Corynebacterium glutamicum gamma-aminobutyric acid efficient synthesis method based on membrane transport engineering
By screening and overexpressing GABA-related transporter genes in Corynebacterium glutamicum, a highly efficient genetically engineered strain was constructed, solving the problems of long fermentation cycle and low yield, and achieving a significant increase in GABA production.
Patent Information
- Application Number
- CN202511378617.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-25
- Publication Date
- 2026-02-10
AI Technical Summary
The existing technology for producing γ-aminobutyric acid (GABA) by Corynebacterium glutamicum microbial fermentation suffers from problems such as long fermentation cycle and low yield, making it difficult to meet the needs of industrial production.
By screening and overexpressing GABA-related transporter genes cg2139, cg2568, cg2339, cg11006, and cg1281, and integrating them into the genome of Corynebacterium glutamicum, a highly efficient genetically engineered strain was constructed, and fermentation conditions were optimized to increase GABA production.
It significantly increased GABA production, reaching 10.73 g/L, a 34% increase compared to the control strain FF10, achieving more efficient GABA production.
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Abstract
Description
Technical Field
[0001] This invention relates to a highly efficient method for synthesizing γ-aminobutyric acid (GABA) from Corynebacterium glutamicum based on membrane transport engineering, belonging to the field of genetic engineering technology. Background Technology
[0002] Gamma-aminobutyric acid (GABA) is a non-protein amino acid widely found in animals, plants, and microorganisms. It possesses various physiological functions, including relieving anxiety, regulating blood pressure, and improving sleep quality. Due to its broad application prospects in the pharmaceutical, food, and chemical industries, it has received increasing attention in recent years. With the accelerated pace of modern life, increased life pressure, and the emergence of various diseases, the number of people in a "sub-healthy" state is constantly increasing. As a potential nutritional factor for the prevention and alleviation of related diseases, the market demand for GABA is showing a continuous growth trend.
[0003] GABA can be obtained through chemical synthesis, biosynthesis, and natural extraction. However, the content of GABA in natural extraction is limited, and chemical synthesis does not meet consumers' demand for green consumption. In contrast, the preparation of GABA through microbial synthesis has the advantages of being safe, pollution-free, and inexpensive.
[0004] Currently, GABA biosynthesis is achieved through genetic engineering, metabolic engineering, and enzyme purification and immobilization. Microbial fermentation methods primarily focus on optimizing fermentation conditions and purifying the GABA product. While offering advantages such as safety, environmental friendliness, and sustainability, the yield remains unsatisfactory, and problems such as low conversion rates and high costs persist. Given GABA's significant nutritional value and the deepening research into the functions of γ-aminobutyric acid (GABA), its market demand continues to expand. Developing strains with higher efficiency in GABA production is in high demand. Existing technologies using Corynebacterium glutamicum microbial fermentation to produce GABA still suffer from long fermentation cycles and low yields. Therefore, further improvements in GABA production are needed to meet the demands of industrial production. Summary of the Invention
[0005] To address the shortcomings of the prior art, this invention provides a highly efficient method for synthesizing γ-aminobutyric acid (GABA) from Corynebacterium glutamicum based on membrane transport engineering, with the aim of enabling Corynebacterium glutamicum to produce GABA more efficiently.
[0006] The first technical solution provided by this invention is the application of GABA-associated transporter genes in improving the ability of Corynebacterium glutamicum to synthesize γ-aminobutyric acid, wherein the GABA-associated transporter genes include genes cg2139, cg2568, cg2339, cg11006, and cg1281.
[0007] In some embodiments, the GenBank accession numbers of the genes cg2139, cg2568, cg2339, cg11006, and cg1281 are CAF20294.1, CAF21003.1, CAF20468.1, BAB98399.1, and CAF19836.1, respectively.
[0008] In some embodiments, the application is to overexpress the GABA-associated transporter gene in Corynebacterium glutamicum.
[0009] The second technical solution provided by the present invention is a genetically engineered bacterium that produces γ-aminobutyric acid (GABA). The genetically engineered bacterium uses Corynebacterium glutamicum as a chassis strain and overexpresses the GABA-related transporter gene in the chassis strain. The GABA-related transporter gene is cg2139, cg2568, cg2339, cg11006, or cg1281.
[0010] In some embodiments, the GenBank accession numbers of the genes cg2139, cg2568, cg2339, cg11006, and cg1281 are CAF20294.1, CAF21003.1, CAF20468.1, BAB98399.1, and CAF19836.1, respectively, and their nucleotide sequences are shown in SEQ ID NO. 1 to 5, respectively.
[0011] In some embodiments, the chassis strain also has the gene cg0280 knocked out. The gene cg0280 has the Genbank accession number CAF18798.1 and its nucleotide sequence is shown in SEQ ID NO.6.
[0012] In some embodiments, the GABA-associated transporter gene is integrated and expressed at the cg0280 site of the chassis strain genome.
[0013] In some embodiments, the chassis strain is Corynebacterium glutamicum FF10, which has been disclosed in patent CN114752544B.
[0014] The present invention provides a third technical solution, which is a method for increasing the production of γ-aminobutyric acid in Corynebacterium glutamicum. The method involves overexpressing the GABA-related transporter gene in Corynebacterium glutamicum. The GABA-related transporter gene is cg2139, cg2568, cg2339, cg11006 or cg1281.
[0015] In some embodiments, the GenBank accession numbers of the genes cg2139, cg2568, cg2339, cg11006, and cg1281 are CAF20294.1, CAF21003.1, CAF20468.1, BAB98399.1, and CAF19836.1, respectively, and their nucleotide sequences are shown in SEQ ID NO. 1 to 5, respectively.
[0016] In some embodiments, the GABA-associated transporter gene is integrated and expressed at the cg0280 site of the Corynebacterium glutamicum genome.
[0017] In some embodiments, the Corynebacterium glutamicum is Corynebacterium glutamicum FF10, which has been disclosed in patent CN114752544B.
[0018] The present invention provides a fourth technical solution, which is a method for biosynthesizing γ-aminobutyric acid (GABA). The method involves inoculating the genetically engineered bacteria described in the second technical solution into a fermentation system with glucose as the carbon source to synthesize GABA.
[0019] In some implementations, the concentration of glucose in the fermentation system is 50 g / L.
[0020] In some embodiments, the genetically engineered bacteria described in the second technical solution are cultured in BHI medium to obtain a seed culture, which is then inoculated into CGXII medium to ferment and generate γ-aminobutyric acid.
[0021] In some implementations, the inoculation rate is 10%, the fermentation temperature is 30°C, the shaking speed is 200 rpm, and the fermentation time is 96 hours.
[0022] The fifth technical solution provided by this invention is the application of the GABA-related transporter protein gene or the genetically engineered bacteria described in the second technical solution in the preparation of γ-aminobutyric acid or products containing γ-aminobutyric acid.
[0023] In some embodiments, the GABA-associated transporter gene is cg2139, cg2568, cg2339, cg11006, or cg1281.
[0024] In some embodiments, the GenBank accession numbers of the genes cg2139, cg2568, cg2339, cg11006, and cg1281 are CAF20294.1, CAF21003.1, CAF20468.1, BAB98399.1, and CAF19836.1, respectively, and their nucleotide sequences are shown in SEQ ID NO. 1 to 5, respectively.
[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0026] This invention screens GABA transporters. First, a transporter library is constructed, and a series of effective GABA transporters, namely genes cg2139, cg2568, cg2339, cg11006, and cg1281, are obtained by flow cytometry screening. The screened transporters are integrated into the genome and fermentation is performed for verification, enabling Corynebacterium glutamicum to produce GABA more efficiently. Compared with the control strain FF10, the yield reached 10.73 g / L, which is 34% higher. Attached Figure Description
[0027] Figure 1 Flowchart for transport protein library construction and flow cytometry (FACS) screening test.
[0028] Figure 2 The graph shows the fluorescence values and corresponding GABA yields during well plate fermentation.
[0029] Figure 3 This is a graph showing the yield of shake-flask fermentation. Detailed Implementation
[0030] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0031] Raw materials used in the examples:
[0032] 1. Strains and plasmids
[0033] (1) The strain FF10 and expression plasmid pCES-GADMUT used in this invention have been disclosed in patent CN114752544B.
[0034] (2) The GABA biosensor used in this invention has been disclosed in patent CN114752589B.
[0035] (3) The pCES plasmid was published in the paper “Development of a high-copy-number plasmid via adaptive laboratory evolution of Corynebacterium glutamicum”.
[0036] 2. Culture medium
[0037] CGXII medium: 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, trace element solution 1 ml / L, pH adjusted to 7.0 with KOH; wherein, the trace element solution: FeSO4·7H2O 10 g / L, MnSO4·1H2O 10 g / L, ZnSO4·7H2O 1 g / L, CuSO4·5H2O 313 mg / L, NiCl·6H2O 20 mg / L.
[0038] BHI medium: peptone 10.0 g / L, dehydrated calf brain extract 12.5 g / L, dehydrated calf heart extract 5.0 g / L, sodium chloride 5.0 g / L, glucose 2.0 g / L, disodium hydrogen phosphate 2.5 g / L.
[0039] The following embodiments involve methods:
[0040] 1. Shake flask fermentation method
[0041] The control strain and recombinant strain carrying the expression plasmid were inoculated into BHI medium and cultured in test tubes at 30°C for 12 hours to obtain seed culture. 50 mL of CGXII medium was added to a 500 mL shake flask and inoculated at a rate of 10%. The fermentation temperature was 30°C and the shaker speed was 200 rpm for 96 hours.
[0042] 2. High-performance liquid chromatography (HPLC) for the determination of GABA yields: Agilent UPLC-DAD 1290 system; Eclipse Plus 95C18 column, 2.1*100mm, 3.5µm; mobile phase A was 0.05M sodium acetate solution, mobile phase B was 100% chromatographic grade methanol; run time was 10 min; flow rate was 0.3 mL / min for 40% A-60% B from 0-3 min, 0.3 mL / min for 10% A-90% B from 3.01-5 min, and 0.3 mL / min for 40% A-60% B from 5.1-10 min; detector wavelengths were set to 228 nm and 330 nm, respectively; column temperature was 30℃; injection volume was 10 μL.
[0043] Example 1: Construction of a GABA-producing test strain
[0044] To screen for efficient GABA transporters, the first step is to construct a chassis strain that integrates a biosensor and GABA production capabilities. The fluorescence intensity of this biosensor is positively correlated with the extracellular GABA concentration; that is, the higher the extracellular GABA concentration, the stronger the fluorescence intensity of the sensor. Therefore, in subsequent screening, reliable GABA transporters can be precisely screened based on the strain's GABA production level (indirectly reflected by fluorescence intensity), thus providing key technical support for optimizing GABA-producing strains. The specific method is as follows:
[0045] 1) By inserting a GABA biosensor into FF10, a recombinant Corynebacterium glutamicum containing a GABA biosensor was obtained and named C1.
[0046] The GABA biosensor contains promoters PgabTDP and PgabR, a reporter gene, and a gabR gene; the promoters PgabTDP and PgabR, the reporter gene, and the gabR gene are located on the same vector or on the genome, constituting the biosensor; PgabTDP regulates the expression of the reporter gene; PgabR and PgabTDP have sequences with GABA-GabR binding sites and regulate the expression of the reporter gene; the promoters PgabTDP and PgabR have opposite transcription directions.
[0047] How GABA biosensors work: GABA binds to the regulatory protein GabR, forming a conjugate that binds to the GABA-GabR binding site, initiating transcription of the PgabTDP promoter and regulating reporter gene expression. Taking superfolded green fluorescent protein (sfgfp) as an example, when GABA is produced in the strain's growth environment, the gabTDP promoter begins transcription, generating green fluorescent protein. The PgabTDP promoter responds to different concentrations of GABA, thereby regulating different intensities of green fluorescent protein expression. High-GABA-producing strains are screened by detecting fluorescence.
[0048] The specific method for inserting the GABA biosensor into FF10 is as follows: The biosensor plasmid Pk18mobsacB-ΔgabTD::sfgfp (disclosed in patent CN114752589B) was transformed into competent Corynebacterium glutamicum FF10. A second recombination was performed using kanamycin resistance selection followed by sucrose tolerance selection. Colony PCR confirmed the successful replacement of gabTD by sfgfp, thus constructing a recombinant Corynebacterium glutamicum containing the GABA biosensor, named C1.
[0049] 2) Construct the recombinant plasmid pK18-Δcg0280::GADMUT to knock out the conserved hypothetical protein cg0280 and enhance the synthesis of the glutamate decarboxylase mutant GADMUT: Using the Corynebacterium glutamicum genome as a template, clone the homologous arms 1000 bp upstream and downstream of the conserved hypothetical protein cg0280 (Genbank accession number: CAF18798.1) gene. Using the plasmid as a template, clone the coding gene (nucleotide sequence as shown in SEQ ID NO.1) of the glutamate decarboxylase mutant GADMUT (sequence disclosed in patent CN114752544B, amino acid sequence as shown in SEQ ID NO.1). As shown in NO.2, the recombinant plasmid pK18-Δcg0280::GADMUT was constructed between the upstream and downstream homologous arms of the cg0280 gene and linked to the Pk18mobsacB backbone using the Gibson method. The obtained recombinant plasmid pK18-Δcg0280::GADMUT was transformed into Corynebacterium glutamicum C1 competent cells, and the recombinant strain C1 / Δcg0280::GADMUT was obtained and named C2.
[0050] 3) C2 was inoculated into BHI medium and cultured in test tubes at 30℃ for 12 hours to obtain seed culture. 50 mL of CGXII medium was added to a 500 mL shake flask, and inoculated at a 10% inoculum rate. Fermentation was carried out at 30℃ and 200 rpm for 96 hours. The GABA yield of C2 was measured to be 1.8 g / L. The construction of the GABA-producing test strain was completed.
[0051] Example 2: Construction of a transporter protein overexpression library
[0052] All 400 membrane transport proteins on the genome of *Corynebacterium glutamicum* ATCC 13032 were predicted using TMHMM 2.0 (a software for predicting transmembrane helical structures). Using *Corynebacterium glutamicum* ATCC 13032 as a template, PCR was performed to amplify the 400 fragments. The amplified products were purified using a DNA purification kit, and then ligated to the backbone fragment of plasmid pCES using Gibson DNA ligation. The ligation products were then transformed into *E. coli* DH5α to construct a transport protein overexpression library. Sequencing analysis was used to confirm the integrity of the library.
[0053] Example 3: Flow Cytometry FACS Screening Test
[0054] The transporter protein overexpression library was introduced into test strain C2 and cultured at 30°C for 30 hours in CGXII medium suitable for GABA production. After culture, cell samples were collected, and flow cytometry (FACS) was used to screen for the top 0.5% of cell strains with the highest green fluorescence intensity. Figure 1As shown, with the increase of sorting times, strains with higher fluorescence intensity gradually accumulated, and the sorting effect was stable and reliable. To further verify the screening results, the sorted cells were diluted and plated, and 500 single colonies were selected and inoculated into 96-well plates for culture. The fluorescence intensity in the wells was detected using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Clones with fluorescence intensity more than 40% higher than the control group were selected, and the GABA production of each clone was detected by high performance liquid chromatography (HPLC). Some fluorescence intensity and GABA production correlation results are shown below. Figure 2 Based on this, the top five clones in terms of yield were identified through shake-flask fermentation and secondary screening. Finally, these five strains with significantly increased yield were sequenced to obtain relevant information about the GABA transporter protein, and the results are shown in Table 1.
[0055] Table 1. Transfer protein-related genes and annotations
[0056]
[0057]
[0058] Example 4: Construction and Fermentation Validation of Recombinant Strains
[0059] 1) The selected transporter protein genes were inserted into the genome for expression. The safe site cg0280 was knocked out in the FF10 substrate bacteria to enhance the selected transporter protein genes. A recombinant strain was constructed using FF10 as the substrate bacteria. Recombinant plasmids pK18-Δcg0280::cg2139, pK18-Δcg0280::cg2568, pK18-Δcg0280::cg2339, pK18-Δcg0280::cg1006, and pK18-Δcg0280::cg1281 were constructed using the same strategy as in Example 1. These recombinant plasmids were then transformed into FF10. In 0 competent cells, the following strains were recombined: FF10 / Δcg0280::cg2139, FF10 / Δcg0280::cg2568, FF10 / cg0280::cg2339, FF10 / Δcg0280::cgl1006, and FF10 / Δcg0280::cg1281, which were named D1, D2, D3, D4, and D5, respectively.
[0060] 2) The expression plasmid pCES-GADMUT was transformed into the successfully constructed recombinant strains D1-D5 and control strain FF10 for fermentation verification. The control strains and recombinant strains with the expression plasmid were inoculated into BHI medium and cultured in test tubes at 30℃ for 12 hours to obtain seed liquid. 50 mL of CGXII medium was added to 500 mL of shake flask and inoculated at a 10% inoculum. The fermentation temperature was 30℃, the shaker speed was 200 rpm, and the fermentation time was 96 hours. Figure 3 The results show the GABA production of each recombinant strain after 96 hours of shake-flask fermentation. The results indicate that strain D2 achieved a GABA production of 10.73 g / L, a 34% increase compared to the control strain FF10. This strongly confirms that a transporter protein screening strategy can successfully identify strains with higher GABA production capacity, thereby significantly improving GABA production efficiency.
[0061] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. The application of GABA-associated transporter genes in enhancing the ability of Corynebacterium glutamicum to synthesize γ-aminobutyric acid, characterized in that, The GABA-related transporter genes include genes cg2139, cg2568, cg2339, cg11006, and cg1281, and the nucleotide sequences of genes cg2139, cg2568, cg2339, cg11006, and cg1281 are shown in SEQ ID NO.1 to 5, respectively.
2. The application according to claim 1, characterized in that, The application involves overexpressing the GABA-associated transporter gene in Corynebacterium glutamicum.
3. A genetically engineered bacterium producing γ-aminobutyric acid, characterized in that, The genetically engineered bacteria use Corynebacterium glutamicum as the chassis strain, and overexpress the GABA-associated transporter gene in the chassis strain. The GABA-associated transporter gene is cg2139, cg2568, cg2339, cg11006 or cg1281, and the nucleotide sequences of the genes cg2139, cg2568, cg2339, cg11006 and cg1281 are shown in SEQ ID NO.1 to 5, respectively.
4. The genetically engineered bacteria according to claim 3, characterized in that, The chassis strain also had the gene cg0280 knocked out, and the nucleotide sequence of the gene cg0280 is shown in SEQ ID NO.
6.
5. The genetically engineered bacterium according to claim 4, characterized in that, The strain in the chassis is Corynebacterium glutamicum FF10.
6. A method for increasing the yield of γ-aminobutyric acid (GABA) from Corynebacterium glutamicum, characterized in that, The method involves overexpressing a GABA-associated transporter gene in Corynebacterium glutamicum FF10. The GABA-associated transporter gene is cg2139, cg2568, cg2339, cg11006, or cg1281, and the nucleotide sequences of the genes cg2139, cg2568, cg2339, cg11006, and cg1281 are shown in SEQ ID NO. 1 to 5, respectively.
7. The method according to claim 6, characterized in that, The GABA-associated transporter gene is integrated and expressed at the cg0280 site of the Corynebacterium glutamicum genome.
8. A method for biosynthesizing γ-aminobutyric acid, characterized in that, The method involves inoculating the genetically engineered bacteria described in any one of claims 3 to 5 into a fermentation system using glucose as a carbon source to synthesize γ-aminobutyric acid.
9. The method according to claim 8, characterized in that, In the fermentation system, the glucose concentration is 50 g / L; the inoculum is 10%, the fermentation temperature is 30℃, the shaking speed is 200 rpm, and the fermentation time is 48-120 hours.
10. The use of a GABA-associated transporter gene or the genetically engineered bacteria according to any one of claims 3 to 5 in the preparation of γ-aminobutyric acid or products containing γ-aminobutyric acid, characterized in that, The GABA-related transporter genes include genes cg2139, cg2568, cg2339, cg11006, and cg1281, whose GenBank accession numbers are CAF20294.1, CAF21003.1, CAF20468.1, BAB98399.1, and CAF19836.1, respectively.