Corynebacterium glutamicum engineering bacterium for producing gamma-aminobutyric acid by utilizing lignocellulose and application of corynebacterium glutamicum engineering bacterium

Abstract

The invention belongs to the technical field of microorganism application, and relates to a corynebacterium glutamicum engineering bacterium for producing gamma-aminobutyric acid (GABA) by utilizing lignocellulose and application of the corynebacterium glutamicum engineering bacterium. A metabolic engineering means is adopted, a secretory expression type glutamate decarboxylase coding gene is integrated in a corynebacterium glutamicum genome, an isocitrate dehydrogenase coding gene and a GABA permease coding gene are knocked out, and the recombinant strain corynebacterium glutamicum GJ09 is obtained. The corynebacterium glutamicum engineering bacterium is the corynebacterium glutamicum engineering bacterium capable of producing gamma-aminobutyric acid by utilizing lignocellulose. The engineering bacterium has the metabolic capability on various biomass-derived sugars, especially lignocellulose-derived glucose and xylose, does not need to add antibiotics to maintain plasmids, and can stably produce gamma-aminobutyric acid in a lignocellulose hydrolysate system.

Description

Technical Field

[0001] This invention belongs to the field of microbial application technology, and in particular relates to an engineered strain of Corynebacterium glutamicum that produces γ-aminobutyric acid (GABA) using lignocellulose and its application. Background Technology

[0002] Gamma-aminobutyric acid (GABA) has great potential as a food additive, plant growth promoter, and in the synthesis of a series of novel polyesteramides. Glucose is a common carbon source for the microbial fermentation production of GABA, but large-scale production of GABA using glucose is costly. Therefore, producing GABA from inexpensive and widely available lignocellulosic biomass is a production route with significant industrial application value.

[0003] Corynebacterium glutamicum is one of the commonly used strains for producing γ-aminobutyric acid (GABA), providing sufficient glutamate for GABA synthesis. Although Corynebacterium glutamicum lacks endogenous glutamate decarboxylase, GABA production can be achieved through heterologous expression of glutamate decarboxylase. Xie Jingjing et al. from Nanjing University of Technology reported a recombinant Corynebacterium glutamicum strain, C. glutamicum NJM6, which incorporated glutamate decarboxylase and produced 36.1 g / L of GABA via fed-batch fermentation (CN 103555647 A). Wang Xiaoyuan et al. from Jiangnan University reported a strain that integrated glutamate decarboxylase derived from Lactobacillus brevis into the genome of Corynebacterium glutamicum ATCC 13032, achieving antibiotic-free and efficient GABA production with a yield of 58.3 g / L via fed-batch fermentation (CN 113583930B). Wang Xiaoyuan et al. from Jiangnan University reported a recombinant strain obtained by modifying the γ-aminobutyric acid (GABA) producing strain C. glutamicum CGY-PG-304 as the starting strain. The strain produced 112.03 g / L of GABA through fed-batch fermentation (CN 118028196A).

[0004] The aforementioned strains only achieved the fermentation production of γ-aminobutyric acid (GABA) using glucose as a carbon source. Furthermore, if the strain contains free plasmids, antibiotics are required during fermentation to maintain plasmid stability, leading to downstream contamination and difficulties in product purification. Currently, there are no reports on plasmid-free stable production of GABA by *Corynebacterium glutamicum* utilizing glucose and xylose from lignocellulose. Therefore, constructing an engineered *Corynebacterium glutamicum* strain that utilizes glucose and xylose from corn cob residue hydrolysate to produce GABA is of great significance for the industrial production of lignocellulose-based GABA.

[0005] The information disclosed in this background section is only intended to enhance understanding of the overall background of the invention and should not be considered as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0006] In view of the shortcomings of the aforementioned strains and production technologies, this invention aims to provide an engineered strain of Corynebacterium glutamicum that produces γ-aminobutyric acid (GABA) from lignocellulose. This strain possesses the ability to efficiently metabolize and stably synthesize γ-aminobutyric acid (GABA) from various biomass sugars, especially glucose and xylose from lignocellulose, and has potential for industrial application.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] The starting strain of this invention is *C. glutamicum* S9114, an industrial glutamate production strain. By integrating the xylose isomerase encoding gene *xylA*, the xylulose kinase encoding gene *xylB*, and the pentose transporter encoding gene *araE* from *E. coli* into the genome, a xylose metabolic pathway was introduced. Subsequently, 110 amino acids at the C-terminus of the glutamate secretion channel protein *MscCG* were knocked out, weakening α-ketoglutarate dehydrogenase in the TCA cycle, thus directing more metabolic flux towards glutamate synthesis. The resulting recombinant strain *C. glutamicum* GJ04 possesses the ability to co-utilize glucose and xylose in the lignocellulose system and can produce high levels of glutamate, providing sufficient precursors for the synthesis of γ-aminobutyric acid (GABA).

[0009] The technical solution employed in this invention to construct the γ-aminobutyric acid (GABA) synthesis pathway in Corynebacterium glutamicum is as follows: A three-stage secretory glutamate decarboxylase is integrated into *C. glutamicum* GJ04 to construct the γ-aminobutyric acid synthesis pathway, allowing for sufficient extracellular accumulation of γ-aminobutyric acid; isocitrate dehydrogenase is knocked out in *C. glutamicum*, causing a greater carbon flux to flow towards the synthesis of α-ketoglutarate, thus enabling more α-ketoglutarate to be used for glutamate synthesis; and GABA permeabilization enzyme is knocked out in *C. glutamicum*, reducing the transmembrane transport of extracellular γ-aminobutyric acid into the cell. (Knocking out the isocitrate dehydrogenase encoding gene aceA and the GABA permeabilization enzyme encoding gene gabP yields the engineered *C. glutamicum* strain capable of producing γ-aminobutyric acid (GABA) using lignocellulose.)

[0010] The present invention provides an engineered strain of Corynebacterium glutamicum that produces γ-aminobutyric acid (GABA) using lignocellulose, which is constructed by the following steps:

[0011] (1) The gene encoding secretory glutamate decarboxylase was first integrated into the ldh gene locus of the recombinant strain of Corynebacterium glutamicum;

[0012] (2) The gene encoding secretory glutamate decarboxylase was integrated for the second time into the gabT gene locus of the recombinant strain of Corynebacterium glutamicum;

[0013] (3) The gene encoding secretory glutamate decarboxylase was integrated for the third time into the gabD gene locus of the recombinant strain of Corynebacterium glutamicum;

[0014] (4) Knock out the aceA gene in recombinant strains of Corynebacterium glutamicum;

[0015] (5) Knock out the gabP gene in recombinant strains of Corynebacterium glutamicum.

[0016] The specific operating method is as follows:

[0017] The first integration of the secretory expression glutamate decarboxylase encoding gene into the ldh gene site of the recombinant strain C. glutamicum GJ04 of Corynebacterium glutamicum: First, an integration plasmid for secretory expression glutamate decarboxylase was constructed. Then, the integration plasmid pK18-Δldh::pTacM-NsgadBmut was transferred into the recombinant strain by electroporation. Strains that successfully underwent homologous recombination were screened by PCR verification and sequencing verification to obtain intermediate recombinant strain No. 1.

[0018] In step (2), the gene encoding secretory glutamate decarboxylase is integrated for the second time into the gabT gene site of intermediate recombinant strain 1 of Corynebacterium glutamicum: First, an integration plasmid for secretory glutamate decarboxylase is constructed. Then, the integration plasmid pK18-ΔgabT::pTacM-NsgadBmut is transferred into intermediate recombinant strain 1 by electroporation. Strains that successfully undergo homologous recombination are screened by PCR verification and sequencing verification to obtain intermediate recombinant strain 2.

[0019] In step (3), the secretory expression type glutamate decarboxylase encoding gene is integrated for the third time into the gabD gene site of intermediate recombinant strain No. 2: First, an integration plasmid containing a bicistronic structure for secretory expression type glutamate decarboxylase is constructed. Then, the integration plasmid pK18-ΔgabD::pTHP7-NsgadBmut is transferred into intermediate recombinant strain No. 2 by electroporation. Strains that successfully undergo homologous recombination are screened by PCR verification and sequencing verification to obtain intermediate recombinant strain No. 3.

[0020] Step (4) knocking out the aceA gene of Corynebacterium glutamicum intermediate recombinant strain 3: First, construct the aceA knockout plasmid, then transfer the knockout plasmid pK18-ΔaceA into intermediate recombinant strain 3 by electroporation, and screen strains that successfully undergo homologous recombination by PCR verification and sequencing verification to obtain intermediate recombinant strain 4.

[0021] Step (5) knocking out the gabP gene in the intermediate recombinant strain of Corynebacterium glutamicum No. 4: First, a gabP knockout plasmid is constructed, and then the knockout plasmid pK18-Δgab is transferred into the intermediate recombinant strain No. 4 by electroporation. Strains that successfully undergo homologous recombination are screened by PCR verification and sequencing verification to obtain recombinant Corynebacterium glutamicum GJ09.

[0022] The present invention provides an engineered strain of Corynebacterium glutamicum that produces γ-aminobutyric acid (GABA) using lignocellulose. The detailed construction steps are as follows:

[0023] (1) Using the C. glutamicum S9114 genome as a template, the upstream homologous arm ldh-up and the downstream homologous arm ldh-down of the lactate dehydrogenase encoding gene ldh were amplified by PCR.

[0024] (2) Using the secretory expression plasmid pTacM-NsgadBmut as a template, the gene encoding secretory glutamate decarboxylase pTacM-NsgadBmut was amplified by PCR.

[0025] (3) After the plasmid pK18sacBmob was pre-linearized with restriction endonuclease BamHⅠ, the vector pK18sacBmob was amplified by PCR using the linearized pK18sacBmob as a template.

[0026] (4) The ldh-up, pTacM-NsgadBmut, ldh-down and vector pK18sacBmob obtained in steps (1)(2)(3) were ligated by the seamless cloning enzyme HB-infusion. The ligation solution was transferred into Escherichia coli DH5α by calcium chloride conversion. Kanamycin was used as the resistance selection marker. Positive transformants were obtained by colony PCR and the integration plasmid pK18-Δldh::pTacM-NsgadBmut was obtained by sequencing.

[0027] (5) Electroporate the integrated plasmid pK18-Δldh::pTacM-NsgadBmut obtained in step (4) into C. glutamicum GJ04. Transformants were obtained after kanamycin resistance screening. The strain was verified by colony PCR to obtain a successful recombinant strain.

[0028] (6) The positive transformants that were verified by colony PCR in step (5) were streaked and cultured for 12 hours. A small amount of colonies were picked and cultured in LB liquid medium for 3 hours. 100 μL of bacterial solution was spread on LBS sucrose plates and cultured for 12-24 hours. The obtained single colonies were streaked on LBS plates and LK plates. Transformants that grew on LBS plates but did not grow on LK plates were selected for colony PCR verification and sequencing verification. The strain that was successfully verified by secondary recombination was obtained and named intermediate recombination strain No. 1.

[0029] (7) Repeat steps (1)-(4) to construct the integration plasmid pK18-ΔgabT::pTacM-NsgadBmut;

[0030] (8) Repeat steps (5) and (6). Using the No. 1 intermediate recombinant strain obtained in step (6) as the starting strain, replace the GABA transaminase encoding gene gabT with the secretory glutamate decarboxylase encoding gene pTacM-NsgadBmut by homologous recombination to obtain the No. 2 intermediate recombinant strain.

[0031] (9) Using the expression plasmid pTacM-NsgadBmut as a template, the TacM promoter and the gene encoding secretory glutamate decarboxylase NsgadBmut were amplified by PCR.

[0032] (10) Using C. glutamicum S9114 as a template, the 62bp high-expression gene HP7 and the upstream homologous arm gabD-up and downstream homologous arm gabD-down of the succinate semialdehyde dehydrogenase encoding gene gabD were amplified by PCR.

[0033] (11) The vector pK18sacBmob obtained in the order of upstream homologous arm gabD-up, TacM promoter, high expression gene HP7, secretory glutamate decarboxylase encoding gene NsgadBmut, and downstream homologous arm gabD-down and step (3) was ligated by HB-infusion seamless cloning enzyme. The ligation solution was transferred into Escherichia coli DH5α by calcium chloride conversion method. Kanamycin was used as the resistance selection marker. Positive transformants were obtained by colony PCR. The integrative plasmid pK18-ΔgabD::pTHP7-NsgadBmut was obtained by sequencing.

[0034] (12) Repeat steps (5) and (6), using the No. 2 intermediate recombinant strain obtained in step (8) as the starting strain, and replace the succinate semialdehyde dehydrogenase encoding gene gabD with the secretory glutamate decarboxylase encoding gene pTHP7-NsgadBmut containing a bicistronic structure through homologous recombination to obtain the No. 3 intermediate recombinant strain.

[0035] (13) Using the genome of Corynebacterium glutamicum S9114 as a template, the upstream homologous arm aceA-up and the downstream homologous arm aceA-down of the isocitrate dehydrogenase encoding gene aceA were amplified by PCR.

[0036] (14) After the plasmid pK18sacBmob was pre-linearized with restriction endonuclease BamHⅠ, the vector pK18sacBmob was amplified by PCR using the linearized pK18sacBmob as a template.

[0037] (15) The aceA-up, aceA-down and vector pK18sacBmob obtained in steps (11) and (12) were ligated by the seamless cloning enzyme HB-infusion. The ligation solution was transferred into Escherichia coli DH5α by calcium chloride conversion. Kanamycin was used as the resistance selection marker. Positive transformants were obtained by colony PCR and the knockout plasmid pK18-ΔaceA was obtained by sequencing.

[0038] (16) Repeat steps (5) and (6), using the No. 3 intermediate recombinant strain obtained in step (12) as the starting strain, knock out the isocitrate dehydrogenase encoding gene aceA by homologous recombination to obtain the No. 4 intermediate recombinant strain.

[0039] (17) Repeat steps (13)-(15) to construct the knockout plasmid pK18-ΔgabP;

[0040] (18) Repeat steps (5) and (6), using the intermediate recombinant strain No. 4 obtained in step (16) as the starting strain, knock out the GABA permease encoding gene gabP by homologous recombination to obtain the recombinant strain C. glutamicum GJ09 (CCTCC M20251199).

[0041] This invention also provides a method for producing γ-aminobutyric acid (GABA) using the engineered strain C. glutamicum GJ09, comprising the following steps:

[0042] (1) Activation of strain: The engineered strain C. glutamicum GJ09 was inoculated into CM2B liquid medium and cultured at 30-35℃ with shaking for 12-16 hours to obtain the strain activation solution.

[0043] (2) Seed culture: The activated culture medium was transferred to the seed culture medium at an inoculation rate of 5-20% (v / v), and the strain was cultured at a constant temperature of 30-35℃ with shaking for 12-16 hours to obtain the primary seed culture medium; the primary seed culture was transferred to the seed culture medium at an inoculation rate of 5-20% (v / v), and the strain was cultured at a constant temperature of 30-35℃ with shaking for 12-16 hours to obtain the secondary seed culture medium.

[0044] (3) Fermentation culture: The secondary seed culture medium is inoculated into a culture medium containing sugars from different biomass sources at an inoculation rate of 5-20% (v / v) to produce γ-aminobutyric acid (GABA). During the fermentation process, acid and alkali are added to adjust the pH, and nutrients need to be added.

[0045] Preferably, the seed culture medium used in step (2) consists of the following components and concentrations: 15–30 g / L glucose, 0.6–1.5 g / L potassium dihydrogen phosphate, 0.3–0.6 g / L magnesium sulfate, 1.2–2.5 g / L urea, 1.0–2.0 mg / L ferrous sulfate, 1.0–2.0 mg / L manganese sulfate, and 2.5–5.0 g / L corn steep liquor.

[0046] Preferably, the fermentation culture used in step (3) consists of two parts: biomass raw materials and nutrients, with the biomass raw materials being a source of fermentable sugars.

[0047] Preferably, the biomass used in step (3) includes, but is not limited to, various grain starches, agricultural waste, energy crops, forestry waste, and industrial biomass. More specifically, it can be wheat straw or corn straw.

[0048] Preferably, the various biomass used in step (3) needs to undergo appropriate pretreatment and enzymatic decomposition to release free fermentable sugars. Furthermore, wheat straw or corn straw is pretreated with dilute acid and saccharified.

[0049] As a preferred option, the nutrient composition and concentration of γ-aminobutyric acid (GABA) fermentation in step (3) are as follows: 1-2 g / L potassium dihydrogen phosphate, 5-10 g / L ammonium sulfate, 10-20 g / L corn steep liquor, 0.6-1.2 g / L magnesium sulfate, 2.0-4.0 mg / L ferrous sulfate, 2.0-4.0 mg / L manganese sulfate, and 0.1-0.2 mM pyridoxal 5'-phosphate (PLP).

[0050] Preferably, in step (3), the acid-base neutralizing agent is 2M sulfuric acid and ammonia water, and the pH of the γ-aminobutyric acid (GABA) fermentation broth is controlled to be 6-7.

[0051] Preferably, the fermentation method in step (3) is stepwise saccharification fermentation, with the following conditions: the solid content of the biomass raw material is 15-30% (w / w), the amount of cellulase is 4-6 mg protein / g corn cob residue (dry basis), the amount of CaCO3 is 20-30 mg / g corn cob residue (dry basis), the pH of the corn cob residue is adjusted to 5.5-6.0, and saccharification is carried out at 45-50℃ and 200 rpm for 48-72 h; after saccharification, the mixture is centrifuged at 8000 rpm for 15 min, and the supernatant is collected for high temperature and high pressure sterilization; after sterilization, the supernatant is collected by filtration in a sterile environment; γ-aminobutyric acid (GABA) is fermented at 30-35℃, 600 rpm, and pH 6-7.

[0052] Compared with the prior art, the present invention has the following positive effects:

[0053] The *Corynebacterium glutamicum* engineered strain C. glutamicum GJ09 provided in this invention can efficiently convert various biomass-derived sugars to produce γ-aminobutyric acid (GABA). Even in lignocellulosic hydrolysate containing inhibitors, batch fermentation and fed-batch fermentation of GABA can be achieved, with yields of 44.3 g / L and 63.4 g / L, and probabilities of 0.45 g / g and 0.42 g / g, respectively. This strain does not require the addition of antibiotics to maintain plasmids and does not experience plasmid loss during fermentation, ensuring stable production of GABA and demonstrating significant application potential.

[0054] The classification name is Corynebacterium glutamicum GJ09.

[0055] The accession number is CCTCC NO: M 20251199.

[0056] The deposit date is May 27, 2025.

[0057] The depository is located at the China Center for Type Culture Collection. Attached Figure Description

[0058] Figure 1 Metabolic engineering strategies for efficient and stable GABA production in Corynebacterium glutamicum. Specific implementation plan

[0059] The following embodiments are provided to better understand the present invention and are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. Corynebacterium glutamicum S9114 (derived from the China Center of Industrial Culture Collection, CICC) http: / / www.china-cicc.org / The accession number is CICC 20935. It has also been disclosed in the following literature: Wen JB, Bao J. Engineering Corynebacterium glutamicum triggers glutamic acid accumulation in biotin-rich corn stover hydrolysate. Biotechnology for Biofuels. 2019, 12:86. Corynebacterium glutamicum GJ04 has been disclosed in the following literature: Jin C, Huang Z, Bao J. High-Tier Glutamic Acid Production from Lignocellulose Using an Engineered Corynebacterium glutamicum with Simultaneous Co-utilization of Xylose and Glucose. ACS Sustainable Chemistry & Engineering. 2020, 8:6315-6322.

[0060] Example 1: Multiple copies of the gene encoding secretory glutamate decarboxylase were integrated into the genome of Corynebacterium glutamicum.

[0061] First, three integrative plasmids for secretory expression of glutamate decarboxylase were constructed. The specific construction methods are as follows: (1) pK18-Δldh::pTacM-NsgadBmut. First, the secretory expression of glutamate decarboxylase gene was amplified from the expression plasmid pTacM-NsgadBmut using primers pTN-F1 / R1 (as shown in the sequence listing pTN-F1 / R1). Using the C. glutamicum S9114 genome as a template, the homologous arms of the lactate dehydrogenase encoding gene ldh, which was to be replaced, were amplified using primers ldh-F1 / R1 (as shown in the sequence listing ldh-F1 / R1) and ldh-F2 / R2 (as shown in the sequence listing ldh-F2 / R2). Then, the plasmid pK18sacBmob was pre-linearized with the restriction endonuclease BamHI, and the vector pK18sacBmob was amplified using primers Vector-F / R (as shown in the sequence listing Vector-F / R). Finally, the three gene fragments were ligated with the vector pK18sacBmob in the order of ldh-up, pTacM-NsgadBmut, ldh-down using HB-infusion seamless cloning enzyme to obtain the plasmid pK18-Δldh::pTacM-NsgadBmut. (2) pK18-ΔgabT::pTacM-NsgadBmut First, the secretory glutamate decarboxylase gene was amplified from the expression plasmid pTacM-NsgadBmut using primers pTN-F1 / R1 (as shown in the sequence listing pTN-F1 / R1). Using the C. glutamicum S9114 genome as a template, the upstream and downstream homologous arms of the gene encoding the GABA transaminase, gabT, were amplified using primers gabT-F1 / R1 (as shown in the sequence listing gabT-F1 / R1) and gabT-F2 / R2 (as shown in the sequence listing gabT-F2 / R2). Then, after pre-linearizing the plasmid pK18sacBmob with the restriction endonuclease BamHI, the vector pK18sacBmob was amplified using the primer Vector-F / R (as shown in the sequence listing Vector-F / R). Finally, the three gene fragments were ligated to the vector pK18sacBmob in the order of gabT-up, pTacM-NsgadBmut, gabT-down using HB-infusion seamless cloning enzyme to obtain the plasmid pK18-ΔgabT::pTacM-NsgadBmut. (3) pK18-ΔgabD::pTHP7-NsgadBmut The third integration selection replaced the succinate semialdehyde dehydrogenase encoding gene gabD with a bicistronic expression cassette containing a secretory glutamate decarboxylase encoding gene.Based on the secretory expression of glutamate decarboxylase gene, the highly expressed gene HP7 is added. HP7, as a procistronic gene, forms a bicistronic expression cassette with the target gene pTacM-NsgadBmut. The TacM promoter was amplified from the expression plasmid pTacM-NsgadBmut using primers TacM-F / R (as shown in the sequence listing). Using the C. glutamicum S9114 genome as a template, the 62 bp highly expressed gene HP7 was amplified using primers HP7-F1 / R1 (as shown in the sequence listing). The secretory glutamate decarboxylase gene NsgadBmut was amplified from the expression plasmid pTacM-NsgadBmut using primers pTHP7N-F / R (as shown in the sequence listing). Using the C. glutamicum S9114 genome as a template, primers gabD-F1 / R1 (as shown in the sequence listing) and gabD-F2 / R2 (as shown in the sequence listing) were used to amplify the gene from C. glutamicum S9114. GJ04 amplified the upstream and downstream homologous arms gabD-up and gabD-down of the succinate semialdehyde dehydrogenase gene gabD. The above five fragments and the vector pK18sacBmob were ligated with HB-infusion seamless cloning enzyme to obtain the plasmid pK18-ΔgabD::pTHP7-NsgadBmut.

[0062] The specific plasmid construction steps are divided into three parts: preparation of E. coli DH5α competent cells, preparation of ligation solution, and transformation. First, prepare E. coli DH5α competent cells: (1) Take out the E. coli DH5α cryovial and streak it on an LB plate for activation. Incubate upside down at 37℃ for 12h. (2) After 12h, take out the plate and inoculate a single colony into 5mL of LB liquid. Incubate at 37℃ and 200rpm in a shaker for 12h. (3) Inoculate the bacterial solution into 100mL of LB liquid and incubate in a shaker for 5-7h. OD of the bacterial solution 600The concentration is 0.3-0.4. (4) Transfer the bacterial solution to a sterile 50mL centrifuge tube and incubate on ice for 30min. (5) Pre-cool the centrifuge at 4℃ and centrifuge the bacterial solution at 4℃ and 4000rpm for 10min. Discard the supernatant, add 10mL of pre-cooled 0.1M calcium chloride, and gently resuspend the bacterial cells by pipetting. (6) Repeat the above step twice. (7) Discard the supernatant and add 2mL of stock solution. After resuspending the cells, aliquot the bacterial solution into sterilized EP tubes at a volume of 100μL. Store the aliquoted competent cells in a -80℃ freezer. Then prepare the ligation solution: Calculate the volume of the target gene and vector according to the instructions of the HB-infusion seamless cloning kit. Mix thoroughly by pipetting 10μL and place in a preheated 50℃ water bath for 30min. Finally, the ligation solution was transformed into E. coli DH5α competent cells: (1) The competent cells, which had been taken out of the -80℃ freezer in advance, were added to 10 μL of ligation solution after being placed in a water bath. The cells were gently pipetted and mixed, and then placed in an ice bath for 30 min. (2) The water bath was set to 42℃ in advance. The bacterial solution after being placed in the water bath was placed in the water bath and heat-shocked at 42℃ for 90 s. After being placed in an ice bath for 2 min, 900 μL of LB medium was immediately added. After being thoroughly mixed, the cells were cultured at 37℃ and 200 rpm for 1 h. (3) After 1 h, the EP tube was removed and centrifuged at 4000 rpm for 2 min. 900 μL of supernatant was aspirated, and the remaining bacterial solution was gently pipetted and mixed before being spread onto LK plates. (4) The spread LK plates were inverted and placed in a 37℃ incubator for overnight culture. (5) After single colonies grew the following morning, the LK plate was removed, and single colonies were picked for colony PCR verification using the verification primers pK18-F / R and TacM-F / R. (6) The positive transformants verified by colony PCR were inoculated into 5 mL of LK liquid medium. The culture was carried out at 37℃ and 200 rpm for 12 h. The plasmid was extracted and verified by sequencing.

[0063] Next, the integrative plasmid pK18-Δldh::pTacM-NsgadBmut was transformed into *C. glutamicum* GJ04 via electroporation. Strains exhibiting correct homologous recombination were then screened using colony PCR and sequencing verification, resulting in intermediate recombinant strain 1. The integrative plasmid pK18-ΔgabT::pTacM-NsgadBmut was then transformed into intermediate recombinant strain 1 via electroporation. Strains exhibiting correct homologous recombination were then screened using colony PCR and sequencing verification, resulting in intermediate recombinant strain 2. The integrative plasmid pK18-ΔgabD::pTHP7-NsgadBmut was then transformed into intermediate recombinant strain 2 via electroporation. Strains exhibiting correct homologous recombination were then screened using colony PCR and sequencing verification, resulting in intermediate recombinant strain 3.

[0064] The specific strain construction method is divided into two parts: preparation of competent Corynebacterium glutamicum cells and electroporation. First, prepare competent Corynebacterium glutamicum cells: (1) Take out the C. glutamicum GJ04 cryopreservation tube and activate it by streaking on a CM2B plate. Incubate in an inverted incubator at 30℃ for 2-3 days. (2) After single cells grow, inoculate them into 5 mL of liquid CM2B medium and incubate in a shaker at 30℃ and 200 rpm for 12 h. (3) Transfer the bacterial solution to 30 mL of LBG competent medium at an inoculation rate of 10%, incubate in a shaker for 4 h, add 60 μL of ampicillin at a concentration of 50 mg / L, and incubate in a shaker for 1 h. (4) Pour the bacterial solution into a 50 mL sterile centrifuge tube and incubate on ice for 15 min. (5) After the ice bath, centrifuge at 4℃ and 5000 rpm for 10 min. After the centrifugation, discard the supernatant, add 30 mL of pre-cooled 10% glycerol, and gently resuspend the bacterial cells by pipetting. Centrifuge again under the same centrifugation conditions to collect bacterial cells. (6) Repeat step 5, resuspend in 10% glycerol and centrifuge once, and pipette as cleanly as possible of the supernatant. (7) Add 2 mL of 10% glycerol (the amount of glycerol added depends on the total amount of bacterial cells), gently pipette to resuspend the bacterial cells, and dispense 100 μL into sterile EP tubes and freeze at -80℃ for later use. Then electroporate the plasmid into competent Corynebacterium glutamicum cells: (1) Gently mix 20 μL of plasmid and 100 μL of pre-thawed competent cells with a pipette and incubate on ice for 30 min. (2) After the ice bath, transfer the bacterial solution to a pre-cooled and dried electroporation cup, being gentle to avoid the generation of bubbles, and cover the electroporation cup. Wipe the water droplets off the outer wall of the electroporation cup with a paper towel before electroporation. The electroporation parameters are voltage 1800V and resistance 200Ω. (3) After electroporation, transfer the bacterial culture to an EP tube, immediately add 1 mL of preheated LBHIS medium at 46℃, mix well, and place the EP tube in a preheated water bath at 46℃ for 8 min. (4) After heat shock, incubate the EP tube at 37℃ and 200 rpm for 1 h, then transfer it to a 30℃ and 200 rpm incubator for 1 h. (5) After incubation, centrifuge the EP tube at 4000 rpm for 3 min. Discard the supernatant, gently mix the remaining bacterial culture, and spread it onto an LBHISK plate. Incubate at 30℃ for 60-72 h. After growing single colonies of appropriate size, perform colony PCR verification. (6) Design upstream and downstream primers VeTacM-F / R for the expression plasmid pTacM-NsgadBmut. Select single colonies grown after electroporation for colony PCR verification. The strain that runs the correct band in gel electrophoresis is the recombinant strain successfully transformed by the plasmid.

[0065] Example 2: Knockout of the aceA and gabP genes in C. glutamicum

[0066] First, construct two knockout plasmids. The specific construction methods are as follows: (1) pK18-ΔaceA. Using the C. glutamicum S9114 genome as a template, the upstream and downstream homologous arms of the isocitrate dehydrogenase encoding gene aceA were amplified using primers aceA-F1 / R1 (as shown in the sequence listing aceA-F1 / R1) and aceA-F2 / R2 (as shown in the sequence listing aceA-F2 / R2). The homologous arms were then ligated to the vector pK18mob using HB-infusion seamless cloning enzyme to obtain plasmid pK18-ΔaceA. (2) pK18-ΔgabP. Using the C. glutamicum S9114 genome as a template, the upstream and downstream homologous arms of the isocitrate dehydrogenase encoding gene gabP were amplified using primers gabP-F1 / R1 (as shown in the sequence listing gabP-F1 / R1) and gabP-F2 / R2 (as shown in the sequence listing gabPF2 / R2). These were then ligated to the vector pK18mob using HB-infusion seamless cloning enzyme to obtain the plasmid pK18-ΔgabP. Specific plasmid construction steps are detailed in Example 1.

[0067] Next, the integration plasmid pK18-ΔaceA was transformed into intermediate recombinant strain 3 via electroporation. Strains exhibiting correct homologous recombination were then screened using colony PCR and sequencing verification, resulting in intermediate recombinant strain 4. The integration plasmid pK18-ΔgabP was then transformed into intermediate recombinant strain 4 via electroporation. Strains exhibiting correct homologous recombination were then screened using colony PCR and sequencing verification, resulting in recombinant Corynebacterium glutamicum, named C. glutamicum GJ09. Specific strain construction steps are detailed in Example 1.

[0068] Example 3: Production of γ-aminobutyric acid (GABA) by fermentation of engineered Corynebacterium glutamicum GJ09 from corn cob residue hydrolysate with a 15% (w / w) solids content.

[0069] The engineered strain *C. glutamicum* GJ09 was inoculated into 5 mL of CM2B liquid medium and cultured at 30°C with shaking for 12 h to obtain an activated culture solution. The activated culture solution was transferred to seed culture medium at a 10% (v / v) inoculation rate and cultured at 30°C with shaking for 12 h to obtain a primary seed culture solution. The primary seed culture solution was then transferred to the seed culture medium at a 10% (v / v) inoculation rate and cultured at 30°C with shaking for 12 h to obtain a secondary seed culture solution. The secondary seed culture solution was then inoculated at a 10% (v / v) inoculation rate into media containing various biomass-derived sugars to produce γ-aminobutyric acid (GABA).

[0070] The specific conditions for stepwise saccharification and fermentation are as follows: the biomass feedstock has a solid content of 15% (w / w), the cellulase dosage is 6 mg protein / g corn cob residue (dry basis), the CaCO3 dosage is 20 mg / g corn cob residue (dry basis), the pH of the corn cob residue is adjusted to 5.5, and saccharification is carried out at 50℃ and 200 rpm for 48 h; after saccharification, the mixture is centrifuged at 8000 rpm for 15 min, the supernatant is collected and sterilized by high temperature and high pressure; after sterilization, the supernatant is collected by filtration in a sterile environment; nutrients are added to the supernatant, including 1 g / L potassium dihydrogen phosphate, 5 g / L ammonium sulfate, 10 g / L corn steep liquor, 0.6 g / L magnesium sulfate, 2.0 mg / L ferrous sulfate, 2.0 mg / L manganese sulfate, and 0.1 mM... 5'-Pyridoxal phosphate (PLP) was used; secondary seed culture was transferred to the medium at a 10% (v / v) inoculation rate; γ-aminobutyric acid (GABA) fermentation was carried out at 30℃, 600 rpm, and pH 7.0. The pH was adjusted to 7.0 using 2M sulfuric acid and ammonia. The fermentation results are shown in Table 1. The highest yield of γ-aminobutyric acid reached 44.3 g / L, and the yield was calculated to be 0.45 g / g based on glucose and xylose consumption.

[0071] Table 1. Example 3 of γ-aminobutyric acid fermentation

[0072] Fermentation time (h) 0 12 24 36 48 60 72 γ-aminobutyric acid (GABA) production (g / L) 0 11.7 27.3 35.3 40.8 44.3 35.3

[0073] Example 4: Production of γ-aminobutyric acid (GABA) by fed-batch fermentation of engineered Corynebacterium glutamicum GJ09 using corn cob residue hydrolysate with a 15% (w / w) solids content.

[0074] For fed-batch fermentation, a 15% (w / w) solids content corn cob residue hydrolysate was concentrated by rotary evaporation into a syrup with a glucose concentration of 778 g / L and a xylose concentration of 120 g / L. Syrup preparation method: The 15% (w / w) corn cob residue hydrolysate was centrifuged at 8000 rpm for 15 min, and the supernatant was collected. The supernatant was collected as much as possible to minimize the incorporation of lower solid residue. The corn cob residue hydrolysate was then concentrated using a rotary evaporator.

[0075] The cultivation of the engineered strain C. glutamicum GJ09 seed culture and the fermentation method of corn cob residue hydrolysate with a solid content of 15% (w / w) were carried out according to Example 3. Syrup was added as feed starting at 36 hours, with feeding every 12 hours for a total of three feedings, and fermentation lasted for 72 hours.

[0076] The fermentation results are shown in Table 2. The highest yield of γ-aminobutyric acid (GABA) reached 63.4 g / L, and the yield was calculated to be 0.42 g / g based on the consumption of glucose and xylose.

[0077] Table 2 γ-aminobutyric acid fermentation example 4

[0078] Fermentation time (h) 0 12 24 36 48 60 72 γ-aminobutyric acid (GABA) production (g / L) 0 13.7 28.1 48.7 53.3 61.7 63.4

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the concept of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0080] Sequence List:

[0081]

[0082]

Claims

1. A strain of Corynebacterium glutamicum that produces γ-aminobutyric acid using lignocellulose, characterized in that, Using metabolic engineering techniques, the xylose isomerase encoding gene xylA, the xylulose kinase encoding gene xylB, and the pentose transporter encoding gene araE were first integrated into the genome of the glutamate model strain *C. glutamicum* S9114, introducing the xylose metabolic pathway. Next, 110 amino acids at the C-terminus of the glutamate secretion channel protein MScCG were knocked out, weakening α-ketoglutarate dehydrogenase in the TCA cycle, thus directing more metabolic flux towards glutamate synthesis. The recombinant strain possessed the ability to produce high levels of glutamate in a lignocellulose system and was named *Corynebacterium glutamicum* GJ04. Finally, the genome of the recombinant strain *C. glutamicum* GJ04 was integrated with a three-stage secretory glutamate decarboxylase encoding gene, and the isocitrate dehydrogenase encoding gene aceA and the GABA permease encoding gene gabP were knocked out, resulting in the engineered *Corynebacterium glutamicum* strain capable of producing γ-aminobutyric acid (GABA) from lignocellulose.

2. The engineered strain of Corynebacterium glutamicum that produces γ-aminobutyric acid using lignocellulose according to claim 1, characterized in that, It is constructed by the following steps: (1) The gene encoding secretory glutamate decarboxylase was first integrated into the ldh gene locus of the recombinant strain of Corynebacterium glutamicum; (2) The gene encoding secretory glutamate decarboxylase was integrated for the second time into the gabT gene locus of the recombinant strain of Corynebacterium glutamicum; (3) The gene encoding secretory glutamate decarboxylase was integrated for the third time into the gabD gene locus of the recombinant strain of Corynebacterium glutamicum; (4) Knock out the aceA gene in recombinant strains of Corynebacterium glutamicum; (5) Knock out the gabP gene in recombinant strains of Corynebacterium glutamicum.

3. The engineered strain of Corynebacterium glutamicum that produces γ-aminobutyric acid using lignocellulose according to claim 2, characterized in that, The specific operating method is as follows: The first integration of the secretory expression glutamate decarboxylase encoding gene into the ldh gene site of the recombinant strain C. glutamicum GJ04 of Corynebacterium glutamicum: First, an integration plasmid for secretory expression glutamate decarboxylase was constructed. Then, the integration plasmid pK18-Δldh::pTacM-NsgadBmut was transferred into the recombinant strain by electroporation. Strains that successfully underwent homologous recombination were screened by PCR verification and sequencing verification to obtain intermediate recombinant strain No.

1. In step (2), the gene encoding secretory glutamate decarboxylase is integrated for the second time into the gabT gene site of intermediate recombinant strain 1 of Corynebacterium glutamicum: First, an integration plasmid for secretory glutamate decarboxylase is constructed. Then, the integration plasmid pK18-ΔgabT::pTacM-NsgadBmut is transferred into intermediate recombinant strain 1 by electroporation. Strains that successfully undergo homologous recombination are screened by PCR verification and sequencing verification to obtain intermediate recombinant strain 2. In step (3), the secretory expression type glutamate decarboxylase encoding gene is integrated for the third time into the gabD gene site of intermediate recombinant strain No. 2: First, an integration plasmid containing a bicistronic structure for secretory expression type glutamate decarboxylase is constructed. Then, the integration plasmid pK18-ΔgabD::pTHP7-NsgadBmut is transferred into intermediate recombinant strain No. 2 by electroporation. Strains that successfully undergo homologous recombination are screened by PCR verification and sequencing verification to obtain intermediate recombinant strain No.

3. Step (4) knocking out the aceA gene of Corynebacterium glutamicum intermediate recombinant strain 3: First, construct the aceA knockout plasmid, then transfer the knockout plasmid pK18-ΔaceA into intermediate recombinant strain 3 by electroporation, and screen strains that successfully undergo homologous recombination by PCR verification and sequencing verification to obtain intermediate recombinant strain 4. Step (5) knocking out the gabP gene in the intermediate recombinant strain of Corynebacterium glutamicum No. 4: First, a gabP knockout plasmid is constructed, and then the knockout plasmid pK18-Δgab is transferred into the intermediate recombinant strain No. 4 by electroporation. Strains that successfully undergo homologous recombination are screened by PCR verification and sequencing verification to obtain recombinant Corynebacterium glutamicum GJ09.

4. The engineered strain of Corynebacterium glutamicum that produces γ-aminobutyric acid using lignocellulose, as described in any one of claims 1-3, is characterized in that... The engineered bacteria is classified as Corynebacterium glutamicum GJ09, with accession number CCTCC M 20251199, accession date May 27, 2025, and accession address China Center for Type Culture Collection.

5. The application of the engineered strain of Corynebacterium glutamicum used to produce γ-aminobutyric acid from lignocellulose as described in claim 1, characterized in that, The application involves using this bacterium to produce γ-aminobutyric acid (GABA) from sugars derived from various biomass sources; the biomass used includes, but is not limited to, various grain starches, agricultural waste, energy crops, forestry waste, and industrial biomass.

6. The application of the engineered Corynebacterium glutamicum strain for producing γ-aminobutyric acid using lignocellulose as described in claim 5, characterized in that, The production of γ-aminobutyric acid (GABA) using the engineered strain C. glutamicum GJ09 from various biomass sources requires the addition of certain nutrients, including 1–2 g / L potassium dihydrogen phosphate, 5–10 g / L ammonium sulfate, 10–20 g / L corn steep liquor, 0.6–1.2 g / L magnesium sulfate, 2.0–4.0 mg / L ferrous sulfate, 2.0–4.0 mg / L manganese sulfate, and 0.1–0.2 mM pyridoxal 5'-phosphate.

7. A method for producing γ-aminobutyric acid using Corynebacterium glutamicum engineered strain according to any one of claims 1-3 from various biomass-derived sugars, characterized in that, The specific steps include: (1) Activation of strain: The engineered strain C. glutamicum GJ09 was inoculated into CM2B liquid medium and cultured at 30-35℃ with shaking for 12-16h to obtain the strain activation solution; (2) Seed culture: The activated culture medium was transferred to the seed culture medium at an inoculation rate of 5-20% (v / v), and the strain was cultured at a constant temperature of 30-35℃ with shaking for 12-16 hours to obtain the primary seed culture medium; the primary seed culture was transferred to the seed culture medium at an inoculation rate of 5-20% (v / v), and the strain was cultured at a constant temperature of 30-35℃ with shaking for 12-16 hours to obtain the secondary seed culture medium; (3) Fermentation culture: The secondary seed culture medium is inoculated into a culture medium containing sugars from different biomass sources at an inoculation rate of 5-20% v / v to produce γ-aminobutyric acid. During the fermentation process, acid and alkali are added to adjust the pH.

8. The method for producing γ-aminobutyric acid (GABA) using the engineered strain of Corynebacterium glutamicum according to claim 7, characterized in that, The fermentation process uses sulfuric acid and ammonia to control the pH at 6-7, and ferments at 30-35℃ for 2-3 days.

9. A strain of Corynebacterium glutamicum that produces γ-aminobutyric acid using lignocellulose, as described in any one of claims 1-4, characterized in that, This strain achieved a maximum yield of 44.3 g / L of γ-aminobutyric acid through batch fermentation of lignocellulose hydrolysate, with a yield of 0.45 g / g.

10. A strain of Corynebacterium glutamicum that produces γ-aminobutyric acid using lignocellulose, as described in any one of claims 1-4, characterized in that, This strain achieved a maximum yield of 63.4 g / L and a yield of 0.42 g / g using fed-batch fermentation of lignocellulose hydrolysate.

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