Recombinant microorganism producing gamma-aminobutyric acid and method for producing gamma-aminobutyric acid using the same

Abstract

The present invention relates to a recombinant microorganism that produces gamma-aminobutyric acid and a method for producing gamma-aminobutyric acid using the same. The recombinant microorganism of the present invention can produce gamma-aminobutyric acid from acetic acid with high efficiency through stepwise metabolic pathway optimization, and can be usefully employed in the production of pharmaceuticals, functional foods, and biodegradable polymers.

Description

Technology Field

[0001] The present invention relates to a recombinant microorganism that produces gamma-aminobutyric acid from acetic acid with high efficiency through stepwise metabolic pathway optimization, and a method for producing gamma-aminobutyric acid using the same. Background Technology

[0003] Gamma-aminobutyric acid (GABA) is a non-protein amino acid composed of four carbon atoms that is synthesized from L-glutamic acid by glutamate decarboxylase. Since GABA acts as an inhibitory neurotransmitter in animals, it is used in pharmaceuticals such as analgesics, diuretics, antidepressants, and blood pressure regulators. In addition, GABA is a high-value-added substance that is very useful industrially because it is a precursor to 2-pyrrolidone, which can be used in the synthesis of biodegradable polymers.

[0004] Until now, the general method used for the biosynthesis of GABA has been to convert L-glutamic acid into GABA using the reaction of glutamate decarboxylase. Recently, however, research has been actively conducted to produce GABA using substrates such as glucose or glycerol instead of L-glutamic acid, which is a relatively expensive substrate. However, despite efforts to produce GABA using substrates such as glucose or glycerol, the process of producing GABA through biosynthesis remains economically unviable, and further research is needed.

[0005] Accordingly, the inventors completed the present invention by selecting points in the metabolic pathways involved in GABA production within E. coli where the carbon flow splits into GABA synthesis and energy production, and by developing a recombinant microorganism that efficiently produces GABA using acetic acid as a substrate through precise stepwise carbon flow control. Prior art literature

[0007] (Patent Document 0001) KR 10-2226445 B1(Patent Document 0002) KR 10-1785150 B1 The problem to be solved

[0008] The object of the present invention is to provide a recombinant microorganism for producing gamma-aminobutyric acid (γ-aminobutyric acid, GABA).

[0009] Another objective of the present invention is to provide a method for producing gamma-aminobutyric acid using the recombinant microorganism. means of solving the problem

[0011] In order to solve the above-mentioned problems,

[0012] The present invention provides a recombinant microorganism for producing gamma-aminobutyrite in which the iclR gene, gabT gene, and puuE gene are deleted, and the gadB mutant gene represented by SEQ ID NO. 5 is introduced.

[0013] In addition, the present invention provides a method for producing gamma-aminobutyric acid using the recombinant microorganism. Effects of the invention

[0015] The present invention relates to a recombinant microorganism that produces gamma-aminobutyric acid and a method for producing gamma-aminobutyric acid using the same. The recombinant microorganism of the present invention can produce gamma-aminobutyric acid from acetic acid with high efficiency through stepwise metabolic pathway optimization, and can be usefully employed in the production of pharmaceuticals, functional foods, and biodegradable polymers. Brief explanation of the drawing

[0017] Figure 1 is a schematic diagram showing the acetic acid metabolic pathway and target gene. Fig. 2a is iclR , gabT , puuE This shows cell growth (OD600), acetic acid consumption, and GABA production capacity in WG0 (control group) into which the pETDuet-1 vector was introduced from gene-deleted E. coli WG. Fig. 2b is iclR , gabT , puuE Wild type in gene-deleted E. coli WG gadB you This shows cell growth (OD600), acetic acid consumption, and GABA production capacity in the WG1 strain into which electrons were introduced. Fig. 2c is iclR , gabT , puuE In gene-deleted E. coli WG gadB This shows the cell growth (OD600), acetic acid consumption, and GABA production capacity of the WG2 strain introduced with the mutation E89Q Δ452-466. Figure 3a shows the J23114 promoter and on the WG2 strain aceA This shows cell growth (OD600), acetic acid consumption, and GABA production capacity in the WGA114 strain into which the gene was introduced. Figure 3b shows the J23110 promoter and on the WG2 strain aceA This shows cell growth (OD600), acetic acid consumption, and GABA production capacity in the WGA110 strain into which the gene was introduced. Fig. 3c shows the WG2 strain with the J23108 promoter and aceA This shows cell growth (OD600), acetic acid consumption, and GABA production capacity in the WGA108 strain into which the gene was introduced. Figure 3d shows the WG2 strain with the J23100 promoter and aceA This shows cell growth (OD600), acetic acid consumption, and GABA production capacity in the WGA100 strain into which the gene was introduced. Figure 3e shows the WG2 strain aceA This is a graph comparing cell growth (OD600), acetic acid consumption, and GABA production in strains in which the gene was expressed with four different promoter strengths. Figure 4a shows the WGA100 strain sucAThis shows cell growth (OD600), acetic acid consumption, and GABA production capacity in the WGAP strain in which the promoter of the gene was substituted with J23108 and the phlF operator. Figure 4b shows cell growth (OD600), acetic acid consumption, and GABA production capacity in a WGAP1 strain into which a vector expressing phlF with strength 1 in a 5' UTR was introduced. Figure 4c shows cell growth (OD600), acetic acid consumption, and GABA production capacity in a WGAP2 strain into which a vector expressing phlF with strength 2 in a 5' UTR was introduced. Figure 4d shows cell growth (OD600), acetic acid consumption, and GABA production capacity in a WGAP3 strain into which a vector expressing phlF with a 5' UTR having strength 3 was introduced. Figure 4e shows cell growth (OD600), acetic acid consumption, and GABA production capacity in a WGAP4 strain into which a vector expressing phlF with a 5' UTR having strength 4 was introduced. Figure 4f shows cell growth (OD600), acetic acid consumption, and GABA production capacity in a WGAP5 strain into which a vector expressing phlF with a 5' UTR having strength 5 was introduced. Figure 4g is a graph comparing cell growth (OD600), acetic acid consumption, and GABA production in recombinant strains into which a vector expressing phlF with various 5' UTRs was introduced into WGAP strains. Specific details for implementing the invention

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

[0020] The present invention iclR gene, gabT Genes and puuE Genes are deleted, gadBA recombinant microorganism for producing gamma-aminobutyric acid is provided in which a mutant gene has been introduced.

[0021] In the present invention, the term 'gene' should be considered in the broadest sense and may encode a structural protein or a regulatory protein. In this case, the regulatory protein includes transcription factors, heat shock proteins, or proteins involved in DNA / RNA replication, transcription, and / or translation. The target gene subject to expression inhibition may exist as an extrachromosomal component. Additionally, two or more of the genes may be operably linked.

[0022] The gene of the present invention is not limited to a nucleic acid molecule encoding a specific amino acid sequence (polypeptide) described above, but is interpreted to include a nucleic acid molecule encoding a polypeptide having an amino acid sequence that exhibits substantial identity with respect to the specific amino acid sequence as described above. The substantial identity means an amino acid sequence that exhibits at least 60% homology, more preferably at least 80% homology, and most preferably at least 90% homology when the amino acid sequence encoded by the gene of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Furthermore, the polypeptide having said identity includes, for example, a polypeptide of an amino acid sequence in which one or more amino acids are lost, substituted, inserted, and / or added. Such a polypeptide comprises a polypeptide related to GABA synthesis that consists of an amino acid sequence in which one or more amino acid residues are lost, substituted, inserted, and / or added, and it is preferable that the number of loss, substitution, insertion, and / or addition of amino acid residues is small.

[0023] The above iclR gene, gabT Genes and puuEThe gene is involved in the GABA competitive metabolic pathway, and the gene was deleted for efficient GABA production. The above iclR The gene is a gene encoding a repressor of the glyoxylate synthesis pathway that directs acetyl CoA toward succinic acid production, thereby reducing carbon flux through acetate and sustaining the recycling of acetyl CoA, and can be represented by the nucleotide sequence of SEQ ID NO. 1. gabT The gene is a gene encoding GABA aminotransferase, which converts GABA to succinic semialdehyde, as described above gabT The degradation of GABA produced by inactivating the gene can be inhibited, and can be represented by the nucleotide sequence of SEQ ID NO. 2. The above puuE The gene is a gene encoding GABA aminotransferase, as described above puuE The degradation of GABA produced by inactivating the gene can be inhibited and can be represented by the nucleotide sequence of SEQ ID NO. 3.

[0024] The above gadB The gene encodes glutamate decarboxylases (Gad), which can convert glutamate into GABA, and is H+ dependently activated; wild-type GadB has optimal activity at pH 4.5 and is inactivated at pH 7.6. gadB By mutating into the mutant gene (gadB E89Q Δ452-466) gadB-mut GadB transcribed by can be activated over a wide pH range and overproduce GABA. The above gadB The mutation can encode glutamate decarboxylase, which is the mutation E89Q, Δ452-466, and can be represented by the nucleotide sequence of SEQ ID NO. 5.

[0026] In the present invention, the recombinant microorganism comprises one promoter selected from the group consisting of promoters represented by SEQ ID NOs. 9 to 12 and aceA additionally introduced genes thing It is possible.

[0027] During acetic acid metabolism, isocitrate is cleaved into succinate and glyoxylate, and this reaction aceA It is promoted by isocitrate lyase encoded by a gene. In the present invention, the above aceA It may be constructed using promoters of varying strengths for the purpose of regulating genes at various levels during the transcription stage. The aforementioned promoters of various strengths aceA Genes can be expressed by precisely regulating them at the transcription level. In a specific embodiment of the present invention, a promoter having the nucleotide sequences represented by SEQ ID NOs. 9 to 12 aceA It can be used to regulate gene expression.

[0028] In the present invention, "promoter" refers to an untranscribed nucleic acid sequence upstream of a coding region that includes a binding site for polymerase and has transcription initiation activity into mRNA of a gene downstream of the promoter, i.e., a DNA region to which polymerase binds to initiate transcription of the gene. The promoter may be located at the 5' region of the mRNA transcription initiation site.

[0029] The promoter nucleic acid molecule of the present invention can be isolated or prepared using standard molecular biology techniques. For example, it can be prepared using standard synthesis techniques utilizing an automated DNA synthesizer, but is not limited thereto.

[0030] In the present invention, the promoters can bring about the expression of a target gene operably linked to a nucleic acid molecule having the promoter activity in a target microorganism.

[0031] In addition, the promoter sequence of the present invention can be easily modified by a person skilled in the art by conventionally known mutagenic methods, such as directional evolution and site-specific mutagenic methods. Accordingly, the promoter may include, without limitation, nucleotide sequences exhibiting homology of 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more, even more specifically 98% or more, and most specifically 99% or more with respect to the nucleotide sequences of SEQ ID NOs. 9 to 13. Furthermore, nucleotide sequences having such homology that possess promoter activity should be interpreted as being included within the scope of the present invention, provided that some of the sequences are deleted, modified, substituted, or added.

[0032] In the present invention, the term "homology" refers to the percentage of identity between two polynucleotide or polypeptide moieties. Homology between sequences from one moiety to another can be determined by known techniques. For example, homology can be determined by aligning sequence information and using readily available computer programs to directly align parameters such as score, identity, and similarity between two polynucleotide molecules or two polypeptide molecules (e.g., BLAST 2.0). Additionally, homology between polynucleotides can be determined by hybridizing the polynucleotides under conditions of stable double strands between homologous regions, then digesting them with a single-strand-specific nuclease to determine the size of the degraded fragment.

[0034] In the present invention, the recombinant microorganism is sucAA promoter located upstream of the gene replaced with the promoter designated as sequence number 10 and the phlF operator designated as sequence number 14. thing It is possible.

[0035] The above sucA The gene is a gene encoding succinyl-CoA synthetase, which can be represented by the nucleotide sequence of SEQ ID NO. 7.

[0037] In the present invention, the recombinant microorganism may further include a phlF gene and a synthetic 5' UTR for regulating the expression of the phlF gene.

[0038] In the present invention, the 5' UTR (untranslated region) is a non-translating region located at the 5' end of mRNA. Generally, the 5' UTR of mRNA performs various functions during the gene expression process, but among these functions, the most significant feature is its involvement in regulating mRNA translation efficiency. It has been reported that the nucleotide sequence of the 5' UTR located adjacent to the translation initiation codon affects the efficiency of the translation step. Furthermore, research results have been reported regarding sequences belonging to the 5' UTR that can be considered ribosome binding site sequences in eukaryotes, even though they are not located at a fixed position like the Shine-Dalgarno sequence, which is known as a ribosome binding site sequence located in the 5' UTR in prokaryotes. In one embodiment of the present invention, the synthetic 5' UTR for regulating the expression of the phlF gene may be one selected from the group consisting of nucleotide sequences represented by SEQ ID NOs 15 to 19. The above phlF gene and 5' UTR can be regulated by a promoter indicated by sequence number 13.

[0039] Microorganisms that can be used to produce the recombinant microorganism of the present invention are bacteria, yeast, mold, etc., and preferably Escherichia coli ( E. coli ), Bacillus genus ( Bacillus sp. ), genus Pseudomonas ( Pseudomonas sp. ), Agrobacterium genus ( Agrobacterium sp. ), inside Rhodobacter ( Rhodobacter sp. ), Erwinia genus ( Erwinia sp. ...etc., and more preferably, it may be E. coli.

[0041] In addition, the present invention provides a method for producing gamma-aminobutyric acid comprising the step of culturing the recombinant microorganism.

[0042] In a specific embodiment of the present invention, any medium used for the cultivation of ordinary microorganisms may be used, and the culture may be performed under aerobic conditions while controlling the temperature, pH, etc., in a conventional medium containing a suitable carbon source, nitrogen source, amino acid, vitamin, etc., but is not limited thereto. Preferably, the medium may be one in which acetic acid is used as a carbon source.

[0043] In a specific embodiment of the present invention, the medium may further comprise an inorganic compound, examples of which include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Additionally, the medium may further comprise amino acids, vitamins, and suitable precursors. These media or precursors may be added to the culture in a batch or continuous manner.

[0044] In addition, during the cultivation of the recombinant microorganism according to the present invention, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during cultivation, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress the formation of bubbles. Furthermore, to maintain an aerobic state of the culture, oxygen or an oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain anaerobic and aerobic states.

[0045] The temperature of the culture medium can usually be set to 27°C to 37°C, preferably 30°C to 35°C. The culture period can be continued until a desired amount of useful substance is obtained, and preferably, the culture can be carried out for 10 to 100 hours.

[0046] In a specific embodiment of the present invention, the method for producing gamma-aminobutyric acid may further include a step of further purifying or recovering gamma-aminobutyric acid produced in a culture step, and the method for recovering gamma-aminobutyric acid from a recombinant microorganism or culture may use methods known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC, but is not limited thereto. The recovery step may include a purification process, and a person skilled in the art may select and utilize one of several known purification processes as needed.

[0048] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0050] <Example 1> Confirmation of GABA production capacity of recombinant microorganisms with eliminated competitive metabolic pathways and overexpression of GABA synthase

[0051] To construct recombinant microorganisms for GABA production We constructed a recombinant microorganism that eliminated a competing metabolic pathway and overexpressed GABA synthase.

[0052] Specifically, known for having excellent acetic acid metabolic ability Escherichia coli To remove competitive metabolic pathways in W strain (KCTC1039) iclR gene, gabT gene, puuEA WG strain with a deleted gene was constructed. Using the pETDuet-1 vector as a template on the above WG strain, the wild-type GABA synthase was produced by utilizing the Tac promoter and a synthetic 5' UTR sequence to maximize gene expression. gadB The WG1 strain was constructed by introducing a gene. In addition, the E89Q Δ452-466 ​​mutation, known to maintain activity even at around pH 7.0, was introduced into the WG strain. gadB_mut WG2 was constructed by introducing the vector. As a control, WG0 was prepared by introducing the pETDuet-1 vector into the WG strain. The constructed strains WG0, WG1, and WG2 are as follows.

[0053] - WG : iclR, gabT, puuE Gene-deleted E. coli W

[0054] - WG0 (Control): WG strain introduced with pETDuet-1 vector

[0055] - WG1 : Wild type gadB WG strains introduced with a gene-containing pET vector

[0056] - WG2: E89Q Δ452-466 ​​mutation applied gadB WG strain introduced with a pET vector containing (Sequence No. 1)

[0058] Among the strains established above (WG0, WG1, WG2), an experiment was conducted to determine the strain with the highest GABA production in a pH 6.5 environment most suitable for E. coli growth by checking the acetic acid consumption and GABA production.

[0059] Specifically, individual colonies were obtained by culturing each of the three strains mentioned above on solid LB agar plates. The obtained individual colonies were cultured in test tubes containing liquid LB medium at 37°C and 200 rpm for approximately 12 hours. The cultured strains were diluted 1 / 100 in test tubes containing production medium and cultured at 37°C and 200 rpm. 600The culture was carried out until the value reached 0.7–1.0. Subsequently, the cultured strain was placed in 25 mL of production medium contained in a 300 mL round-bottom flask with OD. 600 The cells were inoculated to achieve a value of 0.05 and cultured at 37°C and 200 rpm. The production medium used for culture was based on M9 medium, with an additional 2 g / L yeast extract and 5 g / L acetic acid added as a carbon source. Every 6 hours of culture, 1 mL of the culture medium was centrifuged to separate the cells from the supernatant; the supernatant was then collected and quantitatively analyzed using HPLC. HPLC analysis of acetic acid was performed using an Aminex HPX-87H column as the stationary phase and 5 mM aqueous sulfuric acid as the mobile phase at a mobile phase rate of 0.6 mL / min, utilizing a Shodex RI-101 instrument for detection. GABA was analyzed by derivatizing GABA with o-phthaldialdehyde (OPA) and measuring it via HPLC. The OPA reagent for derivatization was prepared by adding 0.2 g / L OPA to 9.0 mL of methanol, followed by 1.0 mL of pH 9.0 borate buffer and 160 μL of 2-mercaptoethanol. For HPLC measurement, 1 μL of the supernatant culture, 5 μL of pH 9.0 borate buffer, and 1 μL of the OPA reagent were mixed and injected into the HPLC. A Zorbax Eclipse AAA column was used as the stationary phase, and measurements were performed using a concentration gradient with two mobile phases. Mobile phase A consisted of 1.4 g / L Na2HPO4, 3.8 g / L Na2B4O7·10H2O, and 8 mg / L NaN3, and the pH was adjusted to 7.2 using hydrochloric acid. Mobile phase B consisted of 45% methanol, 45% acetonitrile, and 10% water. The concentration gradient is as follows.

[0060] - 0-0.5 min: Mobile phase B 0%

[0061] - 0.5-18 min: Increase Mobile Phase B from 0% to 57%

[0062] - 18-26 min: Increase Mobile Phase B from 57% to 100%

[0063] - 26-32 mins: Mobile phase B 100%

[0064] - 32-32.5 min: Mobile phase B from 100% to 0%

[0065] - 32.5-33 min: Mobile phase B 0%

[0066] As a result, as shown in Figures 2a to 2c, when cultured in a production medium at pH 6.5, the control WG0, which did not overexpress the GABA-producing enzyme, and the wild type gadB While WG1, to which it was overexpressed, failed to synthesize GABA at all, gadB_mut In the case of WG2, which expressed [it], it was confirmed that about 0.96 g / L of GABA was synthesized.

[0068] <Example 2> Confirmation of GABA production capacity of recombinant microorganisms by applying carbon flow optimization at the isocitrate node

[0069] Based on the WG2 strain, which was confirmed to have the best GABA production capacity in Example 1, a recombinant strain with optimized carbon flow at the isocitrate node was constructed.

[0070] The isocitrate node is the point in the TCA cycle where the flow of carbon separates into energy production and anaplerosis. At this point, the action leading to anaplerosis is aceA (Sequence No. 4) is activated by the gene. Therefore, after expressing aceA with four different promoter strengths, aceA We aimed to investigate the change in GABA production according to expression intensity. To this end, aceA Strains were constructed by introducing five types of vectors containing synthetic promoters J23100, J23108, J23110, J23114 and synthetic 5' UTR sequences, and these are as follows.

[0071] - WGA114: WG2 strain under the J23114 promoter aceAStrains into which the vector containing was introduced

[0072] - WGA110: WG2 strain under the J23110 promoter aceA Strains into which the vector containing was introduced

[0073] - WGA108: WG2 strain under the J23108 promoter aceA Strains into which the vector containing was introduced

[0074] - WGA100: WG2 strain under the J23100 promoter aceA Strains into which the vector containing was introduced

[0076] The above strains were compared with the control group WG2 in terms of acetic acid consumption and GABA production using the same culture and analysis methods described in Example 1.

[0077] As a result, as shown in Figures 3a to 3e, it can be confirmed that all recombinant strains except WGA110 showed increased GABA production compared to the control WG2. In particular, in the case of WGA100, which was measured to have the highest GABA production, it was observed that GABA production increased by approximately 69% compared to the control WG2.

[0079] <Example 3> Confirmation of GABA production capacity of recombinant microorganisms by applying carbon flow optimization at the alpha-ketoglutarate node

[0080] Based on the WGA100 strain, which was confirmed to have the best GABA production capacity in Example 2, a recombinant strain was constructed with additional carbon flow optimization at the alpha-ketoglutarate node.

[0081] The enzyme that converts alpha-ketoglutarate to succinic acid sucA In order to secure the maximum amount of precursors that can be used for GABA production by inhibiting sucA We inhibited succinic acid at different intensities using a phlF repressor library to identify the succinic acid inhibition site that maximizes GABA production. To this end, located on the chromosome of the WGA100 strain sucA WGAP strains were constructed by substituting the gene promoter with J23108 and the phlF operator. To these constructed WGAP strains, five vectors were created by inserting the phlF (SEQ No. 2) gene into the synthetic promoter J23106 and five synthetic 5' UTRs with different intensities from 1 to 5, and strains were constructed as follows.

[0082] - WGAP: From WGA100 strain sucA Strains in which the gene promoter is substituted with J23108 and the phlF operator

[0083] - WGAP1: A strain into which a vector expressing phlF via a 5' UTR with strength 1 has been introduced into the WGAP strain.

[0084] - WGAP2: A strain into which a vector expressing phlF via a 5' UTR with strength 2 has been introduced into the WGAP strain.

[0085] - WGAP3: A WGAP strain into which a vector expressing phlF via a 5' UTR with strength 3 has been introduced.

[0086] - WGAP4: A WGAP strain into which a vector expressing phlF via a 5' UTR with strength 4 has been introduced.

[0087] - WGAP5: A WGAP strain into which a vector expressing phlF via a 5' UTR with strength 5 has been introduced.

[0088] The above strains were compared with the control WGA100 in terms of acetic acid consumption and GABA production using the same culture and analysis methods described in Example 1.

[0089] As a result, as shown in Figures 4a to 4g, the recombinant strain WGAP2 produced 2.52 g / L of GABA over 24 hours. This represents an increase of approximately 55% compared to WGA100, a strain optimized at the isocitrate node, and 163% compared to WG2, which simply overexpressed gadB_mut. This demonstrates that GABA efficiency in acetic acid can be significantly increased through stepwise and sophisticated carbon flow optimization.

[0090] In summary, the inventors have significantly increased the amount of GABA that can be produced from acetic acid by selecting and optimizing, stepwise, nodes that are important for both GABA synthesis and cell growth within the metabolic pathways of Escherichia coli. Therefore, the recombinant microorganism of the present invention can be utilized in various ways in the field of GABA production.

Claims

Claim 1 (i) iclR gene, gabT Genes and puuE The gene is deleted, and (ii) the gene represented by sequence number 5 gadB Recombinant microorganism for producing gamma-aminobutyric acid (γ-aminobutyric acid, GABA) into which a mutant gene has been introduced. Claim 2 In claim 1, the part indicated by the above sequence number 5 gadB A recombinant microorganism characterized by encoding L-glutamate decarboxylase (GAD), which is the mutation E89Q, Δ452-466. Claim 3 In claim 1, the recombinant microorganism comprises one promoter selected from the group consisting of promoters represented by SEQ ID NOs 9 to 12 and aceA A recombinant microorganism characterized by the additional introduction of a gene. Claim 4 In paragraph 3, the recombinant microorganism sucA The promoter located upstream of the gene is replaced with the promoter represented by sequence number 10 and the phlF operator represented by sequence number 14, and phlF Genes and phlF A recombinant microorganism characterized by comprising a synthetic 5' UTR for regulating gene expression, wherein the synthetic 5' UTR is one selected from the group consisting of nucleotide sequences represented by SEQ ID NOs 15 to 19. Claim 5 delete Claim 6 delete Claim 7 In paragraph 4, the above phlF A recombinant microorganism characterized in that gene expression is regulated by a promoter represented by SEQ ID NO.

13. Claim 8 A recombinant microorganism according to claim 1, characterized in that the recombinant microorganism is Escherichia coli. Claim 9 A method for producing gamma-aminobutyric acid, comprising the step of culturing a recombinant microorganism according to any one of claims 1 to 4, 7 and 8. Claim 10 A method for producing gamma-aminobutyric acid according to claim 9, characterized in that the culture is carried out in a medium having acetic acid as a carbon source.

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