Recombinant bacteria, methods for constructing the same and use thereof in the production of gamma-aminobutyric acid

Abstract

This application relates to recombinant bacteria, their construction method, and their application in the preparation of γ-aminobutyric acid (GABA). The recombinant bacteria are obtained by modifying glutamate-producing bacteria. The modification includes inserting a glutamate decarboxylase gene and an exocrine protein gene into the genome of the glutamate-producing bacteria. The exocrine protein gene includes the cgl0824 gene with the nucleotide sequence shown in SEQ ID NO.1. This application, by inserting a glutamate decarboxylase gene into the genome of a glutamate-producing bacterium and simultaneously inserting the cgl0824 gene with the nucleotide sequence shown in SEQ ID NO.1 as an exocrine protein gene, combines cgl0824-mediated transport enhancement with GAD-mediated synthesis enhancement, thereby significantly increasing the GABA production of the recombinant bacteria, which can be used for GABA preparation.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to recombinant bacteria, their construction methods, and their application in the preparation of γ-aminobutyric acid. Background Technology

[0002] Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid and a major inhibitory neurotransmitter in the mammalian central nervous system. GABA has various physiological functions, including lowering blood pressure, regulating heart rate, improving sleep, reducing anxiety, and promoting kidney function. Therefore, GABA is widely used in functional foods, pharmaceutical preparations, and feed additives.

[0003] Currently, GABA production methods mainly include chemical synthesis, plant enrichment, and microbial fermentation. Among these, microbial fermentation has become the mainstream method for industrial GABA production due to its mild reaction conditions, high safety, and alignment with green and sustainable development principles. This process primarily utilizes glutamate decarboxylase within microorganisms, using L-glutamate (L-Glu) or glutamate-rich substances as substrates to generate GABA through a decarboxylation reaction. Although these methods have some effect on increasing GABA production from Corynebacterium glutamicum, the levels remain low and are insufficient to meet demand. Summary of the Invention

[0004] Based on this, this application provides a recombinant strain, its construction method, and its application in the preparation of γ-aminobutyric acid (GABA). The fermentation broth obtained using this recombinant strain exhibits a high GABA yield.

[0005] In a first aspect, this application provides a recombinant bacterium obtained by modifying a glutamate-producing bacterium, the modification comprising: inserting a glutamate decarboxylase gene and an exocytogenetic protein gene into the genome of the glutamate-producing bacterium; the exocytogenetic protein gene comprising the cgl0824 gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0006] This application research found that existing technologies for increasing GABA production generally suffer from a key problem: a mismatch between intracellular accumulation of the product and transmembrane transport efficiency. This application addresses this issue by inserting a glutamate decarboxylase gene into the genome of a glutamate-producing bacterium, enabling the conversion of glutamate into GABA. Simultaneously, it inserts the cgl0824 gene (with a nucleotide sequence as shown in SEQ ID NO. 1) as an exocrine protein gene. By combining cgl0824-mediated enhanced transport with GAD-mediated enhanced synthesis, a synergistic effect of "1+1>2" is achieved, significantly increasing the GABA production of the recombinant bacteria, making it suitable for GABA preparation. Experimental verification showed that compared to recombinant bacteria FR01, the total GABA production of the recombinant bacteria FR03 constructed in this application increased by approximately 9.5%; compared to recombinant bacteria FR04 (i.e., overexpressing only the GADMUT gene), the total GABA production of the constructed recombinant bacteria FR05 (i.e., simultaneously overexpressing both the GADMUT and cgl0824 genes) increased by approximately 46.7%, opening a new avenue for constructing high-yielding GABA strains.

[0007] In some embodiments, the exported protein comprises a protein with an amino acid sequence as shown in SEQ ID NO.2; And / or, the cgl0824 gene is derived from Corynebacterium glutamicum ATCC13032.

[0008] In some embodiments, the glutamate-producing bacterium is Corynebacterium glutamicum; Furthermore, the glutamate-producing bacterium is the Corynebacterium glutamicum FF10 strain disclosed in patent publication number CN114752544B; and / or, the glutamate decarboxylase mutant GADMUT disclosed in patent publication number CN114752544B.

[0009] In some embodiments, the modification further includes inserting a promoter of the glutamate decarboxylase gene into the genome of the glutamate-producing bacterium, wherein the promoter is the H36 promoter with a nucleotide sequence as shown in SEQ ID NO.3.

[0010] In some embodiments, the recombinant bacteria overexpress the export protein gene; Furthermore, the modification also includes inserting the promoter of the export protein gene into the genome of the glutamate-producing bacterium, wherein the promoter is the H36 promoter with a nucleotide sequence as shown in SEQ ID NO.3.

[0011] In some embodiments, the recombinant bacteria simultaneously overexpress the exocytogen gene and the glutamate decarboxylase gene; Furthermore, the modification also includes inserting the promoter of the export protein gene into the genome of the glutamate-producing bacterium, wherein the promoter is the Ptuf promoter with a nucleotide sequence as shown in SEQ ID NO.4.

[0012] A second aspect of this application provides a method for constructing the recombinant bacteria described above, characterized by comprising the following steps: Genetic modification of glutamate-producing bacteria to obtain the recombinant bacteria; the modification includes inserting a glutamate decarboxylase gene and an exocrine protein gene into the genome of the glutamate-producing bacteria, wherein the exocrine protein gene includes the cgl0824 gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0013] In some embodiments, the glutamate-producing bacterium is the Corynebacterium glutamicum FF10 strain disclosed in patent publication number CN114752544B; and the glutamate decarboxylase gene is the glutamate decarboxylase mutant GADMUT disclosed in patent publication number CN114752544B. The step of inserting the glutamate decarboxylase gene into the genome of the glutamate-producing bacterium includes: Using the glutamate-producing bacteria as a template, amplification was performed using the upstream and downstream amplification primer pairs of gdh to obtain the upstream and downstream homologous arms of gdh. Using a plasmid containing the glutamate decarboxylase mutant GADMUT as a template, amplification was performed using the H36-GADMUT amplification primer pair to obtain a glutamate decarboxylase ligation fragment. The glutamate decarboxylase ligation fragment is a glutamate decarboxylase mutant GADMUT gene fragment linked to the H36 promoter. The nucleotide sequence of the H36 promoter is shown in SEQ ID NO.3. The upstream homologous arm of the gdh, the downstream homologous arm of the gdh, and the glutamate decarboxylase linker fragment were linked to the backbone vector and then transferred into the glutamate-producing bacteria to obtain recombinant bacteria FR01.

[0014] In some embodiments, the upstream amplification primer pair of gdh includes gdh-upF with a nucleotide sequence as shown in SEQ ID NO.5 and gdh-upR with a nucleotide sequence as shown in SEQ ID NO.6; The downstream amplification primer pair of the gdh includes lldd-downF with nucleotide sequences as shown in SEQ ID NO.7 and lldd-downR with nucleotide sequences as shown in SEQ ID NO.8; The H36-GADMUT amplification primer pair includes H36-GADMUT-F with the nucleotide sequence shown in SEQ ID NO.9 and H36-GADMUT-R with the nucleotide sequence shown in SEQ ID NO.10; The backbone plasmid is Pk18mobsacB backbone plasmid.

[0015] In some embodiments, the step of inserting the export protein gene into the genome of the glutamate-producing bacterium includes: The target fragment was amplified using Corynebacterium glutamicum ATCC13032 as a template. The target fragment, the H36 promoter fragment, and the plasmid pCES were ligated and transformed into the recombinant bacterium FR01 to obtain the recombinant bacterium. The nucleotide sequence of the H36 promoter is shown in SEQ ID NO.3.

[0016] In some embodiments, in the step of amplifying with Corynebacterium glutamicum ATCC13032 as a template to obtain the target fragment, the amplification primer pair used includes nucleotide sequences such as 0824F shown in SEQ ID NO.11 and 0824R shown in SEQ ID NO.12; The plasmid pCES is described in the paper Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum.

[0017] In some embodiments, the step of inserting the export protein gene into the genome of the glutamate-producing bacterium includes: The plasmid containing the glutamate decarboxylase mutant GADMUT was used as a template for amplification to obtain the first target fragment; The first target fragment is ligated to the H36 promoter to obtain the first ligation fragment, the nucleotide sequence of which is shown in SEQ ID NO.3; The second target fragment was amplified using Corynebacterium glutamicum ATCC13032 as a template. The second target fragment is ligated to the Ptuf promoter to obtain the second ligation fragment, the nucleotide sequence of which is shown in SEQ ID NO.4; The first ligation fragment, the second ligation fragment, and plasmid pCES were ligated and transferred into the recombinant bacterium FR01 to obtain the recombinant bacterium.

[0018] In some embodiments, in the step of amplifying the plasmid containing the glutamate decarboxylase mutant GADMUT as a template to obtain the first target fragment, the amplification primer pair used includes GADF with the nucleotide sequence shown in SEQ ID NO.13 and GADR with the nucleotide sequence shown in SEQ ID NO.14; In the step of amplifying with Corynebacterium glutamicum ATCC13032 as a template to obtain the second target fragment, the amplification primer pair used includes nucleotide sequences such as 0824F shown in SEQ ID NO.11 and 0824R shown in SEQ ID NO.12; The plasmid pCES is described in the paper Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum.

[0019] In a third aspect, this application provides the use of the recombinant bacteria described above in the preparation of γ-aminobutyric acid.

[0020] The fourth aspect of this application provides a method for preparing γ-aminobutyric acid, comprising the following steps: The recombinant bacteria described above were cultured on a larger scale, and the culture medium was subjected to solid-liquid separation. The supernatant was collected to obtain γ-aminobutyric acid. Attached Figure Description

[0021] Figure 1 GABA yield from shake-flask fermentation of strains FR02 and FR01; Figure 2 The yield of GABA by shake-flask fermentation of strains FR03 and FR01; Figure 3 The yield of GABA by shake-flask fermentation of strains FR04 and FR05; Figure 4 The image shows a map of the plasmid containing the glutamate decarboxylase mutant GADMUT in the examples. Figure 5 This is a map of the Pk18mobsacB backbone plasmid. Detailed Implementation

[0022] The recombinant bacteria, their construction method, and their application in the preparation of γ-aminobutyric acid (GABA) are further described in detail below with reference to specific embodiments. This application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0024] As used herein, the terms “and / or,” “or / and,” and “and / or” may include any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0025] In this article, "one or more" refers to any one, two or more of the listed items.

[0026] In this application, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, terms such as "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0027] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0028] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0029] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0030] Unless otherwise specified, all percentage concentrations mentioned in this application refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0031] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows for temperature fluctuations within the precision range controlled by the instrument.

[0032] In this application, room temperature generally refers to 4℃~30℃, and preferably 20±5℃.

[0033] Gamma-aminobutyric acid (GABA) is a four-carbon non-protein amino acid and a major inhibitory neurotransmitter in the mammalian central nervous system. GABA has various physiological functions, including lowering blood pressure, regulating heart rate, improving sleep, reducing anxiety, and promoting kidney function. Therefore, GABA is widely used in functional foods, pharmaceutical preparations, and feed additives.

[0034] Currently, GABA production methods mainly include chemical synthesis, plant enrichment, and microbial fermentation. Among these, microbial fermentation has become the mainstream method for industrial GABA production due to its mild reaction conditions, high safety, and alignment with green and sustainable development principles. This process primarily utilizes glutamate decarboxylase within microorganisms, using L-glutamate (L-Glu) or glutamate-rich substances as substrates to generate GABA through a decarboxylation reaction.

[0035] However, a key metabolic bottleneck exists in the fermentation production of GABA using microorganisms (such as Escherichia coli, lactic acid bacteria, or yeast): the intracellular accumulation of the product is mismatched with the efficiency of transmembrane transport. Many industrial strains lack efficient GABA-specific transport systems, resulting in a large amount of synthesized GABA remaining inside the cell. High concentrations of intracellular GABA not only inhibit the activity of Glu / GABA reverse transport proteins or glutamate decarboxylase through feedback inhibition mechanisms, reducing the rate of enzymatic reactions, but also cause osmotic pressure imbalance in the cell, thereby inhibiting cell growth and ultimately limiting the overall production intensity and final yield of GABA.

[0036] In view of the above problems, the first aspect of this application provides a recombinant bacterium, which is obtained by modifying a glutamate-producing bacterium, the modification comprising: inserting a glutamate decarboxylase gene and an exocytogenetic protein gene into the genome of the glutamate-producing bacterium; the exocytogenetic protein gene comprising the cgl0824 gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0037] This application research found that existing technologies for increasing GABA production generally suffer from a key problem: a mismatch between intracellular accumulation and transmembrane transport efficiency. This application addresses this issue by using glutamate-producing bacteria as chassis cells and inserting a glutamate decarboxylase gene into these cells, enabling the conversion of glutamate into GABA. Simultaneously, the cgl0824 gene (with a nucleotide sequence as shown in SEQ ID NO. 1) is inserted as an exogenous protein gene. By combining cgl0824-mediated enhanced transport with GAD-mediated enhanced synthesis, a synergistic effect of "1+1>2" is achieved, significantly increasing the GABA production of the recombinant bacteria, making it suitable for GABA preparation. Experimental verification showed that compared to recombinant bacteria FR01, the total GABA production of the recombinant bacteria FR03 constructed in this application increased by approximately 9.5%; compared to recombinant bacteria FR04 (i.e., overexpressing only the GADMUT gene), the total GABA production of the constructed recombinant bacteria FR05 (i.e., simultaneously overexpressing both the GADMUT and cgl0824 genes) increased by approximately 46.7%, opening a new avenue for constructing high-yielding GABA strains.

[0038]

[0039] This application is the first to functionally confirm that the *Corynebacterium glutamicum* cgl0824 gene is a GABA exogenous protein gene, and creatively applies it to co-metabolic engineering, achieving a leapfrog increase in GABA production. This invention not only has significant theoretical value but also possesses enormous industrial application potential.

[0040] In some embodiments, the exported protein comprises a protein with an amino acid sequence as shown in SEQ ID NO.2.

[0041] Such as SEQ ID The sequence shown in NO.2 is: LRYPAMTSGKSTSTRGALDRYFKISERGSSIGTEIRAGVVTFFAMAYIIILNPLILGTTPDVEGNTLGIAQVAAATALAAGVMTIAFGLIARYPFGIAAGLGINTMVAVTLVSGE GLTWPEAMGLVVLDGVVIVILAVSGFRVAVFRAIPASMKAAISVGIGLFIAMIGLVDAGFVRRIPDAAGTTVPVTLGIDGSIASWPTFVFVVGVLLCGILVVRRVRGGLFIGILGTTILAIIAEAI FDSGASFENGEANAEGWSLAVPGLPDSFGGIPDLSIVGAVDLIGAFSRIGVVAATLLIFTLVLANFFDAMGTMTALGKQGNLVDDEGNLPDIKKALVVEGAGAIVGGAFSASSNTVFADSSAGVAD GARTGLANVVTGSLFLAAMFLTPLYEIVPIEAAAPVLVVVGAMMMGQVTEIDFSKFYIAFPAFLTIVIMPFTYSIANGIGVGFIMYAIMAAAAGKAKQVHWLMWLVAGLFVVFFAIDPIMEAVG*.

[0042] It should be noted that the exported proteins are not limited to proteins encoded by the cgl0824 gene, but may also include functional variants of the cgl0824 gene-encoded proteins (i.e. proteins with the same function but with minor sequence differences).

[0043] In some embodiments, the cgl0824 gene is derived from Corynebacterium glutamicum ATCC13032.

[0044] In some embodiments, the glutamate-producing bacterium is Corynebacterium glutamicum.

[0045] Furthermore, the glutamate-producing bacterium is the Corynebacterium glutamicum FF10 strain disclosed in patent publication number CN114752544B; and / or, the glutamate decarboxylase mutant GADMUT disclosed in patent publication number CN114752544B.

[0046] It should be noted that the glutamate-producing bacteria are not limited to the cells mentioned above, but can also be other cells capable of producing GABA. The glutamate decarboxylase gene is not limited to the genes mentioned above, but can also be other glutamate decarboxylase genes.

[0047] In some embodiments, the modification further includes inserting a promoter of the glutamate decarboxylase gene into the genome of the glutamate-producing bacterium, wherein the promoter is the H36 promoter with a nucleotide sequence as shown in SEQ ID NO.3.

[0048] In some embodiments, the recombinant bacteria overexpress the export protein gene; Furthermore, the modification also includes inserting the promoter of the export protein gene into the genome of the glutamate-producing bacterium, wherein the promoter is the H36 promoter with a nucleotide sequence as shown in SEQ ID NO.3.

[0049] Overexpression of the cgl0824 gene can increase the GABA production of recombinant bacteria.

[0050] In some embodiments, the recombinant bacteria simultaneously overexpress the exocytogen gene and the glutamate decarboxylase gene; Furthermore, the modification also includes inserting the promoter of the export protein gene into the genome of the glutamate-producing bacterium, wherein the promoter is the Ptuf promoter with a nucleotide sequence as shown in SEQ ID NO.4.

[0051] By simultaneously overexpressing the exogenous protein gene and the glutamate decarboxylase gene, the GABA production of the recombinant bacteria can be further increased.

[0052] A second aspect of this application provides a method for constructing the recombinant bacteria described above, characterized by comprising the following steps: Genetic modification of glutamate-producing bacteria to obtain the recombinant bacteria; the modification includes inserting a glutamate decarboxylase gene and an exocrine protein gene into the genome of the glutamate-producing bacteria, wherein the exocrine protein gene includes the cgl0824 gene with a nucleotide sequence as shown in SEQ ID NO.1.

[0053] In some embodiments, the glutamate-producing bacterium is the Corynebacterium glutamicum FF10 strain disclosed in patent publication number CN114752544B; and the glutamate decarboxylase gene is the glutamate decarboxylase mutant GADMUT disclosed in patent publication number CN114752544B. The step of inserting the glutamate decarboxylase gene into the genome of the glutamate-producing bacterium includes S110-S130: S110. Using the glutamic acid-producing bacteria as a template, amplification was performed using the upstream amplification primer pair and the downstream amplification primer pair of gdh to obtain the upstream homologous arm and the downstream homologous arm of gdh. S120. Using a plasmid containing the glutamate decarboxylase mutant GADMUT as a template, amplification was performed using the H36-GADMUT amplification primer pair to obtain a glutamate decarboxylase ligation fragment. The glutamate decarboxylase ligation fragment is a glutamate decarboxylase mutant GADMUT gene fragment linked to the H36 promoter. The nucleotide sequence of the H36 promoter is shown in SEQ ID NO.3. S130. The upstream homologous arm of the gdh, the downstream homologous arm of the gdh, and the glutamate decarboxylase linker fragment are linked to the backbone vector and transferred into the glutamate-producing bacteria to obtain recombinant bacteria FR01.

[0054] The recombinant bacterium FR01 was obtained by inserting the glutamate decarboxylase mutant GADMUT into the chassis cells.

[0055] The upstream amplification primer pair for gdh includes gdh-upF with the nucleotide sequence shown in SEQ ID NO.5 and gdh-upR with the nucleotide sequence shown in SEQ ID NO.6; The downstream amplification primer pair of the gdh includes lldd-downF with nucleotide sequences as shown in SEQ ID NO.7 and lldd-downR with nucleotide sequences as shown in SEQ ID NO.8; The H36-GADMUT amplification primer pair includes H36-GADMUT-F with the nucleotide sequence shown in SEQ ID NO.9 and H36-GADMUT-R with the nucleotide sequence shown in SEQ ID NO.10; The backbone plasmid is Pk18mobsacB backbone plasmid.

[0056] In some embodiments, the step of inserting the export protein gene into the genome of the glutamate-producing bacterium includes S210-S220: S210. Using Corynebacterium glutamicum ATCC13032 as a template, the target fragment was amplified to obtain the target fragment. S220. The target fragment, the H36 promoter fragment, and the plasmid pCES are ligated and transferred into the recombinant bacterium FR01 to obtain the recombinant bacterium. The nucleotide sequence of the H36 promoter is shown in SEQ ID NO.3.

[0057] In recombinant bacteria, by combining cgl0824-mediated transport enhancement with GAD-mediated synthesis enhancement, a synergistic effect of "1+1>2" was achieved. The yield increase (compared to overexpressing only the GADMUT gene, the total GABA yield of strains that simultaneously overexpress both the GADMUT and cgl0824 genes increased by approximately 46.7%) far exceeded the effect of single-pathway modification, opening up a new avenue for the construction of high-yield GABA strains.

[0058] In the step of amplifying Corynebacterium glutamicum ATCC13032 as a template to obtain the target fragment, the amplification primer pair used includes nucleotide sequences such as 0824F shown in SEQ ID NO.11 and 0824R shown in SEQ ID NO.12; The plasmid pCES is described in the paper Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum.

[0059] In some embodiments, the step of inserting the export protein gene into the genome of the glutamate-producing bacterium includes S310-S350: S310. Using the plasmid containing the glutamate decarboxylase mutant GADMUT as a template, amplification is performed to obtain the first target fragment; S320. The first target fragment is ligated to the H36 promoter to obtain a first ligation fragment, wherein the nucleotide sequence of the H36 promoter is shown in SEQ ID NO.3; S330, using Corynebacterium glutamicum ATCC13032 as a template, was amplified to obtain the second target fragment; S340. The second target fragment is ligated to the Ptuf promoter to obtain the second ligation fragment, wherein the nucleotide sequence of the Ptuf promoter is shown in SEQ ID NO.4; S350. The first ligation fragment, the second ligation fragment, and plasmid pCES are ligated and transferred into the recombinant bacteria FR01 to obtain the recombinant bacteria.

[0060] Compared with overexpressing only the GADMUT gene, the recombinant strain that simultaneously overexpressed both the GADMUT and cgl0824 genes achieved a significant increase in GABA production of up to 46.7%.

[0061] In the step of amplifying the plasmid containing the glutamate decarboxylase mutant GADMUT as a template to obtain the first target fragment, the amplification primer pair used includes GADF with the nucleotide sequence shown in SEQ ID NO.13 and GADR with the nucleotide sequence shown in SEQ ID NO.14. In the step of amplifying with Corynebacterium glutamicum ATCC13032 as a template to obtain the second target fragment, the amplification primer pair used includes nucleotide sequences such as 0824F shown in SEQ ID NO.11 and 0824R shown in SEQ ID NO.12; The plasmid pCES is described in the paper Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum.

[0062] The recombinant bacteria obtained above can be used to prepare γ-aminobutyric acid.

[0063] This application provides a method for preparing γ-aminobutyric acid, comprising the following steps: The recombinant bacteria described above were cultured on a larger scale, and the culture medium was subjected to solid-liquid separation. The supernatant was collected to obtain γ-aminobutyric acid.

[0064] The culture medium for expanding the recombinant bacteria can be, for example, CGXII medium. The composition of CGXII medium is: glucose 50 g / L, (NH4)2SO4 20 g / L, urea 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.25 g / L, CaCl2·2H2O 13.3 mg / L, MOPS 42 g / L, biotin 0.2 mg / L, and trace element solution 1 ml / L. The pH is adjusted to 7.0 using KOH. The trace element solution contains: FeSO4·7H2O 10 g / L, MnSO4·1H2O 10 g / L, ZnSO4·7H2O 1 g / L, CuSO4·5H2O 313 mg / L, and NiCl·6H2O 20 mg / L. It should be noted that the culture medium used for expansion culture is not limited to CGXII medium, but can also be other media.

[0065] Solid-liquid separation can be achieved through methods such as centrifugation. In a specific example, the centrifugation speed is 12,000 rpm, and the rotation time is 1 minute. It should be noted that solid-liquid separation is not limited to centrifugation; other methods, such as filtration, can also be used.

[0066] Methods for detecting γ-aminobutyric acid in the supernatant include, for example, liquid chromatography.

[0067] The recombinant bacteria described in this application can produce high levels of γ-aminobutyric acid (GABA), laying the foundation for large-scale industrial production of GABA.

[0068] This application finds that existing methods for increasing γ-aminobutyric acid (GABA) yield mainly have the following problems: ① Low product efflux efficiency (“rate-limiting step”): The main function of antitransporters in existing technologies is substrate uptake. Their affinity for the product GABA and their efflux flux are usually insufficient to match the intracellular GABA synthesis rate during high-intensity fermentation. This leads to the rapid accumulation of GABA intracellularly, reaching toxic concentrations.

[0069] ② Feedback inhibition and metabolic flux obstruction: High intracellular GABA concentrations can have feedback inhibition or transmembrane inhibition effects on upstream pathways, reducing the glutamate / GABA concentration gradient across the cell membrane. This leads to the stagnation of concentration gradient-dependent reverse transport processes, and the reaction kinetic equilibrium shifts towards the reverse reaction direction.

[0070] ③ Trade-off between cell growth and product synthesis: The accumulation of intracellular GABA increases the osmotic pressure burden on cells, leading to excessive consumption of ATP to maintain intracellular homeostasis rather than for cell growth or product synthesis, resulting in a trade-off between biomass and yield.

[0071] ④ Downstream extraction difficulties: Due to the high proportion of intracellular GABA, existing processes often require cell wall disruption to extract the product, which significantly increases the cost and energy consumption of downstream separation and purification.

[0072] To address the technical problems in existing technologies, such as severe product feedback inhibition due to GABA intracellular accumulation, impaired metabolic flux, low efflux efficiency, and high extraction costs, the purpose of this invention is: ① Relieving product feedback inhibition: This invention provides a highly efficient GABA transporter and its application. By rapidly and actively transporting intracellularly synthesized GABA to the extracellular space, the intracellular GABA concentration is reduced, thereby relieving product feedback inhibition on the Glu / GABA transport system and glutamate decarboxylase, and maintaining a high rate of enzymatic reaction.

[0073] ② Improve bioconversion efficiency and yield: By utilizing this transporter protein to enhance the transmembrane recycling of substrate L-glutamate, the molar conversion rate, volumetric production intensity and final fermentation titer of GABA are significantly improved while reducing intracellular toxicity.

[0074] ③ Simplified separation process: By achieving efficient extracellular secretion of GABA, the target product is mainly present in the fermentation broth, avoiding complex cell disruption steps and reducing the industrial cost of downstream separation and purification.

[0075] ④ Enhance the industrial adaptability of strains: improve the physiological state of engineered strains in the later stage of fermentation, alleviate the inhibitory effect of high concentration of products on cell growth, and achieve decoupling or positive coupling between cell growth and product synthesis.

[0076] This application's technical solution improves GABA production based on the following core principles: First, gene knockout completely inactivates the gene. If the gene encodes a GABA efflux protein, its loss of function will block the GABA transport pathway from the cell to the extracellular space, significantly reducing the total amount of GABA secreted into the fermentation broth. Second, plasmid overexpression enhances the expression of the target gene. If the gene encodes a GABA efflux protein, its overexpression will increase the number of functional transport proteins on the cell membrane, thereby increasing the rate of GABA efflux and ultimately resulting in an increase in the total GABA production in the fermentation broth.

[0077] The technical solution of this application has the following beneficial effects: (1) It provides a novel and functionally confirmed GABA export protein gene: The cgl0824 gene is a GABA export protein whose function has been clearly verified in Corynebacterium glutamicum, providing a key molecular target and theoretical basis for research in this field.

[0078] (2) Breaking the yield bottleneck and achieving synergistic effect: By combining cgl0824-mediated transport enhancement with GAD-mediated synthesis enhancement, a synergistic effect of "1+1>2" was achieved. The yield increase (compared to overexpressing only the GADMUT gene, the total GABA yield of strains that simultaneously overexpress both the GADMUT and cgl0824 genes increased by about 46.7%) far exceeded the effect of single-pathway modification, opening up a new path for the construction of high-yield GABA strains.

[0079] (3) Effectively alleviate product inhibition and improve cell physiological performance: The efficient outflow mechanism reduces the accumulation of intracellular GABA, reduces the feedback inhibition on key enzymes and cell metabolism, and helps maintain high cell activity and long-term stable production.

[0080] (4) Reduce downstream production costs: Promote the secretion of GABA into the extracellular space, simplify the separation and purification process of fermentation broth, and are expected to eliminate the energy-intensive cell disruption step, thereby significantly reducing the industrial production cost of GABA.

[0081] In summary, this application is the first to functionally confirm that the *Corynebacterium glutamicum* cgl0824 gene is a GABA export protein gene, and creatively applies it to co-metabolic engineering, achieving a leapfrog increase in GABA production. This invention not only has significant theoretical value but also possesses enormous industrial application potential.

[0082] The following is a specific embodiment.

[0083] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0084] The raw materials and reagents involved in the following specific embodiments can be obtained commercially or prepared by those skilled in the art using known methods.

[0085] Unless otherwise specified, in these examples, the amplification enzyme used was Novizan PCR amplification enzyme, catalog number: P515-01, and the amplification reaction system was set up according to the enzyme's instructions for use; the ligation kit (i.e., the kit used for Gibson method ligation) was assembled by Gibson. ® The cloning kit was from NEB, catalog number: E5510S; the DNA purification kit was from TIANGEN, catalog number: DP214-02.

[0086] Unless otherwise specified, the PCR amplification reaction system in the following examples is shown in Table 1, and the PCR amplification reaction procedure is shown in Table 2. In Table 1, template DNA* represents a bacterial strain, plasmid, or gene fragment, etc.

[0087] Table 1 PCR amplification reaction system

[0088] Table 2 PCR amplification reaction procedure

[0089] Example 1: Construction of GABA-producing test strain FR01 Glutamate decarboxylase (GAD) is a normally synthesized enzyme protein in living organisms that removes one molecule of CO2 from the α-carboxyl group of L-glutamic acid to obtain γ-aminobutyric acid (GABA). In this embodiment, strain FF10 (i.e., FF9 Δlldd::Ptufgdh, the construction process of which can be found in the published patent CN114752544B) was used as the starting strain. Referring to the construction process of strain FF10 (FF9 Δlldd::Ptufgdh) in published patent CN114752544B, the glutamate decarboxylase mutant gene GADMUT (the nucleotide sequence of the glutamate decarboxylase mutant GADMUT is shown in SEQ ID NO. 15, and its detailed description can be found in published patent CN114752544B) was used to insert the glutamate decarboxylase mutant gene GADMUT after the gdh gene, enabling the resulting recombinant strain FR01 (i.e., FF10 / pk18::GADMUT, also named FT01) to produce GABA.

[0090] The sequence shown in SEQ ID NO.15 is as follows: ATGCCTCAAT GGCATCCGCA TCGTGAACAA AAAAATTTGC CTGATGAATT TCCTGTTAATCCGCTTTTTT CTCGACAAGG AGAAGTGACA ATTCCAAGAC TGCGTATCGG TAATCAAGGT ATGCTTCCGGAAACGGCTTA TCAAATCATT CATGACGAAA TTGCTTTAGA CGGAAATGCC CGCTTGAATT TAGCTACGTTTGTTACTACG TGGATGGAGC CTGATGCAAA GCGTTTGTAC GGAGAATCTT TTGATAAAAA TATGGTCGATAAAAATCAGT ATCCGCAGAC AGCGGCTATT GAAGAGAGAT GTGTACGTAT TTTAGCGGAT TTGTGGAATTCACCTAATCC TGATACCACG ATGGGCGTTT CTACTACAGG TTCATCTGAA GCATGTATGC TTGGTGGACTAGCGTTAAAA AGACGATGGC AGAAACTGCG TAAAAGTAAA GGGCTAACAACGGACCGCCC CAATATTGTATTTAGTTCAT CGGTTCAAGT GGTATGGGAG AAGTTCGCAA ACTATTGGGA CGTAGAGCCT CGTTATGTGAATATTAATCC AGATCATCCT TATTTAGATG CAGAAGGCGT GATTAATGCG GTTAATGAAA ATACAATTGGCGTCGTACCG ATTCTTGGAG TCACGTATAC AGGGGGTTAC GAACCAATAG CTGCTATCGC AAAAGCATTAGATGAGTTAC AGGAAAAAAC AGGGTTGGAT ATTCCTATCC ATGTGGATGC TGCTTCTGGA GGTTTTATCGCTCCATTTCT TCAACCAGAC CTTATCTGGG ATTTCCGCTT GACGCGAGTA AAGTCCATTA ACGTGTCAGGACACAAGTAT GGTTTAGTTT ACCCTGGCTT GGGATGGGTG ATTTGGAGAA GAAAAGAGGA CTTGCCTGAAAATCTTATTT TCCGCGTTTCTTATTTAGGG GGCAACATGC CAACTTTTGC GCTCAACTTC TCTAGACCAGGAGCACAAGT CCTTTTGCAG TACTACAATT TCTTGCGTTT AGGTAAAGAC GGCTATTATG CCGTGCAAAAAACCTCCCAA GAAAACGCGC TGTATCTTAG CAAAGAAATTGGAGAAATGG ACGCATTCGA AATTCTTGCTGATGGTTCAG ATATCCCGGT TCTTGCTTGG AAACTGAAAG AAGACTATAC ACCAAACTGG ACTCTTTATGATTTGTCTAG ACAACTGCGTACGTACGGAT GGCAAGTTCC AGCTTACCCA CTCCCAGCAAG ACATGGAAGAAATCACAATC ATGCGCATTG TTGTTAGAAA TGGGTTTTCA AGAAACCTTG CTCAATTATTTATGGTTAATTTCAAACAAG CCGTTGAATT TCTTAACTCG TAG.

[0091] The specific construction process of the recombinant strain FR01 (i.e., FF10 / pk18::GADMUT) is as follows: 1. Obtaining the upstream and downstream homologous arms: Using strain FF10 as a template, amplification enzymes were employed, and upstream and downstream homologous arms of the insertion site (1000 bp each) were cloned using upstream and downstream homologous arm amplification primer pairs, respectively. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction program is shown in Table 2.

[0092] The upstream homologous arm amplification primer pair is as follows: gdh-upF: gtaaaacgacggccagtgccaagcttgcatgTAAAAACTCCCTAACCGGCCTGCCAATC (as shown in SEQ IDNO.5), gdh-upR: ccccgtttagggcaccagatagaTTAGATGACGCCCTGTGCCAGCATCGCGTCAGCTA (as shown in SEQ ID NO. 6).

[0093] The downstream homologous arm amplification primer pairs are as follows: lldd-downF:CAAGCCGTTGAATTTCTTAACTCGTAGAAGTTTCTCTCCTTAGCTATTAAAAGGTGCCC (as shown in SEQ ID NO.7), lldd-downR:gattacgaattcgagctcggtacccgggAAGTCCTAGGTGGGATGCGAGGCTTTCAAG (as shown in SEQ ID NO. 8).

[0094] 2. Obtaining the GADMUT gene fragment: Use the already saved plasmid (see plasmid map for details). Figure 4 For plasmid sequences, please refer to [link / reference]. https: / / benchling.com / s / seq-3pKulrjUCDjxMkfnqbpK?m=slm-zeSceWXxqoIHpKYpBDbx Using a template, the H36-GADMUT amplification primer pair was used to clone the GADMUT gene, a glutamate decarboxylase mutant ligated with the H36 promoter, i.e., the glutamate decarboxylase ligation fragment. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction procedure is shown in Table 2.

[0095] The H36-GADMUT amplification primer pair includes: H36-GADMUT-F:CGATGCTGGCACAGGGCGTCATCTAAtctatctggtgccctaaacgggggaatattaac (as shown in SEQ ID NO. 9). H36-GADMUT-R:GGCACCTTTTAATAGCTAAGGAGAGAAACTTCTACGAGTTAAGAAATTCAACGGCTTG (as shown in SEQ ID NO.10).

[0096] 3. Using a ligation kit, the glutamate decarboxylase ligation fragment and its upstream and downstream homologous arms were ligated to the Pk18mobsacB backbone (its nucleotide sequence is shown in SEQ ID NO.20, and its plasmid map is shown in...). Figure 5 (As shown) The plasmid was ligated using the Gibson method and transformed into E. coli DH5α. Sequencing confirmed successful construction, yielding the recombinant plasmid pk18::GADMUT. The obtained recombinant plasmid pk18::GADMUT was transformed into Corynebacterium glutamicum FF10 competent cells, resulting in the strain FF10 / pk18::GADMUT, named strain FR01.

[0097] Example 2: Constructing a plasmid and recombinant bacteria with the cgl0824 gene knocked out, and performing shake-flask fermentation tests. 1. Constructing the recombinant plasmid pK18-Δcgl0824: Using the genome of Corynebacterium glutamicum (ATCC13032) as a template, amplification enzymes were used, and upstream and downstream homologous arm amplification primers were used to clone the upstream and downstream homologous arms of the cgl0824 gene (Genbank accession number: BAB98217.1; PCR amplification reaction system is shown in Table 1; PCR amplification reaction program is shown in Table 2; nucleotide sequence is shown in SEQ ID NO.1; amino acid sequence is shown in SEQ ID NO.2). The homologous arms of the cgl0824 gene (Genbank accession number: BAB98217.1; PCR amplification reaction system is shown in Table 1; PCR amplification reaction program is shown in Table 2; nucleotide sequence is shown in SEQ ID NO.1; amino acid sequence is shown in SEQ ID NO.2) were cloned, and then ligated to the Pk18mobsacB backbone using the Gibson method (using a ligation kit). The cloned gene was then transformed into E. coli DH5α, and sequencing confirmed successful construction.

[0098] The upstream homologous arm amplification primers include: 0824-upF:cgacggccagtgccaagcttgcatgCTCCATAGGTAGTCATTGACTTCAGAAAACGTCG (as shown in SEQ ID NO. 16); 0824-upR:GCGGGATCAGAAATTACCGTGCGCGTTGTCATTTCTATTCAAGTTGTGAGAAAGCTGG (as shown in SEQ ID NO.17).

[0099] Downstream homologous arm amplification primers include: 0824-downF:CCAGCTTTCTCACAACTTGAATAGAAATGACAACGCGCACGGTAATTTCTGATCCCGC (as shown in SEQ ID NO.18); 0824-downR:catgattacgaattcgagctcggtacccgggGGCACCCGATCGAGTGGTGTCCAAGCGC (as shown in SEQ ID NO. 19).

[0100] 2. Construction of recombinant strain FR01 / pK18-Δcgl0824: The recombinant plasmid pK18-Δcgl0824 was transformed into Corynebacterium glutamicum FR01 competent cells, and the resulting strain FR01 / pK18-Δcgl0824 was named FR02.

[0101] 3. Shake-flask fermentation test: (1) The FR02 strain was used to produce GABA from glucose via a one-step fermentation process. The specific fermentation process was as follows: The FR02 strain was cultured in BHIS medium (from Guangdong Huankai Microbial Technology Co., Ltd., catalog number: 024053) in test tubes at 30℃ for 16 hours to obtain seed culture; 30 mL of CGXII medium was added to a 250 mL shake flask, inoculated at a rate of 10% (v / v), fermented at 30℃, and shaker speed of 220 rpm for 96 hours. After fermentation, the fermentation broth was separated into solid and liquid components, the supernatant was collected, and the GABA yield in the supernatant was measured. The solid-liquid separation method was centrifugation at 12000 rpm for 1 min.

[0102] The composition of CGXII medium is as follows: glucose 50 g / L, (NH4)2SO4 20 g / L, urea 5 g / L, KH2PO4 1 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.25 g / L, CaCl2·2H2O 13.3 mg / L, MOPS 42 g / L, biotin 0.2 mg / L, and trace element solution 1 ml / L. The pH is adjusted to 7.0 using KOH. Among them, the trace element solution contains: FeSO4·7H2O 10 g / L, MnSO4·1H2O 10 g / L, ZnSO4·7H2O 1 g / L, CuSO4·5H2O 313 mg / L, and NiCl·6H2O 20 mg / L.

[0103] (2) Detection of GABA yield using liquid chromatography: Agilent HPLC 1290, equipped with photodiode array detector; column: ZORBAX RR Eclipse Plus C18, 95Å, 2.1 x 100 mm, 3.5 µm; mobile phase A was 95% (v / v) water-5% (v / v) acetonitrile (containing 0.1% (v / v) formic acid), and mobile phase B was methanol; derivatizing agent: 10 mg OPA (o-phthalaldehyde) was weighed, dissolved in 0.5 mL of pure methanol, 30 µL of β-mercaptoethanol was added, and 1.5 mL of 0.1 mol / L borate buffer (0.31 g boric acid and 0.19 g sodium hydroxide, dissolved in water and diluted to 50 mL); detection wavelength: 330 nm; flow rate: 0.3 mL / min; column temperature: 30 °C; injection volume: 1 μL; pre-column derivatization.

[0104] Sample preparation method: Draw 10 μL of derivatization reagent, clean the injection port for 3 seconds, draw 1 μL of sample, mix 10 times in air (quantitative loop), let stand for 0.5 min, and inject the sample; mobile phase gradient (as shown in Table 3 below, all percentages in Table 3 are volume percentages, i.e., v / v).

[0105] Table 3 Mobile phase gradient for GABA production detection by liquid chromatography

[0106] Test results as follows Figure 1 As shown. Figure 1 The yield of GABA from shake-flask fermentation of FR02 and FR01.

[0107] Figure 1 This reflects the GABA production of recombinant strains FR02 and FR01 after 96 hours of shake-flask fermentation. Figure 1 It was found that the GABA production of FR02 (4.22 g / L) was significantly lower than that of FR01 (6.62 g / L), with a total reduction of approximately 36%. This significant change is highly consistent with the theoretical expectation of loss of function. This result provides strong reverse evidence that cgl0824 is a gene essential for GABA efflux.

[0108] Example 3: Constructing a plasmid and recombinant bacteria overexpressing the cgl0824 gene, and performing shake-flask fermentation tests. 1. Construct the recombinant plasmid pCES-H36-cgl0824: Using Corynebacterium glutamicum ATCC13032 as a template, amplification was performed using primers 0824F and 0824R, and the H36 promoter with the nucleotide sequence shown in SEQ ID NO.3 was ligated. The target fragment was amplified by PCR using an amplification enzyme (PCR amplification reaction system is shown in Table 1, and PCR amplification reaction procedure is shown in Table 2). After purification with a DNA purification kit, the PCR amplification product and the backbone fragment of plasmid pCES were ligated using a ligation kit via Gibson, and transformed into E. coli DH5α. Sequencing verified the successful construction, yielding the recombinant plasmid pCES-H36-cgl0824.

[0109] Among them, 0824F:cttggttggtaggagtagcatgggatccTTGAGGTATCCGGCAATGACGTCAGGG (as shown in SEQ ID NO.11).

[0110] 0824R:ccgcgctactgccgccaggcagcggccgcTTAGCCGACAGCTTCCATGATGGGATC (as shown in SEQ ID NO.12).

[0111] The sequence shown in SEQ ID NO.3 is as follows: tctatctggtgccctaaacgggggaatattaacgggcccagggtggtcgcaccttggttggtaggagtagcatgggatcc.

[0112] The plasmid pCES was published in the paper Choi, JW, Yim, SS, & Jeong, KJ (nd). Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum . Applied Microbiology and Biotechnology . https: / / yimlabkaist.github.io / assets / pdf / 20.%20Cg%20high%20copy%20number%20plasmid_ supp.pdf . 2. Construction of recombinant strain FR01 / pCES-H36-cgl0824: The expression vector pCES-H36-cgl0824 carrying the coding sequence of cgl0824 was transformed into Corynebacterium glutamicum FR01 to obtain the recombinant strain FR01 / pCES-H36-cgl0824, which was named FR03.

[0113] 3. Shake-flask fermentation test: GABA was produced from glucose via a one-step fermentation process using strain FR03. Strains FR03 were cultured in BHIS medium (see Example 2 for details) in test tubes at 30°C for 16 hours to obtain a seed culture. 30 mL of CGXII medium (see Example 2 for details) was added to a 250 mL shake flask, inoculated at a 10% (v / v) inoculation rate, and fermented at 30°C and 220 rpm for 96 hours. The method for detecting GABA yield using liquid chromatography was the same as in Example 2. The detection results are as follows: Figure 2 As shown. Figure 2 The yield of GABA from shake-flask fermentation of FR03 and FR01.

[0114] Figure 2 The results showed the GABA production of recombinant strains FR03 and FR01 after 96 hours of shake-flask fermentation. The GABA production of FR03 (7.25 g / L) was significantly higher than that of FR01 (6.62 g / L), with a total yield increase of approximately 9.5%. This significant positive phenotypic change is consistent with theoretical expectations and provides direct positive evidence that cgl0824 has the function of promoting GABA excretion.

[0115] Example 4: Application of the cgl0824 gene in co-metabolic engineering, and further verification of the function of the cgl0824 gene. This strategy aims to simultaneously enhance the two core processes of GABA production: "synthesis" and "export," by simultaneously overexpressing the cgl0824 and GAD genes to increase GABA yield.

[0116] 1. Construction of strain FR04: Construct the recombinant plasmid pCES-H36-GADMUT, overexpress the gene GADMUT, and design amplification primers: GADF:ggagtagcatgggatccATGCCTCAATGGCATCCGCATCG (as shown in SEQ ID NO.13); GADR:ctactgccgccaggcagcggccgCTACGAGTTAAGAAATTCAACGGC (as shown in SEQ ID NO.14); Using the H36 promoter, with the already saved plasmid (see plasmid map for details) Figure 4 Using plasmid sequences (see https: / / benchling.com / s / seq-3pKulrjUCDjxMkfnqbpK?m=slm-zeSceWXxqoIHpKYpBDbx) as templates, the target fragment was amplified by PCR using amplification enzymes (PCR amplification reaction system is shown in Table 1, and PCR amplification reaction procedure is shown in Table 2). After purification with a DNA purification kit, the PCR amplification product and the backbone fragment of plasmid pCES (see Example 3 for details) were ligated using a ligation kit via the Gibson method. This ligation was then transformed into E. coli DH5α, and sequencing verified successful construction, yielding the expression vector pCES-H36-GADMUT carrying the GAD coding sequence. The expression vector pCES-H36-GADMUT carrying the GAD coding sequence was transformed into Corynebacterium glutamicum FR01 to obtain the recombinant strain FR01 / pCES-H36-GADMUT, named FR04.

[0117] 2. Construction of strain FR05: The recombinant plasmid pCES-H36-GADMUT-Ptuf-cgl0824 was constructed to simultaneously overexpress the GADMUT gene and cgl0824: The GADMUT gene and cgl0824 genes were selected as described above. The GADMUT gene used the H36 promoter (mentioned above), and the cgl0824 gene used the Ptuf promoter (nucleotide sequence shown in SEQ ID NO.4). The specific process is as follows: (1) Using a plasmid containing the glutamate decarboxylase mutant GADMUT (see plasmid map for details) Figure 4 For plasmid sequences, please refer to [link / reference]. https: / / benchling.com / s / seq-3pKulrjUCDjxMkfnqbpK?m=slm- zeSceWXxqoIHpKYpBDbxUsing a template, an amplification enzyme and amplification primer pair (the nucleotide sequence GADF shown in SEQ ID NO. 13 and the nucleotide sequence GADR shown in SEQ ID NO. 14) were used for PCR amplification to obtain the first target fragment. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction program is shown in Table 2. A ligation kit (Gibson assembly kit from NEB) was used. ® Cloning kit (catalog number: E5510S) ligates the first target fragment to the H36 promoter to obtain the first ligation fragment; (2) Using Corynebacterium glutamicum ATCC13032 as a template, amplification enzyme and amplification primer pair (i.e., primers 0824F and 0824R mentioned above) were used for PCR amplification to obtain the second target fragment. The PCR amplification reaction system is shown in Table 1, and the PCR amplification reaction program is shown in Table 2. A ligation kit (Gibson kit from NEB) was used for assembly. ® Cloning kit (catalog number: E5510S) ligates the second target fragment to the Ptuf promoter to obtain the second ligated fragment; (3) After the first and second ligation fragments were purified by a DNA purification kit, the PCR amplification product and the backbone fragment of plasmid pCES (see Example 3 for details) were ligated using a ligation kit (Gibson Assembly® Cloning Kit from NEB; catalog number: E5510S) via the Gibson method. The ligation product was then transformed into E. coli DH5α, and sequencing was used to verify the successful construction, resulting in the expression vector pCES-H36-GADMUT-Ptuf-cgl0824 carrying the GAD coding sequence and the cgl0824 coding sequence.

[0118] (4) The expression vector pCES-H36-GADMUT-Ptuf-cgl0824 carrying the GAD coding sequence and the cgl0824 coding sequence was transformed into Corynebacterium glutamicum FR01 to obtain the recombinant strain FR01 / pCES-H36-GAD-Ptuf-cgl0824, which was named FR05.

[0119] The sequence shown in SEQ ID NO.4 is as follows: CAGATGTTATTGCTGAGCGCAACGGCACCGCTTCCTAAAGATCGTTTAGATCCGAAGGAAAACGTCGAAAAGCAATTTGCTTTTCGACGCCCCACCCCGCGCGTTTTAGCGTGTCAGTAGGCGCGTAGGGTAAGTGGGGTAGCGGCTTGTTAGATATCTTGAAATCGGCTTTCAACAGCATTGATTTCGATGTATTTAGC TGGCCGTTACCCTGCGAATGTCCACAGGGTAGCTGGTAGTTTGAAAATCAACGCCGTTGCCCTTAGGATTCAGTAACTGGCACATTTTGTAATGCGCTAGATCTGTGTGCTCAGTCTTCCAGGCTGCTGATCACAGTGAAAGCAAAACCAATTCGTGGCTGCGAAAGTCGTAGCCACCACGAAGTCCAGGAGGACATACA.

[0120] 3. Shake-flask fermentation test: The FR04 and FR05 strains constructed in Example 4 were used to produce GABA from glucose via a one-step fermentation method. Strains FR04 and FR05 were cultured in BHIS medium (see Example 2 for details) in test tubes at 30°C for 16 hours to obtain seed culture. 30 mL of CGXII medium (see Example 2 for details) was added to a 250 mL shake flask, inoculated at a 10% (v / v) inoculation rate, and fermented at 30°C and 220 rpm for 96 hours. GABA yield was detected using liquid chromatography, following the method described in Example 2. The detection results are as follows: Figure 3 As shown, Figure 3 The yield of GABA by shake-flask fermentation of FR04 and FR05 bacteria.

[0121] Figure 3 The results show the GABA yields of FR04 and FR05 strains after 96 hours of shake-flask fermentation. Compared to the strain that only overexpressed the GADMUT gene (i.e., FR04, with a GABA yield of 11.79 g / L), the recombinant strain that simultaneously overexpressed both the GADMUT and cgl0824 genes (i.e., FR04, with a GABA yield of 17.3 g / L) achieved a significant increase in GABA yield of up to 46.7%. This increase not only verifies that the transport link is indeed a key bottleneck when increasing the synthesis throughput of the cgl0824 gene, but also, from an application perspective, demonstrates the core pivotal role of cgl0824 in GABA transport.

[0122] In summary, this application is the first to functionally confirm that the *Corynebacterium glutamicum* cgl0824 gene is a GABA export protein gene, and creatively applies it to co-metabolic engineering, achieving a leapfrog increase in GABA production. This invention not only has significant theoretical value but also possesses enormous industrial application potential.

[0123] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A recombinant bacterium, characterized in that, The recombinant bacteria were obtained by modifying glutamate-producing bacteria. The modification included inserting a glutamate decarboxylase gene and an exocrine protein gene into the genome of the glutamate-producing bacteria. The exocrine protein gene included the cgl0824 gene with a nucleotide sequence as shown in SEQ ID NO.

1.

2. The recombinant bacteria according to claim 1, characterized in that, The exported protein includes proteins with amino acid sequences as shown in SEQ ID NO.2; And / or, the cgl0824 gene is derived from Corynebacterium glutamicum ATCC13032.

3. The recombinant bacteria according to claim 1, characterized in that, The glutamate-producing bacterium is Corynebacterium glutamicum; Furthermore, the glutamate-producing bacterium is the Corynebacterium glutamicum FF10 strain disclosed in patent publication number CN114752544B; and / or, the glutamate decarboxylase mutant GADMUT disclosed in patent publication number CN114752544B.

4. The recombinant bacteria according to any one of claims 1-3, characterized in that, The modification further includes inserting the promoter of the glutamate decarboxylase gene into the genome of the glutamate-producing bacterium, wherein the promoter is the H36 promoter with a nucleotide sequence as shown in SEQ ID NO.

3.

5. The recombinant bacteria according to claim 4, characterized in that, The recombinant bacteria overexpress the exogenous protein gene; Furthermore, the modification also includes inserting the promoter of the export protein gene into the genome of the glutamate-producing bacterium, wherein the promoter is the H36 promoter with a nucleotide sequence as shown in SEQ ID NO.

3.

6. The recombinant bacteria according to claim 4, characterized in that, The recombinant bacteria simultaneously overexpress the exogenous protein gene and the glutamate decarboxylase gene; Furthermore, the modification also includes inserting the promoter of the export protein gene into the genome of the glutamate-producing bacterium, wherein the promoter is the Ptuf promoter with a nucleotide sequence as shown in SEQ ID NO.

4.

7. A method for constructing the recombinant bacteria according to any one of claims 1-6, characterized in that, Includes the following steps: Genetic modification of glutamate-producing bacteria to obtain the recombinant bacteria; the modification includes inserting a glutamate decarboxylase gene and an exocrine protein gene into the genome of the glutamate-producing bacteria, wherein the exocrine protein gene includes the cgl0824 gene with a nucleotide sequence as shown in SEQ ID NO.

1.

8. The recombinant bacteria according to claim 7, characterized in that, The glutamate-producing bacterium is Corynebacterium glutamicum FF10 strain disclosed in patent publication number CN114752544B; the glutamate decarboxylase gene is the glutamate decarboxylase mutant GADMUT disclosed in patent publication number CN114752544B. The step of inserting the glutamate decarboxylase gene into the genome of the glutamate-producing bacterium includes: Using the glutamate-producing bacteria as a template, amplification was performed using the upstream and downstream amplification primer pairs of gdh to obtain the upstream and downstream homologous arms of gdh. Using a plasmid containing the glutamate decarboxylase mutant GADMUT as a template, amplification was performed using the H36-GADMUT amplification primer pair to obtain a glutamate decarboxylase ligation fragment. The glutamate decarboxylase ligation fragment is a glutamate decarboxylase mutant GADMUT gene fragment linked to the H36 promoter. The nucleotide sequence of the H36 promoter is shown in SEQ ID NO.

3. The upstream homologous arm of the gdh, the downstream homologous arm of the gdh, and the glutamate decarboxylase linker fragment were linked to the backbone vector and then transferred into the glutamate-producing bacteria to obtain recombinant bacteria FR01.

9. The recombinant bacteria according to claim 8, characterized in that, The upstream amplification primer pair for gdh includes gdh-upF with the nucleotide sequence shown in SEQ ID NO.5 and gdh-upR with the nucleotide sequence shown in SEQ ID NO.6; The downstream amplification primer pair of the gdh includes lldd-downF with nucleotide sequences as shown in SEQ ID NO.7 and lldd-downR with nucleotide sequences as shown in SEQ ID NO.8; The H36-GADMUT amplification primer pair includes H36-GADMUT-F with the nucleotide sequence shown in SEQ ID NO.9 and H36-GADMUT-R with the nucleotide sequence shown in SEQ ID NO.10; The backbone plasmid is Pk18mobsacB backbone plasmid.

10. The recombinant bacteria according to any one of claims 8-9, characterized in that, The step of inserting the export protein gene into the genome of the glutamate-producing bacterium includes: The target fragment was amplified using Corynebacterium glutamicum ATCC13032 as a template. The target fragment, the H36 promoter fragment, and the plasmid pCES were ligated and transformed into the recombinant bacterium FR01 to obtain the recombinant bacterium. The nucleotide sequence of the H36 promoter is shown in SEQ ID NO.

3.

11. The recombinant bacteria according to claim 10, characterized in that, In the step of amplifying with Corynebacterium glutamicum ATCC13032 as a template to obtain the target fragment, the amplification primer pair used includes nucleotide sequences such as 0824F as shown in SEQ ID NO.11 and 0824R as shown in SEQ ID NO.12; The plasmid pCES is described in the paper Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum.

12. The recombinant bacteria according to any one of claims 8-9, characterized in that, The step of inserting the export protein gene into the genome of the glutamate-producing bacterium includes: The plasmid containing the glutamate decarboxylase mutant GADMUT was used as a template for amplification to obtain the first target fragment; The first target fragment is ligated to the H36 promoter to obtain the first ligation fragment, the nucleotide sequence of which is shown in SEQ ID NO.3; The second target fragment was amplified using Corynebacterium glutamicum ATCC13032 as a template. The second target fragment is ligated to the Ptuf promoter to obtain the second ligation fragment, the nucleotide sequence of which is shown in SEQ ID NO.4; The first ligation fragment, the second ligation fragment, and plasmid pCES were ligated and transferred into the recombinant bacterium FR01 to obtain the recombinant bacterium.

13. The recombinant bacteria according to claim 12, characterized in that, In the step of amplifying the plasmid containing the glutamate decarboxylase mutant GADMUT as a template to obtain the first target fragment, the amplification primer pair used includes GADF with nucleotide sequences as shown in SEQ ID NO.13 and GADR with nucleotide sequences as shown in SEQ ID NO.14; In the step of amplifying with Corynebacterium glutamicum ATCC13032 as a template to obtain the second target fragment, the amplification primer pair used includes nucleotide sequences such as 0824F as shown in SEQ ID NO.11 and 0824R as shown in SEQ ID NO.12; The plasmid pCES is described in the paper Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum.

14. The use of the recombinant bacteria according to any one of claims 1-6 in the preparation of γ-aminobutyric acid.

15. A method for preparing γ-aminobutyric acid, characterized in that, Includes the following steps: Expand the culture of the recombinant bacteria according to any one of claims 1-6, perform solid-liquid separation on the culture medium, collect the supernatant, and obtain γ-aminobutyric acid.

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