Acid-specific promoter from corynebacterium glutamicum and application thereof
By exploring and strengthening the acid-specific promoter Pcg2796 of Corynebacterium glutamicum, the problems of low growth ability and fermentation efficiency of the strain under acid stress were solved, efficient fermentation and high product yield were achieved in an acidic environment, and production costs were reduced.
Patent Information
- Application Number
- CN202510810352.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-17
- Publication Date
- 2025-09-12
AI Technical Summary
The existing Corynebacterium glutamicum has low growth ability and fermentation efficiency under acid stress conditions, resulting in a decrease in product synthesis rate and cell death, affecting fermentation efficiency and cost.
The acid-specific promoter Pcg2796 of Corynebacterium glutamicum was discovered and strengthened. By constructing a recombinant plasmid and expression cassette, the promoter was used to strongly activate the expression of the target gene under acidic conditions, thereby improving the acid tolerance of the strain.
The growth ability and survival rate of Corynebacterium glutamicum in an acidic environment are significantly improved, the fermentation efficiency is enhanced, the production cost is reduced, and the yield and quality of the target product are improved.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and particularly relates to an acid-specific promoter newly derived from Corynebacterium glutamicum and application thereof. Background Art
[0002] Corynebacterium glutamicum is a Gram-positive, facultatively anaerobic industrial microorganism widely used in the fermentation production of amino acids, organic acids, and other high-value-added metabolites. It plays a particularly dominant role in the industrial synthesis of amino acids such as glutamic acid and lysine. Its stable growth, strong genetic manipulability, and high carbon source utilization efficiency make it an important model strain in metabolic engineering and industrial fermentation.
[0003] In the actual fermentation process, with the gradual accumulation of target products, especially the continuous synthesis and secretion of amino acids and organic acids such as glutamic acid and lactic acid, the pH value in the fermentation environment often decreases significantly, thereby causing the cells to suffer from acid stress. Acid stress can destroy the integrity of the cell membrane, inhibit the activity of important enzymes, disrupt the proton gradient inside and outside the cell and the balance of energy metabolism, ultimately leading to suppressed cell growth, a decrease in the rate of product synthesis, and even cell death, which seriously affects fermentation efficiency and industrial production capacity. Therefore, improving the tolerance of Corynebacterium glutamicum to acid stress is of great significance for optimizing fermentation processes, improving target product yields, and reducing production costs.
[0004] Currently, methods to improve the acid stability of strains mainly include rational metabolic engineering, whole-genome random mutagenesis and screening, and the discovery and functional enhancement of tolerance-related regulatory elements. Among them, by identifying and modifying genetic factors such as key genes, promoters, transporters or signal transduction elements that regulate acid stress response, it is possible to effectively enhance the acid tolerance of Corynebacterium glutamicum. In particular, the exploration and utilization of natural acid-resistant elements in prokaryotes that are closely related to acid stress response, such as two-component regulatory systems, acid-sensitive promoters and their downstream genes, can provide stable and efficient genetic tools and strategic support for the construction of highly acid-resistant and high-yield engineered strains.
[0005] Therefore, systematically screening and validating acid-tolerant regulatory elements in Corynebacterium glutamicum, particularly promoters or regulatory sequences that are strongly activated under acid stress conditions, holds significant scientific research value and industrial application prospects. By enhancing the function and synthetically modifying these genetic elements, it is expected that the strain's growth capacity and synthetic efficiency under low pH conditions will be significantly improved, providing key technical support for the large-scale, efficient production of target products such as amino acids and organic acids. Summary of the Invention
[0006] The invention mines the acid-specific promoter of Corynebacterium glutamicum from the transcriptome data of Corynebacterium glutamicum ATCC 13032.
[0007] Therefore, the present invention provides an acid-specific promoter of Corynebacterium glutamicum, the nucleotide sequence of which is shown in SEQ ID NO.1.
[0008] The present invention also provides an expression cassette containing the acid-specific promoter of Corynebacterium glutamicum.
[0009] The present invention provides a recombinant plasmid comprising the acid-specific promoter of Corynebacterium glutamicum or the expression cassette.
[0010] Specifically, the acid-specific promoter of Corynebacterium glutamicum can be operably linked to the target gene.
[0011] Particularly, it is a prokaryotic expression plasmid or a eukaryotic expression plasmid.Preferably, it is an expression plasmid that is applicable to Corynebacterium glutamicum, for example, pXMJ19 is a framework.
[0012] The present invention provides a recombinant bacterium containing the acid-specific promoter of Corynebacterium glutamicum, which contains the acid-specific promoter of Corynebacterium glutamicum, or the expression cassette, or the recombinant plasmid.
[0013] Specifically, it is a Corynebacterium, in particular Corynebacterium glutamicum.
[0014] The present invention also provides the use of the acid-specific promoter of Corynebacterium glutamicum in expressing a target gene, specifically, Corynebacterium, specifically Corynebacterium glutamicum, to express the target gene.
[0015] Specifically, it is to express the target gene under acidic stress conditions.
[0016] By applying the above technical solution, the present invention has the following advantages compared with the prior art: The acid-resistant promoter obtained by the present invention is an acid-specific promoter, which significantly improves the growth ability and survival rate of Corynebacterium glutamicum in an acidic environment, especially when the treatment time is long, it can still maintain normal metabolic activity, which is significantly better than the existing unmodified strain. Due to the enhanced acid resistance of the strain, it can continue to ferment efficiently in a lower pH environment, which helps to reduce the use of alkaline neutralizers, reduce production costs, and improve the yield and quality of the target product. Specific experiments show that the expression level of the acid-specific promoter is higher than that of the control at 20min, 2h and 16h acid stimulation. Corynebacterium glutamicum ATCC 13032 were upregulated by approximately 1.3-1.5 times, 7.0-7.4 times, and 80-83 times, respectively. The transcriptome prediction was verified by fluorescence spectrophotometry, and the fluorescence intensity of the acid-specific promoter of the present invention was approximately 40-45 times higher than that of the control. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 、P cg2796 The relative expression levels of the acid-specific promoter treated at pH 5.5 for 20 min, 2 h and 16 h (compared to Corynebacterium glutamicum ATCC 13032).
[0018] Figure 2 、P cg2796 Fluorescence assay for acid-specific promoters. DETAILED DESCRIPTION
[0019] Example 1: Transcriptomic analysis of Corynebacterium glutamicum Selection of parent strains Corynebacterium glutamicum ATCC 13032 was cultured in BHI medium. After the logarithmic phase, the OD 600 = 0.1, transfer. The experimental setup consisted of three treatment times: 20 minutes, 2 hours, and 16 hours. Two pH conditions were also established: acidic (pH 5.5) and neutral (pH 7.0). The treated samples were sequenced at Beijing Novogene Technology Co., Ltd. Sequencing data showed high correlation between replicate samples, indicating good consistency in expression profiles. Transcriptome data have been uploaded to the NCBI SRA database, accession number: PRJNA1068289.
[0020] This example uses transcriptome analysis to systematically screen key metabolic pathways and regulatory factors in Corynebacterium glutamicum under low pH stress, identifying core regulatory elements and signal response modules related to acid adaptation. The screening criteria for acid response elements are: expression levels increased by more than 50 times at pH 5.5 for 16 hours, and the 2-hour treatment group was higher than the 20-minute treatment group. Based on the above criteria, a highly matched candidate acid response element was finally screened. cg2796 , used for subsequent functional verification.
[0021] The P cg2796 Acid-specific promoter, the upstream gene of which encodes a protein belonging to the ABC transporter superfamily, is speculated to be involved in the transport of extracellular iron-siderophore. Figure 1 As shown in the transcriptome data, the expression levels of α-glucose-1-phosphate dehydrogenase (α-glucose) were significantly higher than those of α-glucose-1-phosphate dehydrogenase (α-glucose) at 20 min, 2 h, and 16 h after acid stimulation. Corynebacterium glutamicum ATCC 13032 were upregulated by approximately 1.3-1.5-fold, 7.0-7.4-fold, and 80-83-fold, respectively, indicating that the promoter has a significant response ability to low pH environment and has the potential to serve as an acid-specific regulatory element.
[0022] Example 2: Construction method of recombinant plasmid 1. Extraction of Corynebacterium glutamicum genome In this example, a commercial genomic DNA extraction kit (provided by Beijing Tiangen Biochemical Technology Co., Ltd.) was used to isolate chromosomal DNA from Corynebacterium glutamicum. The procedure was as follows, following the product instructions: Take 1 to 5 mL of bacterial culture and centrifuge at 12,000 rpm for 1 minute to collect the cells. Remove the supernatant. Then add sterile buffer for washing. Centrifuge again and discard the supernatant. Reserve the precipitate for later use.
[0023] Add 110 μL of lysis pretreatment buffer (containing 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1.2% Triton X-100) and 70 μL of lysozyme (mass concentration of 50 mg / mL) to the cell pellet and place it at a constant temperature of 37°C for more than 0.5 h to fully destroy the cell wall structure.
[0024] Add 20 μL of proteinase K, mix gently, then add 220 μL of lysis buffer GB, shake vigorously for about 15 seconds, and heat at 70°C for 10 minutes until the reaction system becomes clear.
[0025] An equal volume of anhydrous ethanol (220 μL) was added and mixed thoroughly. The entire mixture was added to a centrifugal adsorption column (CA type), centrifuged at 12,000 rpm for 1 min, and the effluent was discarded.
[0026] The washing steps are as follows: add 500 μL of washing buffer GD and centrifuge for 1 minute; after discarding the flow-through, add 600 μL of washing buffer PW and centrifuge for 1 minute; repeat the PW wash once, and extend the centrifugation time to 2 minutes to completely remove residual alcohol.
[0027] After washing, perform an empty centrifugation for 2 minutes to ensure that there is no residual liquid in the filter column.
[0028] Transfer the treated adsorption column to a clean 1.5 mL collection tube, add 50 μL of elution buffer, and let it sit for 2 minutes to allow the DNA to fully dissolve. Centrifuge at 12,000 rpm for 1 minute, repeating this step twice to improve recovery efficiency. The resulting eluate is the chromosomal DNA of C. glutamicum.
[0029] The final product should be stored at -20°C for subsequent experimental operations.
[0030] 2. Construction of Overexpression Plasmid In this example, a recombinant strain of Corynebacterium glutamicum was established by using pXMJ19 as the expression vector backbone, combining seamless cloning and electroporation techniques. The specific operations are as follows: First, the genomic DNA of the wild-type strain of Corynebacterium glutamicum ATCC 13032 extracted above was used as a template to amplify P cg2796 The nucleotide sequences of the primers for the amino acid-specific promoter and GFP fragment are shown in SEQ ID Nos. 2 and 3, and 4 and 5, respectively. The amplified sequence was designed to cover the target gene body, the first 30 bases of the start codon, and the putative ribosome binding site (RBS) to ensure expression efficiency.
[0031] The amplified fragment obtained above was integrated into the pXMJ19 plasmid vector using seamless cloning technology, and the recombinant plasmid was transformed into competent E. coli cells. During the culture process, after colonies appeared on the resistant plates, single colonies were selected for plasmid extraction and the construction was verified by Sanger sequencing. Sequences that matched the designed sequence were considered to be successfully constructed overexpression vectors.
[0032] Next, the confirmed recombinant plasmid was electroporated into the competent cells of Corynebacterium glutamicum and incubated at 32°C for 48 hours to complete the transformation process. After the transformation, colonies appeared on the plate, and they were streaked and inoculated into LBHIS liquid medium containing chloramphenicol. The culture was continued at 32°C overnight to expand the culture and preserve it. Finally, a stable carrier of P was obtained. cg2796 Acid-specific promoter and GFP expression construct pXMJ19-P cg2796 and pXMJ19-GFP recombinant strains.
[0033] Example 3: Fluorescence activity detection of the Corynebacterium glutamicum acid-specific promoter Will carry pXMJ19-P cg2796 The constructed Corynebacterium glutamicum was activated in LBHIS liquid medium, with pXMJ19-GFP as a negative control, until the logarithmic growth phase, and then transferred to pH 5.5 and pH 7.0 buffers for different treatment times of 20 min, 2 h, and 16 h. The cells were collected and the OD was adjusted. 600 = 10, and the cells were broken. The fluorescence intensity of each sample was then measured using a fluorescence spectrophotometer to evaluate the expression activity of the promoter under different pH conditions and stimulation times.
[0034] like Figure 2 As shown in Figure 2, under 16 h acid stress conditions, P cg2796 The fluorescence intensity of GFP expression driven by the acid-specific promoter increased 40-45 times compared with the control. The results showed that the promoter was highly specific to low pH stimulation and had the potential to be used as an excellent acid-responsive regulatory element.
Claims
1. The acid-specific promoter of Corynebacterium glutamicum, characterized in that Its nucleotide sequence is shown in SEQ ID NO.
1.
2. An expression cassette comprising the acid-specific promoter of Corynebacterium glutamicum according to claim 1.
3. A recombinant plasmid, characterized in that: The method comprises the acid-specific promoter of Corynebacterium glutamicum according to claim 1 or the expression cassette according to claim 2.
4. The recombinant plasmid according to claim 3, wherein The acid-specific promoter of Corynebacterium glutamicum can be operably linked to a target gene.
5. The recombinant plasmid according to claim 3 or 4, characterized in that It is a prokaryotic expression plasmid or a eukaryotic expression plasmid.
6. The recombinant plasmid according to claim 5, wherein It is an expression plasmid suitable for Corynebacterium glutamicum, for example, using pXMJ19 as the framework.
7. A recombinant bacterium containing the acid-specific promoter of Corynebacterium glutamicum according to claim 1, characterized in that: It contains the acid-specific promoter of Corynebacterium glutamicum according to claim 1, or the expression cassette according to claim 2, or the recombinant plasmid according to any one of claims 3 to 6.
8. The recombinant bacterium according to claim 7, characterized in that It is a Corynebacterium, specifically Corynebacterium glutamicum.
9. Use of the acid-specific promoter of Corynebacterium glutamicum according to claim 1 in expressing a target gene, specifically, Corynebacterium, specifically Corynebacterium glutamicum, to express the target gene.
10. The use according to claim 9, characterized in that It expresses the target gene under acidic stress conditions.