out in a medium that does not contain glucose as a carbon source, and the productivity of 3-hydroxypropionic acid is 2 g / L / h or more. A method for producing 3-hydroxypropionic acid (3-HP), comprising the steps of: The method includes producing a culture solution of a 3-hydroxypropionic acid-producing strain, the culture solution containing 3-hydroxypropionic acid at a concentration of 45 g / L or more and a concentration of a by-product selected from the group consisting of acetate and lactic acid at a concentration of 0.1% (w / v) or less.

2. The method according to claim 1, wherein the step (1) is carried out by a fed-batch culture method, and the step (2) is carried out by fermentation culture.

3. The method described in claim 1 or 2, wherein the (1) step is carried out in a medium containing glucose as a carbon source.

4. The step (1) is carried out in a medium not containing glycerol as a carbon source. The method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, wherein the (2) step is carried out in a medium containing glycerol as a carbon source.

6. The method according to any one of claims 1 to 5, wherein the yield of 3-hydroxypropionic acid is 80% or more.

7. The method according to any one of claims 1 to 5, wherein the production amount of 3-hydroxypropionic acid when step (2) is performed for 29 hours is 41 g / L or more.

8. A method for producing a composition for producing 3-hydroxypropionic acid, comprising the method described in any one of claims 1 to 7.

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