Two-step method 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] Mutual citation with related applications (etc.) This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0096238 filed on Jul. 31, 2020, and all the contents disclosed in the literature of the Korean Patent Application are incorporated herein by reference in part.

[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. More specifically, the present invention relates to a two-step production method including a first step of culturing cells at a high concentration and a second step of producing 3-HP using the cells cultured at the high concentration as a catalyst.

Background Art

[0003] 3-Hydroxypropionic acid is a platform compound that can be converted into various chemical substances such as acrylic acid, methyl acrylate, and acrylamide. After 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 studied in the academic and industrial fields.

[0004] The production of 3-HP is mainly carried out by two methods: a chemical method and a biological method. In the case of the chemical method, it has been pointed out that it is not environmentally friendly due to the high cost of starting materials and the generation of toxic substances during the production process. Therefore, the environmentally friendly bio-process has been in the spotlight.

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

[0006] However, in the one-step production process described above, the substrate for 3-HP production is also used for cell proliferation, generating by-products such as acetate and lactate, which reduces the yield and production capacity of 3-HP. Due to these low yields and production capacity, despite the potential of 3-HP, it has not yet been commercialized. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] One example of this application provides a method for producing 3-hydroxypropionic acid (3-HP) and / or a method for improving the production capacity of 3-HP, comprising the steps of (1) culturing a 3-hydroxypropionic acid (3-HP) producing strain at a high concentration; and (2) separating the high-concentration cultured cells, inoculating them into a medium for 3-hydroxypropionic acid production and / or culturing them in a medium for 3-hydroxypropionic acid production to produce 3-hydroxypropionic acid.

[0008] The aforementioned two-step manufacturing method can improve the reduction in 3-HP production capacity 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 manufacturing method described earlier. The culture medium can be used for the production of 3-hydroxypropionic acid. [Means for solving the problem]

[0010] One example is, (1) The step of culturing a 3-hydroxypropionic acid (3-HP) producing strain at high cell concentration; and (2) The step of separating the high-concentration cultured cells and inoculating them into a medium for 3-hydroxypropionic acid production and / or culturing them in a medium for 3-hydroxypropionic acid production to produce 3-hydroxypropionic acid. The present invention provides a method for producing 3-hydroxypropionic acid (3-HP) and / or a method for improving the production capacity of 3-HP, including the above. Another example provides a culture medium of a 3-hydroxypropionic acid-producing bacterial strain containing a high concentration of 3-hydroxypropionic acid. This culture medium can be used for the production of 3-hydroxypropionic acid. This culture medium may be produced by the method described above.

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

[0012] Two-stage 3-HP production (1) High-concentration culture stage of 3-HP producing strain A method for producing 3-HP and / or a method for improving the production capacity of 3-HP provided herein comprises (1) a step of culturing a 3-hydroxypropionic acid-producing strain at a high concentration.

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

[0014] 3-hydroxypropionic acid-producing strains may contain genes encoding one or more proteins selected from the group consisting of glycerol dehydratase and aldehyde dehydrogenase, or both of the aforementioned proteins. In one example, a 3-HP-producing strain may additionally contain a gene encoding glycerol dehydrate reactivating enzyme (GdrAB) (gdrAB). In another example, a 3-HP-producing strain may additionally contain a gene encoding vitamin B 12 It may be a strain of bacteria capable of biosynthesizing [the substance].

[0015] Glycerol dehydratase may, but is not limited to, be encoded by the dhaB gene (GenBank accession no. U30903.1). The dhaB gene may, but is not limited to, be derived from the enzyme Klebsiella pneumonia. Genes encoding glycerol dehydratase may include genes encoding dhaB1, dhaB2, and / or dhaB3. Glycerol dehydratase proteins and the genes encoding them may contain gene and / or amino acid sequence mutations within the range that maintains enzymatic activity in breaking down glycerol into 3-hydroxypropanal (3-HPA) and water (H2O).

[0016] The gene encoding aldehyde dehydrogenase (ALDH) (aldH) may be, for example, the aldH (GenBank Accession no. U00096.3; EaldH) gene derived from Escherichia coli or E. coli K12 MG1655 cell line, the puuC gene derived from Klebsiella pneumonia, and / or the KGSADH gene derived from Azospirillum brasilense. The aldehyde dehydrogenase protein and the gene encoding it may contain gene and / or amino acid sequence mutations within the range that maintains activity for producing 3-HP from 3-HPA.

[0017] The gene encoding glycerol dehydration reactivating enzyme (GdrAB) may be the gdrAB gene derived from Klebsiella pneumonia.

[0018] The 3-HP producing strain can contain a recombinant vector containing 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.

[0019] The recombinant vector can be used by replacing the promoter and regulatory sites within the target range for intracellular expression of the 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 by methods known in the art.

[0020] High-concentration culture can be carried out using methods known in the art without limitation within the target range for ensuring a large quantity of the 3-HP producing strain. In one example, the culture can be a fed-batch culture.

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

[0022] In one example, the pH during the high-concentration 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 can be selected and used without limitation from monosaccharides, disaccharides, and / or polysaccharides within the target range for high-concentration culture. For example, the carbon source can be one or more, two or more, three or more, four or more, five or more, ten or more, or all combinations of the eleven kinds 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 can be one that does not contain glycerol as a carbon source. Thus, when the medium used during high-concentration culture does not contain glycerol, 3-HP production may not occur during the high-concentration culture stage.

[0024] In one example, the cell concentration after high-concentration culture is 600 based on the OD value, and can be 10 or more, 30 or more, 50 or more, 70 or more, 100 or more, or 110 or more. For example, at 20 hours of culture, when it is 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, the OD 600 value can be 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, and can be, for example, 120, but is not limited thereto.

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

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

[0027] The isolation of highly cultured cells may be performed by one or more, or all, of the following steps, selected from the group consisting of the steps 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, PBS (Phosphate-buffered saline).

[0028] The medium for 3-hydroxypropionic acid production can be used without restriction within the desired range to prevent the additional growth of the 3-HP-producing strain while enabling the strain to produce 3-HP.

[0029] In one example, the carbon source of the medium for 3-hydroxypropionic acid production may be, but is not limited to, glycerol. In one example, the production medium may contain vitamin B 12 It may also include the following. The culture medium for 3-hydroxypropionic acid production may not contain glucose as a carbon source.

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

[0031] Inoculation of highly cultured cells can be appropriately adjusted by a standard technician to the appropriate cell inoculation concentration within the target range for 3-HP production. For example, the cell concentration at inoculation (based on dry cell weight (DCW) per liter of medium) may 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 can be any culture method known in the art within the desired range for producing 3-HP, and in one example, the culture step may be carried out by fermentation.

[0033] During the 3-HP production stage, proliferation of the inoculated cells may not occur. For example, the number of cells at the end of the production stage may be, but is not limited to, 150%, 130%, 100%, 90%, 80%, 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 stage.

[0034] The 3-HP yield of the methods for producing 3-HP and / or methods for improving the production capacity of 3-HP provided herein may be, but are not limited to, 80% or more, 85% or more, 90% or more, 93% or more, or 95% or more, based on a 29-hour culture (3-HP production). The 3-HP yield may be calculated in step (2) above as the amount of 3-HP produced in the medium relative to the amount of glycerol used in the medium (in moles), and in one example it may be calculated as shown in Formula 1 below.

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

[0036] The 3-HP production capacity (3-HP production per hour and per L of culture medium (g)) of the 3-HP production method and / or method for improving the production capacity of 3-HP provided herein may be, but are 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, when 3-HP was produced using the two-step 3-HP production method of the present invention, the production capacity 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 yield of the 3-HP production method and / or method for improving the production capacity of 3-HP provided herein is, for example, 40 g / L or more, 41 g / L or more, 42 g / L or more, 43 g / L or more, based on the 3-HP content (g) per liter of culture medium during a 20-30 hour (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hour) culture (3-HP production), respectively. It may be 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 special restrictions from 60 to 1000g / L, for example, it may be 1000g / L, 500g / L, 100g / L, 90g / L, 80g / L, 70g / L, or 60g / L, but is not limited to these).

[0039] The 3-HP production method and / or method for improving the production capacity of 3-HP provided by the present invention may not produce any by-products from 3-HP production. The by-products may be acetate and / or lactate. In one example, the by-product 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 medium (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 at a concentration of 0%(w / v) (the by-product may not be produced at a detectable concentration).

[0040] The two-step 3-HP production method provided herein may yield a 3-HP yield (%) that is 1.05 times or 1.1 times higher than a one-step fermentation method in which cell proliferation and 3-HP production occur simultaneously. For example, it may be 1.05 to 10 times, 1.05 to 5 times, 1.05 to 2 times, 1.05 to 1.7 times, 1.05 to 1.5 times, 1.05 to 1.2 times, 1.1 to 10 times, 1.1 to 5 times, 1.1 to 2 times, 1.1 to 1.7 times, 1.1 to 1.5 times, or 1.1 to 1.2 times higher, and may be approximately 1.13 times higher.

[0041] The two-stage 3-HP production method provided by the present invention may have a 3-HP production capacity (g / L / h) that is 1.1 times or more, 1.3 times or more, or 1.5 times or more higher than the one-stage fermentation method described above. For example, it may be 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, 1.3 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, it may be about 1.96 times higher.

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

[0043] Culture medium containing 3-HP Another example of this application provides a culture medium of a 3-hydroxypropionic acid-producing strain containing a high concentration of 3-hydroxypropionic acid. The culture medium may or may not contain cells (3-hydroxypropionic acid-producing strain). The culture medium may not contain glucose.

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

[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 higher, 42 g / L or higher, 43 g / L or higher, 44 g / L or higher, 45 g / L or higher, 46 g / L or higher, 47 g / L or higher, 48 g / L, 49 g / L or higher, 50 g / L or higher, 51 g / L or higher, 52 g / L or higher, or 53 g / L or higher (the upper limit may be selected without special restrictions within the range of 60 to 1000 g / L, 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 to these). The culture medium may have a concentration of by-products 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) or 0.00001%(w / v) or less (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 it may be 0%(w / v).

[0047] The culture medium can be used for the production of 3-hydroxypropionic acid.

[0048] Another example provides a composition for the production of 3-hydroxypropionic acid, comprising the culture medium.

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

[0050] The two-step 3-HP production method provided by the present invention significantly improves 3-HP production capacity and yield compared to one-step culture, and can be usefully used for the commercialization of 3-HP. [Brief explanation of the drawing]

[0051] [Figure 1] This graph shows the change in OD over time as a result of high-concentration cell culture of a 3-HP-producing strain according to one embodiment of the present invention. [Figure 2] This graph shows 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 one embodiment of the present invention into a 3-HP production medium (0h). [Modes for carrying out the invention]

[0052] The present invention will be described in more detail below based on examples. However, the following examples are merely illustrative of the content of the present invention, and the scope of the rights of the present invention is not limited by the following examples.

[0053] Unless otherwise specified in this specification, all temperatures are given in Celsius, and nucleic acid sequences are written from the 5' end to the 3' end unless otherwise specified. [Examples]

[0054] Example 1.3-HP-producing cell culture Production of 1-1.3-HP-producing strains

[0055] Recombinant vectors were 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 prepared recombinant vectors were introduced into E. coli W3110 strain to create a 3-HP producing strain.

[0056] Specifically, the genes encoding glycerol dehydrogenase (dhaB), aldehyde dehydrogenase (aldH), and glycerol dehydrogenase reactivation enzyme (gdrAB) were cloned into plasmid pCDF to construct a recombinant vector for 3HP production (pCDF_J23101_dhaB_gdrAB_J23100_aldH).

[0057] The pCDFDuetJ23 vector used to produce the recombinant vector is a vector in which the promoter portion of the pCDFDuet-1 vector is replaced with the J23101 and J23100 promoters. The dhaB (U30903.1; approximately 2.7kb; containing dhaB1, dhaB2, and dhaB3) and gdrAB genes (approximately 2.2kb; gdrA, gdrB) inserted into the vector were amplified on the chromosome of Klebsiella pneumoniae (ATCC 25955) using the primers shown in Table 1 below. The dhaB123 and gdrA genes are located side by side on the Klebsiella pneumoniae chromosome and were amplified together, while gdrB is located in the opposite direction from dhaB123 and gdrA, so gdrB was amplified separately.

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

[0059] [Table 1]

[0060] After amplification of each gene, the dhaB123 and gdrA genes were cloned downstream of the J23101 promoter in 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. aldH was cloned under the J23108 promoter using the restriction enzymes KpnI and NdeI. The cloning methods for each gene were performed using methods known in the art. The plasmid was introduced into E. coli W3110 (KCCM 40219) by electroporation to produce a 3HP-producing strain.

[0061] 1-2. High-concentration cell culture The 3-HP-producing strains produced were subjected to high-concentration cell culture using a fed-batch culture method in a 5L fermenter (working volume 2L).

[0062] Specifically, MR medium (1L contains 6.67g of KH2PO4, 4g of (NH4)2HPO4, 0.8g of MgSO4·7H2O, 0.8g of citric acid, and 5mL of trace metal solution; where the trace metal solution is 5mL of 5M HCl, 10g of FeSO4·7H2O, 2g of CaCl2, 2.2g of ZnSO4·7H2O, 0.5g of MnSO4·4H2O, 1g of CuSO4·5H2O, 0.1g of (NH4)6Mo7O2·4H2O, and Na2B4O2·10H2O 0.02 g) of the culture medium was prepared by adding 20 g / L of glucose and 25 mg / L of the 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 up to 900 rpm, and the aeration rate was maintained at 1 vvm.

[0063] Fed-batch culture was performed using the pH-stat feeding method, with glucose added at a concentration of 3 g / L.

[0064] Cell concentration was measured by measuring the absorbance (Optical Density, OD) using a UV-Spectrometer over time. Figure 1 shows the OD over time. 600 A graph showing the change in values ​​is shown. 20 hours after the start of culture, OD 600 The value was approximately 120 (dry cell weight 30 g / L).

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

[0066] Example 2.3 - HP Production Stage The culture medium for 3-HP production is M9 medium without glucose, with 70 g / L of glycerol and vitamin B 12 The medium was prepared by adding 50 microM. The cell suspension prepared in Examples 1-2 was inoculated into the 3-HP production medium to a cell inoculation volume of 5 g / L (based on dry cell weight), and the 3-HP production stage was carried out in a 5 L fermenter (working volume 2 L).

[0067] The culture conditions for 3-HP production were a temperature of 35 degrees Celsius, a stirring speed of 300 rpm, an aeration rate of 1 vvm, and a pH of 7.0 maintained using Ca(OH)2.

[0068] During the 3-HP production process described above, the concentrations of 3-HP, glycerol, acetate, and lactate were measured over time using high-pressure liquid chromatography (HPLC), and the results are shown in Table 2 and Figure 2 below. Table 2 shows the results of measuring the concentrations (g / L) of 3-HP, glycerol, acetate, and lactate according to the fermentation time after inoculation at the 3-HP production culture stage (two-stage culture).

[0069] [Table 2]

[0070] As can be seen in Figure 2, after inoculating the 3-HP production medium with the bacterial strain, the 3-HP concentration was shown to be 50.0 g / L or higher 17 hours later, and the production capacity was confirmed to be 2.94 g / L / h, which is the highest 3-HP production capacity reported in the literature to date.

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

[0072] Comparative Example 1.1: Comparison with a 3-HP production system To compare the 3-HP production capacity of a two-stage production method using the glucose-free 3-HP production medium provided in this application with that of an existing one-stage production method, the 3-HP-producing strain produced in Example 1-1 was cultured using a fed-batch culture method, and glucose necessary for cell growth and glycerol, a substrate, were simultaneously added to the culture medium to promote cell proliferation and 3-HP production simultaneously (one-stage production method).

[0073] The one-step production process performed for comparison is described in more detail below. The culture medium used was the MR medium from Example 1-2 with 20 g / L of glucose and 25 mg / L of the antibiotic streptomycin for selection added, 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] Once all of the glucose initially added to the culture medium was consumed, glucose was added at a rate of 3 g / L using a continuous feeding method, and glycerol was added at a rate of 70 g / L 20 hours after the start of culture. The subsequent process was carried out in the same manner as in Example 2.

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

[0076] [Table 3]

[0077] As confirmed in Table 3 above, the two-stage 3-HP production method is superior to the existing one-stage production method in terms of 3-HP yield and production capacity. Furthermore, unlike the one-stage production method, the two-stage 3-HP production method does not produce by-products such as acetate and lactate, thus confirming the superiority of the two-stage 3-HP production method.

Claims

1. (1) The step of culturing a 3-hydroxypropionic acid (3-HP) producing strain in high-concentration cell cultures; and (2) The step of separating the high-concentration cultured cells and inoculating them into a medium for 3-hydroxypropionic acid production to produce 3-hydroxypropionic acid; The aforementioned 3-HP-producing strain is an E. coli strain into which a recombinant vector has been introduced, containing genes encoding glycerol dehydrogenase and aldehyde dehydrogenase proteins, and in which the promoter portion is replaced with the J23101 and J23100 promoters. The above step (1) is carried out in a culture medium containing glucose as a carbon source. The above step (2) is carried out in a culture medium in which no cell proliferation occurs, which contains glycerol as a carbon source but does not contain glucose as a carbon source, and the production capacity of 3-hydroxypropionic acid is 2 g / L / h or more. A method for producing 3-hydroxypropionic acid (3-HP), A method for producing a culture solution of a 3-hydroxypropionic acid-producing bacterial strain, wherein the culture solution contains 3-hydroxypropionic acid at a concentration of 45 g / L or more, and the concentration of a by-product selected from the group consisting of acetate and lactic acid is 0.1% (w / v) or less.

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

3. The above step (1) is carried out in a culture medium that does not contain glycerol as a carbon source. The method according to claim 1 or 2.

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

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

6. A method for producing a composition for producing 3-hydroxypropionic acid, comprising the method according to any one of claims 1 to 5.

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