Recombinant microorganism having enhanced 3-hydroxypropionic acid production capability

Abstract

The present invention relates to a recombinant microorganism having enhanced 3-hydroxypropionic acid production capability, the microorganism having 3-hydroxypropionic acid biosynthesis pathways in which a pathway for converting 3-hydroxypropionaldehyde to 3-hydroxypropionic acid and a pathway for converting acetate to acetyl-CoA are enhanced. In addition, the present invention relates to a method for producing 3- hydroxypropionic acid by using the recombinant microorganism for producing 3-hydroxypropionic acid.

Description

Recombinant microorganism with enhanced 3-hydroxypropionic acid production

[0001] The present invention relates to a recombinant microorganism with enhanced 3-hydroxypropionic acid production ability.

[0002] 3-Hydroxypropionic acid (3-HP) can be produced through chemical synthesis and microbial fermentation processes. Recently, research has been conducted on methods for producing 3-HP using bacteria such as Escherichia coli and Klebsiella. For example, Korean Patent Publication No. 10-2020-0051375 discloses a technology for producing 3-HP using a 3-HP-producing microorganism transformed with a gene encoding adenosyltransferase.

[0003] However, the method of producing 3-HP through a microbial fermentation process has not yet reached a commercial level in terms of productivity, yield, and economic feasibility of the medium, and further research is needed on methods to increase the productivity of 3-HP using glycerol as a substrate.

[0004] The present inventors, while studying a recombinant microorganism with high 3-hydroxypropionic acid production ability, completed the present invention by producing a recombinant microorganism with low by-product production and high 3-hydroxypropionic acid production ability.

[0005] The purpose of the present invention is to provide a recombinant microorganism having low by-product production and high 3-hydroxypropionic acid production capacity.

[0006] To achieve the above object, the present invention provides a recombinant microorganism having enhanced 3-hydroxypropionic acid production ability, wherein the pathway for converting 3-hydroxypropionaldehyde into 3-hydroxypropionic acid and the pathway for converting acetate into acetyl CoA are enhanced in a microorganism having a 3-hydroxypropionic acid biosynthetic pathway.

[0007] The present invention also provides a method for producing 3-hydroxypropionic acid using the recombinant microorganism.

[0008] The recombinant microorganism of the present invention has the advantage of not only having a high 3-hydroxypropionic acid production ability but also having a low by-product production ability, making it easy to produce and recover 3-hydroxypropionic acid.

[0009] Figure 1 shows changes in metabolic pathways through gene overexpression in a recombinant microorganism of the present invention. It shows a process of simultaneously overexpressing the puuC gene and the ydcW gene to promote the pathway of converting 3-hydroxypropionaldehyde to 3-hydroxypropionic acid, and a process of overexpressing the acs gene to promote the pathway of converting acetate to acetyl CoA. In addition, it shows an example in which the trc-puuC and trc-ydcW genes are inserted into the positions of the major byproduct-producing genes dhaT, yqhD, ldhA, and glpK to increase the copy numbers of the puuC and ydcW genes.

[0010] Figure 2 shows the pathway for producing 3-hydroxypropionic acid from glycerol and the results of analysis of the production amount and yield of 3-hydroxypropionic acid according to the location on the genome for overexpressing the puuC gene and the ydcW gene and the type of each promoter in the recombinant microorganism of the present invention.

[0011] Figure 3 shows the results of analysis of the production amount and yield of 3-hydroxypropionic acid according to the increase in the number of copies of the puuC gene and the ydcW gene in the genome of the recombinant microorganism of the present invention.

[0012] Figure 4 shows the results of analyzing the amount and yield of 3-hydroxypropionic acid and the amount of acetate produced according to the location and promoter on the genome for overexpressing the acs gene in the recombinant microorganism of the present invention.

[0013] The present invention,

[0014] In microorganisms having a 3-hydroxypropionic acid biosynthetic pathway,

[0015] Converting 3-hydroxypropionaldehyde to 3-hydroxypropionic acid

[0016] This relates to a recombinant microorganism with enhanced 3-hydroxypropionic acid production capacity and an enhanced pathway.

[0017] In addition, the present invention,

[0018] In microorganisms having a 3-hydroxypropionic acid biosynthetic pathway,

[0019] This relates to a recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, which has an enhanced pathway for converting acetate to acetyl CoA.

[0020] In addition, the present invention,

[0021] This relates to a recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the genes puuC and ydcW encoding aldehyde dehydrogenase involved in the pathway for converting to 3-hydroxypropionic acid and the gene acs encoding acetyl CoA synthetase involved in the pathway for converting to acetyl CoA are each overexpressed.

[0022] In addition, in the present invention, a gene encoding an enzyme having at least 90% amino acid sequence homology with the expression product of the gene puuC (SEQ ID NO: 1) or ydcW (SEQ ID NO: 2) encoding the aldehyde dehydrogenase may also be preferably used, and furthermore, a gene encoding an enzyme having at least 90% amino acid sequence homology with the expression product of the gene acs (SEQ ID NO: 47) encoding the acetyl CoA synthetase may also be preferably used. The puuC, ydcW and acs genes of the present invention may be characterized in that they are derived from Klebsiella pneumoniae, but are not limited to those derived from a specific microorganism, and it will be apparent that any gene derived from a microorganism with a similar function to these may be used in the present invention.

[0023] In addition, the present invention,

[0024] This relates to a recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the genes of puuC and ydcW are simultaneously overexpressed within the genome and the number of copies of the simultaneous overexpression is arranged in a repeating manner of 1 to 20 times.

[0025] In addition, the present invention,

[0026] This relates to a recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the puuC, ydcW and acs genes are each operably linked to a trc promoter.

[0027] In addition, the present invention,

[0028] A step of inoculating a recombinant microorganism of the present invention into a medium;

[0029] A step of preparing a culture solution by culturing a medium inoculated with the recombinant microorganism; and

[0030] A method for producing 3-hydroxypropionic acid, comprising a step of recovering 3-hydroxypropionic acid from the above culture solution.

[0031]

[0032] The present invention is described in detail below.

[0033] In the present invention, the main metabolic pathways of the wild type strain were confirmed, and among them, the puuC gene and the ydcW gene, which are key genes for the production of 3-hydroxypropionic acid, were confirmed, which are genes encoding aldehyde dehydrogenase involved in the conversion of 3-hydroxypropionaldehyde to 3-hydroxypropionic acid. The puuC gene utilizes NAD+ with high efficiency, and the ydcW gene utilizes NADP+ with high efficiency. When these two genes were co-expressed in the genome, it was found that the genome stability increased and the 3-hydroxypropionic acid productivity also increased. In particular, when the two genes were continuously and repeatedly arranged in the genome and then expressed, it was found that the 3-hydroxypropionic acid productivity increased further. In addition, the acs gene, which converts acetate into acetyl CoA, was identified, and when it was overexpressed, it was found that the production of acetate, a byproduct, was reduced and the production of 3-hydroxypropionic acid was further increased, thereby completing the present invention.

[0034]

[0035] Microorganisms with 3-hydroxypropionic acid biosynthetic pathway

[0036] The microorganism of the present invention has a 3-hydroxypropionic acid biosynthetic pathway. The microorganism may be a microorganism having the aforementioned 3-hydroxypropionic acid biosynthetic pathway, and is not particularly limited thereto. The 3-hydroxypropionic acid biosynthetic pathway refers to a pathway by which 3-hydroxypropionic acid is synthesized from a specific metabolite in the microorganism. The 3-hydroxypropionic acid biosynthetic pathway of the present invention may be a pathway by which 3-hydroxypropionic acid (3-HP) is synthesized from 3-hydroxypropionaldehyde (3-HPA). Preferably, the microorganism of the present invention has a pathway by which 3-hydroxypropionic acid is synthesized from a carbon source such as glycerol or glycerin.

[0037] In addition, the microorganism of the present invention may be a microorganism having a 3-hydroxypropionic acid biosynthetic pathway in a wild type or a recombinant microorganism obtained by genetic recombination. The microorganism may be selected from the group consisting of the genus Klebsiella, the genus Enterobacter, the genus Corynebacterium, the genus Clostridium, and the genus Lactobacillus, preferably the genus Klebsiella, and more preferably the genus Klebsiella, and more preferably the genus Klebsiella pneumoniae.

[0038]

[0039] Pathway for converting 3-hydroxypropionaldehyde to 3-hydroxypropionic acid

[0040] The recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid in which the pathway for converting 3-hydroxypropionaldehyde into 3-hydroxypropionic acid is enhanced. In this case, the pathway for converting 3-hydroxypropionaldehyde into 3-hydroxypropionic acid may be a pathway in which the conversion of 3-hydroxypropionaldehyde into 3-hydroxypropionic acid is controlled by aldehyde dehydrogenase.

[0041] Aldehyde dehydrogenase regulates the conversion of 3-hydroxypropionaldehyde to 3-hydroxypropionic acid. By increasing the activity of aldehyde dehydrogenase, the pathway for converting 3-hydroxypropionaldehyde to 3-hydroxypropionic acid can be promoted. The increase in aldehyde dehydrogenase activity can be achieved by amplifying the expression of the aldehyde dehydrogenase gene, increasing the enzymatic activity of aldehyde dehydrogenase, etc. For example, a person skilled in the art can increase the activity of aldehyde dehydrogenase by selecting an appropriate method, such as introducing puuC or ydcW, a gene encoding aldehyde dehydrogenase, into a microorganism, causing a mutation in the gene (mutation such as changing, substituting, or deleting some bases or introducing some bases to amplify the expression of the gene), regulating gene expression during the transcription or translation process, replacing or modifying a promoter, etc.

[0042]

[0043] Pathway that converts acetate to acetyl CoA

[0044] The recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid with an enhanced pathway for converting acetate to acetyl CoA. In this case, the pathway for converting acetate to acetyl CoA may be a pathway in which the conversion of acetate to acetyl CoA is regulated by acetyl CoA synthetase.

[0045] Acetyl CoA synthase regulates the conversion of acetate into acetyl CoA. By increasing the activity of the acetyl CoA synthase, the pathway for converting acetate into acetyl CoA can be promoted. The increase in the activity of the acetyl CoA synthase can be achieved by amplifying the expression of the acetyl CoA synthase gene, increasing the enzymatic activity of the acetyl CoA synthase, etc. For example, a person skilled in the art can increase the activity of the acetyl CoA synthase by selecting an appropriate method, such as introducing the gene acs encoding the acetyl CoA synthase into a microorganism, causing a mutation in the gene (mutation such as changing, substituting, or deleting some bases, or introducing some bases to amplify the expression of the gene), regulating gene expression during the transcription or translation process, replacing or modifying the promoter, etc.

[0046]

[0047] Recombinant microorganism for producing 3-hydroxypropionic acid

[0048] The recombinant microorganism with enhanced 3-hydroxypropionic acid (3-HP) production ability of the present invention may be a recombinant microorganism with enhanced 3-hydroxypropionic acid production ability characterized by enhanced pathways for converting 3-hydroxypropionaldehyde (3-HPA) into 3-hydroxypropionic acid (3-HP) and for converting acetate into acetyl CoA.

[0049] As described above, the genes that promote the pathway for converting 3-HPA into 3-HP are puuC and ydcW, and the gene that promotes the pathway for converting acetate into acetyl CoA is acs, as described above. In the present specification, the genes that increase the productivity of 3-HP refer to the three genes puuC, ydcW, and acs. Hereinafter, these three genes may also be referred to as 3-HP productivity enhancing genes.

[0050] The number of copies of the 3-hydroxypropionate (3-HP) production enhancing gene inserted into the genomic DNA is preferably 1 copy or more, more preferably 2 copies or more of the combination of ydcW and puuC, and most preferably 3 copies or more and 20 copies or less.

[0051] In one example, the 3-HP production enhancing genes being replicated are puuC and ydcW, and the acs gene may be inserted alone at the end of a certain copy number, but the location of the inserted acs gene may be anywhere in the genome.

[0052] In this specification, “aldehyde dehydrogenase” is an enzyme that converts 3-hydroxypropionaldehyde into 3-hydroxypropionic acid, and the enzyme may be encoded by the puuC gene or encoded by the ydcW gene, but is not limited thereto.

[0053] The above puuC gene or ydcW gene may be derived from Klebsiella pneumoniae and may include a mutation in the gene and / or amino acid sequence within the range of maintaining the enzymatic activity that converts 3-HPA into 3-HP.

[0054]

[0055] In this specification, "acetyl CoA synthase" is an enzyme that converts acetate into acetyl CoA, and the enzyme may be encoded by the acs gene and may be derived from Klebsiella pneumoniae.

[0056] In one example, the recombinant microorganism of the present invention may additionally comprise an inducible promoter and a 3-HP production enhancing gene operably linked thereto, i.e., a puuC gene, a ydcW gene, and / or an acs gene. The inducible promoter may be one that induces expression of a gene by linking to a promoter through the addition (treatment) of an inducer, and the transcription timing of the genes may be controlled using the inducible promoter. In one example, the inducible promoter may be BAD, LTTR, trc, and / or tac, and in one embodiment, the microorganism may additionally comprise a puuC gene, a ydcW gene, and / or an acs gene operably linked to a trc promoter. The trc promoter and the genes may be cloned into a single vector and introduced into a cell. The genes may use not only an inducible promoter but also a constitutive promoter for the purpose of increasing the expression amount.

[0057] The above "promoter" refers to an untranslated nucleic acid sequence upstream of a coding region, which includes a binding site for a polymerase and has transcription initiation activity into mRNA of a gene downstream of the promoter, i.e., a DNA region to which a polymerase binds to initiate transcription of a gene, and may be located at the 5' site of the mRNA transcription initiation site.

[0058] In the present invention, in order to increase the productivity of 3-hydroxypropionic acid, a method is used in which the copy number of a gene (polynucleotide) encoding aldehyde dehydrogenase is increased and integrated into the genome, and a gene encoding acetyl CoA synthetase is also inserted, and each gene is operably linked to a trc promoter.

[0059]

[0060] The recombinant microorganism with enhanced 3-hydroxypropionic acid production ability of the present invention may have higher productivity (amount of 3-hydroxypropionic acid produced per unit time, per unit volume), productivity (amount of 3-hydroxypropionic acid produced per unit volume, i.e., concentration of 3-hydroxypropionic acid in the culture medium), and yield (production amount of 3-hydroxypropionic acid relative to the amount of carbon source) of 3-hydroxypropionic acid, and in particular, the recombinant microorganism may have a higher concentration of 3-hydroxypropionic acid in the fermentation medium than a wild-type microorganism during fermentation. In the present invention, productivity is defined and used as a broad concept that includes yield in addition to the general concentration concept of productivity.

[0061] When culturing the recombinant microorganism of the present invention, the amount of acetate produced may be lower than that of a wild-type microorganism.

[0062] The biosynthetic pathway of 3-hydroxypropionic acid and the strategy for producing a recombinant microorganism in the recombinant microorganism of the present invention are shown in Figure 1.

[0063] The recombinant microorganism of the present invention integrated the puuC gene and the ydcW gene, or the ydcW gene and the puuC gene, which are involved in encoding the enzyme for increasing the activity of aldehyde dehydrogenase, which converts 3-hydroxypropionaldehyde to 3-hydroxypropionic acid, into the genome so that they were simultaneously expressed regardless of the order, thereby confirming that the genome stability was also increased. In addition, both puuC and ydcW genes can utilize NAD+ and NADP+ as cofactors, but puuC is known to utilize NAD+ with high efficiency (high cofactor preference) and ydcW is known to utilize NADP+ with high efficiency. Therefore, utilizing both genes simultaneously can improve 3-HP production by utilizing cofactors efficiently and increase genome stability by reducing the number of copies of the same gene in the genome (Reference: Luo, LH, Seo, JW., Heo, SY. et al. Identification and characterization of Klebsiella pneumoniae aldehyde dehydrogenases increasing production of 3-hydroxypropionic acid from glycerol. Bioprocess Biosyst Eng 36, 1319-1326 (2013). https: / doi.org / 10.1007 / s00449-012-0880-4). As shown in Fig. 2, the introduction of two genes for simultaneous expression, puuC and ydcW, is most preferable in terms of the production amount and yield of 3-hydroxypropionic acid. In addition, various constitutive promoters can be used in addition to inducible promoters such as trc and tac for the expression of the two genes.

[0064] Furthermore, in the recombinant microorganism of the present invention, it was found that the more the puuC / ydcW gene or ydcW / puuC gene was repeated in the genome, the more the production amount and yield of 3-hydroxypropionic acid increased. It was most preferable that the puuC and ydcW genes were alternately repeated two or more times, rather than the repetition of the same puuC gene or the repetition of the same ydcW gene (see Figure 3).

[0065] In addition, in the recombinant microorganism of the present invention, in order to reduce acetate, a by-product, the acs gene involved in encoding acetyl CoA synthetase, which converts acetate into acetyl CoA, was overexpressed to increase the activity of this enzyme. As shown in the lower part of Fig. 4, the strain with an enhanced 3-HP production pathway was designed to express the trc promoter and acs gene, and as a result, it was found that the 3-HP production ability was the best.

[0066]

[0067] Recombinant microorganism and method for producing 3-hydroxypropionic acid using the same

[0068] The present invention relates to a method for producing 3-hydroxypropionic acid, comprising the steps of: inoculating a medium with a recombinant microorganism for producing 3-hydroxypropionic acid of the present invention; culturing the medium inoculated with the recombinant microorganism to produce a culture solution; and recovering 3-hydroxypropionic acid from the culture solution.

[0069] The above cultivation may be performed as a flask culture without DO (dissolved oxygen) control, or may be performed under aerobic conditions, preferably microaerobic conditions. For example, the above cultivation may be performed while supplying oxygen, i.e. air, during the cultivation, and as a specific example, this may be accomplished through stirring, but is not limited thereto. The recombinant microorganism of the present invention may be cultured in a complex medium, and the type of the complex medium is not particularly limited, and it is obvious that a person skilled in the art can appropriately select and use a commercially available, commonly used complex medium.

[0070] In one embodiment, the culture may be cultured under conditions that do not control DO, or may be performed under conditions of DO of 0 to 10%, 0 to 10%, 1 to 8%, 2 to 7%, 2.5 to 6.5%, or 5%.

[0071] In one embodiment, the culturing can be performed for 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 20 hours or more, 25 hours or more, 30 hours or more, 35 hours or more, 40 hours or more, 45 hours or more, or 48 hours or more.

[0072] In one embodiment, the culturing can be performed for 95 hours or less, 90 hours or less, 85 hours or less, 80 hours or less, 70 hours or less, 65 hours or less, 60 hours or less, 55 hours or less, 50 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, 25 hours or less, 20 hours or less, or 15 hours or less.

[0073] In one embodiment, the culture can be performed in a flask for 1 to 24 hours under conditions of initial pH 7.00, 200 rpm, and 37°C. The data shown in Figures 2 to 4 are the results of culture under these conditions.

[0074] The step of recovering 3-hydroxypropionic acid from the above culture solution is not particularly limited and may be performed by a conventional method for recovering a specific product from a culture solution using microorganisms.

[0075] The above method for producing 3-hydroxypropionic acid may additionally include a step of purifying the culture solution. The purification may be performed using a method commonly used for purifying 3-hydroxypropionic acid, and is not particularly limited.

[0076] The carbon source during the above cultivation can be glycerol, glycerin, etc.

[0077] The recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid in which the pathway for converting 3-hydroxypropionaldehyde into 3-hydroxypropionic acid is enhanced among microorganisms having a 3-hydroxypropionic acid biosynthetic pathway.

[0078] In addition, the recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid with an enhanced pathway for converting acetate into acetyl CoA.

[0079] In addition, the recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid with increased 3-hydroxypropionic acid production ability.

[0080]

[0081] In one embodiment, the recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid in which the pathway for converting 3-hydroxypropionaldehyde into 3-hydroxypropionic acid is promoted and / or the pathway for converting acetate into acetyl CoA is promoted in a microorganism having a 3-hydroxypropionic acid biosynthetic pathway (see FIG. 1).

[0082] In one embodiment, the recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid, which has a 3-hydroxypropionic acid concentration in a culture medium of 1 g / L or more, 2 g / L or more, 3 g / L or more, 4 g / L or more, 5 g / L or more, 6 g / L or more, 7 g / L or more, 8 g / L or more, 9 g / L or more, 10 g / L or more, 11 g / L or more, 12 g / L or more, 13 g / L or more, 14 g / L or more, or 15 g / L or more when cultured in a flask for 24 hours under conditions of initial pH 7.00, 200 rpm, and 37°C.

[0083] In one embodiment, the recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid, which has a 3-hydroxypropionic acid concentration in a culture medium of 30 g / L or less, 29 g / L or less, 28 g / L or less, 27 g / L or less, 26 g / L or less, 25 g / L or less, 24 g / L or less, 23 g / L or less, 22 g / L or less, 21 g / L or less, 20 g / L or less, 19 g / L or less, 18 g / L or less, 17 g / L or less, or 16 g / L or less when cultured in a flask for 24 hours under initial pH 7.00, 200 rpm, and 37°C conditions.

[0084] In one embodiment, the recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid, which has an acetate concentration in a culture medium of 15 g / L or less, 10 g / L or less, 5 g / L or less, 4.5 g / L or less, 4.0 g / L or less, 3.5 g / L or less, 3.0 g / L or less, 2.5 g / L or less, 2.0 g / L or less, 1.5 g / L or less, or 1.0 g / L or less when cultured in a flask for 24 hours under conditions of initial pH 7.00, 200 rpm, and 37°C.

[0085] In one embodiment, the recombinant microorganism of the present invention may be a recombinant microorganism for producing 3-hydroxypropionic acid, which has an acetate concentration in a culture medium of 0.1 g / L or more, 0.15 g / L or more, 0.2 g / L or more, 0.25 g / L or more, 0.3 g / L or more, 0.35 g / L or more, 0.4 g / L or more, 0.45 g / L or more, 0.5 g / L or more, 0.55 g / L or more, 0.6 g / L or more, 0.65 g / L or more, 0.7 g / L or more, or 0.75 g / L or more when cultured in a flask for 24 hours under conditions of initial pH 7.00, 200 rpm, and 37°C.

[0086] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims.

[0087]

[0088] Materials and Methods

[0089] 1) Microorganisms

[0090] Klebsiella pneumoniae GSC123 (KCTC12133BP) was used.

[0091] 2) Production capacity of metabolites

[0092] In the present invention, the metabolic product production capacity of microorganisms was calculated as follows.

[0093] -Product concentration (g / L): the amount of a specific product produced per unit volume

[0094] - Product yield (g / g): Amount of a specific product produced (g) / Carbon source (g)

[0095] 3) Medium, culture conditions and operating conditions

[0096] In the following experimental example, (1) medium, (2) culture conditions, and (3) operating conditions are as follows.

[0097]

[0098] (1) Badge

[0099] Seed cultures are prepared in test tubes and are as shown in Table 1 below.

[0100] [Table 1]

[0101]

[0102] (2) Culture conditions

[0103] The culture conditions for the main culture are as shown in Tables 2 and 3.

[0104] [Table 2]

[0105]

[0106] [Table 3]

[0107]

[0108] (3) Driving conditions

[0109] The operating conditions of this culture are as shown in Table 4. Here, the operating conditions refer to the basic conditions of flask culture.

[0110] [Table 4]

[0111]

[0112] Experimental Example 1: Production of a strain to enhance 3-HP production.

[0113] To construct a strain to enhance 3-HP production, genes encoding aldehyde dehydrogenase were amplified from the genome of Klebsiella pneumoniae GSC123 strain. Specifically, the expression levels of the puuC and ydcW genes present in the genome were enhanced by expressing them through the strong trc promoter. The trc-puuC and trc-ydcW fragments were obtained through overlapping PCR, and the fragments were fused with the homologous region at the desired insertion site to obtain the final integration fragment. The trc-puuC or trc-ydcW fragment was inserted into the puuC or ydcW site in the genome to produce puuC::trc-puuC, puuC::trc-ydcW, ydcW::trc-puuC, ydcW::trc-ydcW strains, thereby confirming the effect of simultaneous expression of the puuC and ydcW genes (Examples 1 to 8). To further enhance the expression of puuC and ydcW, the major byproduct-producing genes identified in the previously applied patent (KR Application No.: 10-2023-0017522), such as dhaT, yqhD, ldhA, glpK, pta-ack, and poxB, were utilized (reference: Fig. 1). By inserting the puuC or ydcW gene in combination with a strong promoter such as trc or tac at the corresponding position, we aimed to increase the expression levels of the puuC (SEQ ID NO: 1) and ydcW (SEQ ID NO: 2) genes along with suppression of the major by-product production pathway. Specifically, the following strains were constructed.

[0114] [Table 5]

[0115]

[0116] Example 1: Construction of puuC::trc-puuC strain, ydcW::trc-ydcW strain, and puuC::trc-puuC ydcW::trc-ydcW strain

[0117] To construct the puuC::trc-puuC strain, the left homologous region was obtained using the primer of SEQ ID NO. (3 / 4), and the right homologous region was obtained using the primer of SEQ ID NO. (5 / 6). An overlapping fragment was obtained using the primer of SEQ ID NO. (3 / 6), and the overlapping fragment was treated with the restriction enzyme (Xba1 / Not1) and then introduced into the pKOV plasmid to obtain pKOV-puuC::trc-puuC. The pKOV-puuC::trc-puuC plasmid was introduced into the strain using electroporation (electroporation, 25 uF, 200 Ω, 2.5 kV / cm), and finally, the puuC gene in the genome was expressed under the trc promoter (Example 1-1). In addition, the ydcW::trc-ydcW strain was also produced using the same method (using SEQ ID NOs. 7 / 8 and 9 / 10) (Example 1-2). In addition, the puuC::trc-puuC ydcW::trc-ydcW strain was also produced using pKOV-puuC::trc-puuC and pKOV-ydcW::trc-ydcW (Example 1-3).

[0118] In order to introduce additional trc-puuC or trc-ydcW, the trc-puuC or trc-ydcW region was amplified by PCR in the genome and inserted into the desired insertion site through homologous recombination.

[0119]

[0120] Example 2: Construction of puuC::trc-puuC ydcW::trc-puuC strain

[0121] To produce the above strain, trc-puuC was inserted into the ydcW site present in the genome, and trc-puuC was amplified as SEQ ID NO: 11 / 12, the left homologous arm was amplified with the SEQ ID NO: 13 / 14 primer, and the right homologous arm was amplified with the SEQ ID NO: 15 / 16 primer. A homologous recombination site was produced through overlapping PCR using the SEQ ID NO: 11 / 16 primer. The produced site was introduced into the pKOV plasmid and used for strain production. The overlapping fragment was introduced into the pKOV plasmid after restriction enzyme treatment with (Xba1 / Xma1), thereby obtaining pKOV-ydcW::trc-puuC, and this was introduced into the puuC::trc-puuC strain produced in Example 1-1 using the electroporation method described above, thereby producing the GSC123 puuC::trc-puuC ydcW::trc-puuC strain.

[0122]

[0123] Example 3: Construction of ydcW::trc-ydcW puuC::trc-ydcW strain

[0124] In order to create the above strain, trc-ydcW was inserted into the puuC site in the genome, and trc-ydcW was amplified as SEQ ID NO: 17 / 18, the left homologous arm was amplified with the SEQ ID NO: 19 / 20 primer, and the right homologous arm was amplified with the SEQ ID NO: 21 / 22 primer. A homologous recombination site was created through overlapping PCR using the SEQ ID NO: 17 / 22 primer. The created site was introduced into the pKOV plasmid and used for strain creation. The overlapping fragment was introduced into the pKOV plasmid after restriction enzyme treatment with (Xba1 / Xma1), thereby obtaining pKOV-puuC::trc-ydcW, and this was introduced into the ydcW::trc-ydcW strain produced in Example 1-2 using the electroporation method described above, thereby producing the GSC123 ydcW::trc-ydcW puuC::trc-ydcW strain.

[0125]

[0126] Example 4: Construction of puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC strains

[0127] To produce the above strain, trc-puuC was inserted into the dhaT site of the byproduct-producing gene present in the genome, and trc-puuC was amplified as SEQ ID NO: 23 / 24, the left homologous arm was amplified with the SEQ ID NO: 25 / 26 primer, and the right homologous arm was amplified with the SEQ ID NO: 27 / 28 primer. A homologous recombination site was produced through overlapping PCR using the SEQ ID NO: 23 / 28 primer. The produced site was introduced into the pKOV plasmid and used for strain production. The overlapping fragment was introduced into the pKOV plasmid after restriction enzyme treatment with (Xba1 / Not1), thereby obtaining pKOV-dhaT::trc-puuC, which was then introduced into the puuC::trc-puuC ydcW::trc-ydcW strain produced in Example 1-3 using the electroporation method described above, thereby producing the GSC123 puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC strain.

[0128]

[0129] Example 5: Construction of puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC yqhD::trc-ydcW strains

[0130] To produce the above strain, trc-ydcW was inserted into the yqhD site of the byproduct-producing gene present in the genome, and trc-ydcW was amplified as SEQ ID NO: 29 / 30, the left homologous arm was amplified with the SEQ ID NO: 31 / 32 primer, the right homologous arm was amplified with the SEQ ID NO: 33 / 34 primer, and a homologous recombination site was produced through overlapping PCR using the SEQ ID NO: 29 / 34 primer. The produced site was introduced into the pKOV plasmid and used for strain production. The overlapping fragment was introduced into the pKOV plasmid after restriction enzyme treatment with (Xba1 / Not1), thereby obtaining pKOV-yqhD::trc-ydcW, which was then introduced into the strain produced in Example 4 using the electroporation method described above, thereby producing the GSC123 puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC yqhD::trc-ydcW strain.

[0131]

[0132] Example 6: Construction of puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC yqhD::trc-ydcW glpK::trc-puuC strains

[0133] To produce the above strain, trc-puuC was inserted into the byproduct-producing gene glpK site in the genome, and trc-puuC was amplified as SEQ ID NO: 35 / 36. The homologous left arm was amplified with the SEQ ID NO: 37 / 38 primers, and the homologous right arm was amplified with the SEQ ID NO: 39 / 40 primers. A homologous recombination site was produced through overlapping PCR using the SEQ ID NO: 35 / 40 primers. The produced site was introduced into the pKOV plasmid and used for strain production. The overlapping fragment was introduced into the pKOV plasmid after restriction enzyme treatment with (BamH1 / Not1), thereby obtaining pKOV-glpK::trc-puuC, and then introduced into the strain produced in Example 5 using the electroporation method described above, thereby producing the GSC123 puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC yqhD::trc-ydcW glpK::trc-puuC strain.

[0134]

[0135] Example 7: Construction of puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC yqhD::trc-ydcW glpK::trc-puuC ldhA::trc-ydcW strains

[0136] To produce the above strain, trc-ydcW was inserted into the ldhA site of the byproduct-producing gene present in the genome, and trc-ydcW was amplified as SEQ ID NO: 41 / 42, the left homologous arm was amplified with the SEQ ID NO: 43 / 44 primer, and the right homologous arm was amplified with the SEQ ID NO: 45 / 46 primer. A homologous recombination site was produced through overlapping PCR using the SEQ ID NO: 41 / 46 primer. The produced site was introduced into the pKOV plasmid and used for strain production. The overlapping fragment was introduced into the pKOV plasmid after restriction enzyme treatment with (BamH1 / Not1), thereby obtaining pKOV-ldhA::trc-ydcW, which was then introduced into the strain produced in Example 6 using the electroporation method described above, thereby producing the GSC123 puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC yqhD::trc-ydcW glpK::trc-puuC ldhA::trc-ydcW strain.

[0137]

[0138] Example 8: Construction of puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC yqhD::trc-ydcW glpK::trc-puuC ldhA::trc-ydcW acs::trc-acs strains

[0139] In order to produce the above strain, trc-acs was inserted into the acs position, which is an acetyl CoA synthetase gene present in the genome, and the left homologous region of trc-acs was obtained using the primer of sequence number (48 / 49), and the right homologous region was obtained using the primer of sequence number (50 / 51) (see sequence number 47 for the base sequence of the acs gene). An overlapping fragment was obtained using the primers of sequence number (48 / 51), and the overlapping fragment was treated with a restriction enzyme (Xba1 / Not1) and introduced into the pKOV plasmid to obtain pKOV-acs::trc-acs, which was then introduced into the strain produced in Example 7 using the electroporation method described above to produce the GSC123 puuC::trc-puuC ydcW::trc-ydcW dhaT::trc-puuC yqhD::trc-ydcW glpK::trc-puuC ldhA::trc-ydcW acs::trc-acs strain.

[0140]

[0141] The base sequences used to produce the above strains are summarized in Table 6 below.

[0142] [Table 6]

[0143]

[0144]

[0145]

[0146]

[0147] Comparative Example 1:

[0148] The GSC123 / pTrc99C strain was constructed by inserting the pTrc99C empty vector into the GSC123 strain. The empty vector has no function and exhibits the same performance as the wild-type strain.

[0149]

[0150] Experimental Example 2: Evaluation of 24-hour culture of recombinant strains

[0151]

[0152] *First, in order to increase the productivity of 3-HP, as in Examples 1 to 3, the puuC gene and the ydcW gene encoding aldehyde dehydrogenase, which regulates the conversion of 3-hydroxypropionaldehyde to 3-hydroxypropionic acid, were linked together on the genome, and the 3-HP productivity of recombinant strains produced by changing the linking order and the type of promoter was compared.

[0153] As a result, it was found that the recombinant strain, in which the puuC gene and the ydcW gene were arranged in that order as shown in Fig. 2 and the trc promoter was used in front of each gene, had the best 3-HP production ability in a 24-hour culture.

[0154] Next, the 3-HP production ability of recombinant strains in which the puuC gene and the ydcW gene were repeatedly arranged on the genome as in Examples 4 to 7 was compared. As shown in Fig. 3, it was found that the 3-HP production ability of the recombinant strain with a higher number of repetitions became increasingly superior as a result of 24-hour culture.

[0155] In addition, in order to reduce the by-product acetate as in Example 8, the puuC gene and ydcW gene were repeated three times in the genome, and then the acs gene encoding acetyl CoA synthetase that converts acetate to acetyl CoA was expressed more strongly as shown in Fig. 4. As a result, it was found that 3-HP productivity was improved and the production of acetate as a by-product was also significantly reduced. Specifically, the amount of acetate as a by-product was reduced by about 35%, while the amount of the target 3-hydroxypropionic acid increased by about 11.6% and the yield also increased by about 7.8%. It is presumed that environmental changes such as pH and toxicity in the culture medium due to the reduced acetate production may have affected the production amount and yield of 3-hydroxypropionic acid.

[0156] On the other hand, the strain of Comparative Example 1 and the wild-type strain did not produce 3-hydroxypropionic acid at all, and it was confirmed that production of 3-hydroxypropionic acid was possible only when the puuC gene or ydcW gene was overexpressed, as in the recombinant strains of Examples 1 to 3. However, it was also found that 3-hydroxypropionic acid was not produced when both promoters were native promoters even if the puuC gene or ydcW gene was present in the genome.

[0157] The above results are summarized in Table 7 below. nat represents the native promoter and trc represents the trc promoter.

[0158] From the results in Table 7 below, it was found that 3-HP production capacity could be maximized by strengthening the promoter, reducing the copy number of the gene encoding aldehyde dehydrogenase, and reducing the byproduct acetate.

[0159] [Table 7]

[0160]

[0161] <Sequence List Free Text>

[0162] Sequence number 1 is the base sequence of the puuC gene.

[0163] Sequence number 2 is the base sequence of the ydcW gene.

[0164] Sequence number 3 is the base sequence of a primer for amplifying the left arm of the trc-puuC gene of Example 1 by PCR. In addition, Sequence number 3 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0165] Sequence number 4 is the base sequence of a primer for amplifying the left arm of the trc-puuC gene of Example 1 by PCR.

[0166] Sequence number 5 is the base sequence of a primer for amplifying the right arm of the trc-puuC gene of Example 1 by PCR.

[0167] Sequence number 6 is the base sequence of a primer for amplifying the right arm of the trc-puuC gene of Example 1 by PCR. In addition, sequence number 6 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0168] Sequence number 7 is the base sequence of a primer for amplifying the left arm of the trc-ydcW gene of Example 1 by PCR. In addition, sequence number 7 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0169] Sequence number 8 is the base sequence of a primer for amplifying the left arm of the trc-ydcW gene of Example 1 by PCR.

[0170] Sequence number 9 is the base sequence of a primer for amplifying the right arm of the trc-ydcW gene of Example 1 by PCR.

[0171] Sequence number 10 is the base sequence of a primer for amplifying the right arm of the trc-ydcW gene of Example 1 by PCR. In addition, sequence number 10 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0172] Sequence number 11 is the base sequence of a primer for amplifying the trc-puuC gene of Example 2 by PCR. In addition, Sequence number 11 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0173] Sequence number 12 is the base sequence of a primer for amplifying the trc-puuC gene of Example 2 by PCR.

[0174] Sequence number 13 is the base sequence of a primer for amplifying the left arm of the trc-puuC gene of Example 2 by PCR.

[0175] Sequence number 14 is the base sequence of a primer for amplifying the left arm of the trc-puuC gene of Example 2 by PCR.

[0176] Sequence number 15 is the base sequence of a primer for amplifying the right arm of the trc-puuC gene of Example 2 by PCR.

[0177] Sequence number 16 is the base sequence of a primer for amplifying the right arm of the trc-puuC gene of Example 2 by PCR. In addition, Sequence number 16 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0178] Sequence number 17 is the base sequence of a primer for amplifying the trc-ydcW gene of Example 3 by PCR. In addition, sequence number 17 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0179] Sequence number 18 is the base sequence of a primer for amplifying the trc-ydcW gene of Example 3 by PCR.

[0180] Sequence number 19 is the base sequence of a primer for amplifying the left arm of the trc-ydcW gene of Example 3 by PCR.

[0181] Sequence number 20 is the base sequence of a primer for amplifying the left arm of the trc-ydcW gene of Example 3 by PCR.

[0182] Sequence number 21 is the base sequence of a primer for amplifying the right arm of the trc-ydcW gene of Example 3 by PCR.

[0183] Sequence number 22 is the base sequence of a primer for amplifying the right arm of the trc-ydcW gene of Example 3 by PCR. In addition, Sequence number 22 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0184] Sequence number 23 is the base sequence of a primer for amplifying the trc-puuC gene of Example 4 by PCR. In addition, Sequence number 23 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0185] Sequence number 24 is the base sequence of a primer for amplifying the trc-puuC gene of Example 4 by PCR.

[0186] Sequence number 25 is the base sequence of a primer for amplifying the left arm of the trc-puuC gene of Example 4 by PCR.

[0187] Sequence number 26 is the base sequence of a primer for amplifying the left arm of the trc-puuC gene of Example 4 by PCR.

[0188] Sequence number 27 is the base sequence of a primer for amplifying the right arm of the trc-puuC gene of Example 4 by PCR.

[0189] Sequence number 28 is the base sequence of a primer for amplifying the right arm of the trc-puuC gene of Example 4 by PCR. In addition, Sequence number 28 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0190] Sequence number 29 is the base sequence of a primer for amplifying the trc-ydcW gene of Example 5 by PCR. In addition, sequence number 29 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0191] Sequence number 30 is the base sequence of a primer for amplifying the trc-ydcW gene of Example 5 by PCR.

[0192] Sequence number 31 is the base sequence of a primer for amplifying the left arm of the trc-ydcW gene of Example 5 by PCR.

[0193] Sequence number 32 is the base sequence of a primer for amplifying the left arm of the trc-ydcW gene of Example 5 by PCR.

[0194] Sequence number 33 is the base sequence of a primer for amplifying the right arm of the trc-ydcW gene of Example 5 by PCR.

[0195] Sequence number 34 is the base sequence of a primer for amplifying the right arm of the trc-ydcW gene of Example 5 by PCR. In addition, Sequence number 34 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0196] Sequence number 35 is the base sequence of a primer for amplifying the trc-puuC gene of Example 6 by PCR. In addition, Sequence number 35 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0197] Sequence number 36 is the base sequence of a primer for amplifying the trc-puuC gene of Example 6 by PCR.

[0198] Sequence number 37 is the base sequence of a primer for amplifying the left arm of the trc-puuC gene of Example 6 by PCR.

[0199] Sequence number 38 is the base sequence of a primer for amplifying the left arm of the trc-puuC gene of Example 6 by PCR.

[0200] Sequence number 39 is the base sequence of a primer for amplifying the right arm of the trc-puuC gene of Example 6 by PCR.

[0201] Sequence number 40 is the base sequence of a primer for amplifying the right arm of the trc-puuC gene of Example 6 by PCR. In addition, Sequence number 40 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0202] Sequence number 41 is the base sequence of a primer for amplifying the trc-ydcW gene of Example 7 by PCR. In addition, sequence number 41 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0203] Sequence number 42 is the base sequence of a primer for amplifying the trc-ydcW gene of Example 7 by PCR.

[0204] Sequence number 43 is the base sequence of a primer for amplifying the left arm of the trc-ydcW gene of Example 7 by PCR.

[0205] Sequence number 44 is the base sequence of a primer for amplifying the left arm of the trc-ydcW gene of Example 7 by PCR.

[0206] Sequence number 45 is the base sequence of a primer for amplifying the right arm of the trc-ydcW gene of Example 7 by PCR.

[0207] Sequence number 46 is the base sequence of a primer for amplifying the right arm of the trc-ydcW gene of Example 7 by PCR. In addition, sequence number 46 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0208] Sequence number 47 is the base sequence of the acs gene.

[0209] Sequence number 48 is the base sequence of a primer for amplifying the left arm of the trc-acs gene of Example 8 by PCR. In addition, Sequence number 48 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0210] Sequence number 49 is the base sequence of a primer for amplifying the left arm of the trc-acs gene of Example 8 by PCR.

[0211] Sequence number 50 is the base sequence of a primer for amplifying the right arm of the trc-acs gene of Example 8 by PCR.

[0212] Sequence number 51 is the base sequence of a primer for amplifying the right arm of the trc-acs gene of Example 8 by PCR. In addition, Sequence number 51 is the base sequence of a primer for producing a fusion fragment by overlapping PCR of the amplified fragment.

[0213]

[0214] [Accession number]

[0215] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0216] Accession number: KCTC12133BP

[0217] Date of acceptance: February 8, 2012

[0218]

Claims

In microorganisms having a 1.3-hydroxypropionic acid biosynthetic pathway, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, wherein the pathway for converting 3-hydroxypropionaldehyde to 3-hydroxypropionic acid is promoted.

2. In paragraph 1, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the pathway for converting the above 3-hydroxypropionaldehyde into 3-hydroxypropionic acid is a pathway in which the conversion of 3-hydroxypropionaldehyde into 3-hydroxypropionic acid is controlled by aldehyde dehydrogenase.

3. In paragraph 2, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that it overexpresses aldehyde dehydrogenase involved in the pathway for conversion to 3-hydroxypropionic acid, in order to promote the production of the above 3-hydroxypropionic acid.

4. In paragraph 1, In a microorganism having the above 3-hydroxypropionic acid biosynthetic pathway, A recombinant microorganism with enhanced 3-hydroxypropionic acid production by promoting the pathway that converts acetate to acetyl CoA.

5. In paragraph 4, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that it overexpresses acetyl CoA synthetase involved in the pathway of converting acetate into acetyl CoA, in order to suppress the production of the above acetate.

6. In paragraph 3, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the gene encoding the above aldehyde dehydrogenase is puuC or ydcW.

7. In paragraph 5, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the gene encoding the above acetyl CoA synthetase is acs.

8. In any one of paragraphs 1 to 7, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that puuC and ydcW are simultaneously overexpressed in the genome and the gene set is arranged repeatedly several times to increase the above 3-hydroxypropionic acid production ability.

9. In paragraph 8, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the simultaneous overexpression copy number of the puuC and ydcW genes is arranged 1 to 20 times in the genome.

10. In paragraph 8, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the puuC and ydcW genes in the above gene set are each operably linked to a trc promoter.

11. In paragraph 8, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized by overexpressing the acs gene encoding acetyl CoA synthetase together with the repeated overexpression of the puuC and ydcW genes.

12. In paragraph 11, A recombinant microorganism having enhanced 3-hydroxypropionic acid production ability, characterized in that the above acs gene is operably linked to a trc promoter.

13. In paragraph 1, A recombinant microorganism with enhanced 3-hydroxypropionic acid production, characterized by increased 3-hydroxypropionic acid production and suppressed acetate production.

14. In paragraph 1, A recombinant microorganism with enhanced 3-hydroxypropionic acid production ability, characterized in that the concentration of acetate in the culture medium during cultivation of the recombinant microorganism is 15 g / L or less.

15. Step of inoculating the recombinant microorganism of paragraph 1 into a medium; A step of preparing a culture solution by culturing a medium inoculated with the recombinant microorganism; and A method for producing 3-hydroxypropionic acid, comprising a step of recovering 3-hydroxypropionic acid from the above culture solution.

16. In paragraph 15, A method for producing 3-hydroxypropionic acid, characterized in that it further comprises a step of purifying the culture solution.

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