Acid-resistant element of corynebacterium glutamicum and application thereof

Abstract

The invention discloses an acid-resistant element derived from corynebacterium glutamicum and application of the acid-resistant element. The acid-resistant element is derived from Corynebacterium glutamicum, and is obtained by carrying out transcriptomic analysis under an acidic condition. The remarkably up-regulated acid-resistant elements obtained under the transcriptome treatment condition are sigE and cg2888. Recombinant bacteria of which the two genes are respectively knocked out are treated and knocked out under the condition that the pH value is 5.5, the OD600 is respectively reduced by 92.05% and 93.68% compared with that of wild bacteria, and after sigE and cg2888 are supplemented, the cg2888 is basically recovered to the growth level of the wild bacteria, so that the cg2888 and the SigE are key elements which remarkably influence the acid adaptation condition in corynebacterium glutamicum.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to an acid-resistant element newly derived from Corynebacterium glutamicum and application thereof. Background Art

[0002] Corynebacterium glutamicum ( Corynebacterium glutamicum ) is an important industrial microorganism and is widely used in the fermentation production of amino acids, organic acids and related fine chemical products. This strain has the characteristics of high metabolic efficiency, strong culture adaptability and good genetic operability, and is an ideal microbial factory for a variety of bio-based products. However, during large-scale fermentation, with the accumulation of products and the continuation of metabolic activities, the pH value in the fermentation system usually shows a downward trend, forming a certain degree of acidic stress environment. Acidic conditions will interfere with the cell membrane structure, inhibit the activity of key enzymes, and affect the intracellular pH homeostasis, thereby significantly reducing the viability of the bacteria and the product yield, becoming one of the main obstacles to fermentation efficiency and strain stability.

[0003] Currently, there are few reports on acid-tolerance elements in C. glutamicum. The only reported application is Chinese patent application 202410600089.9, "Method for Overexpressing the Acid-Tolerance Gene mshA of Corynebacterium glutamicum to Produce Succinic Acid," which mentions an acid-tolerance element for improving succinic acid yield. However, there is no in-depth research, such as data on strain growth. To improve the viability and production performance of C. glutamicum in acidic environments, in-depth identification and analysis of key response genes associated with low pH stress has become an important research direction. Summary of the Invention

[0004] Based on transcriptome analysis, this study systematically compared global gene expression in strains under varying pH conditions and treatment durations, identifying specific response genes whose expression was significantly upregulated under acid stress but remained relatively unchanged under neutral and alkaline conditions. These genes showed a continuous trend of increased expression during the short-term response, mid-term adaptation, and long-term tolerance phases, suggesting that they play an important role in the cellular acid response and are potential key components of acid tolerance.

[0005] Compared to screening methods based on mutations or physiological indicators, responsive gene mining based on whole transcriptome data offers advantages such as comprehensive information, high screening efficiency, and strong targeting. Further verification and functional analysis of these elements will help reveal the acid-tolerance mechanism of Corynebacterium glutamicum and provide genetic resources and theoretical foundations for constructing high-tolerance production strains and developing acid-inducible regulatory systems, possessing significant scientific value and application prospects.

[0006] The present invention first provides an acid-resistant element, wherein the acid-resistant element is selected from Corynebacterium glutamicum say or cg2888,or a homologous gene thereof; preferably, the nucleotide sequence thereof is as shown in SEQ ID NO.1-SEQ ID NO.2 or a degenerate sequence thereof, or a homologous gene thereof having more than 99% identity therewith and derived from Lactobacillus brevis.

[0007] The screening criteria for acid-resistant elements set by the present invention are as follows: during the 20-min acid stress response period, cg2888 and say The acid-resistant element cg2888 and say , from Corynebacterium glutamicum ATCC 13032, was mined from transcriptome data, say yes cg2888 downstream transcription factors.

[0008] The acid-resistant element cg2888 , the gene name is PhoR, in Corynebacterium glutamicum, it can form a two-component signaling system with PhoS, in which cg2888 It is a response regulator that transmits acid signals to downstream genes. Two-component systems are a type of signal regulation mechanism widely distributed in prokaryotic microorganisms. They primarily consist of a receptor kinase and a response regulator, capable of sensing changes in the external environment and initiating corresponding cellular response regulation pathways. This type of system is widely used in bacteria to adapt to changes in external conditions and constitutes an important regulatory mechanism for microorganisms to rapidly recognize and respond to complex environments.

[0009] The acid-resistant element say , belonging to the Sigma factor family, are key transcriptional regulators in bacteria, mediating RNA polymerase recognition of specific promoter sequences, thereby initiating transcription of target genes. Different types of Sigma factors can sense a variety of stress signals and, by binding to promoters in regulatory regions, confer environmental response specificity to RNA polymerase. This type of factor is widely involved in bacterial adaptation to adverse environments, playing a core regulatory role under various stress conditions such as oxidative stress, acid-base fluctuations, and heat shock, and is an important component in regulating cellular transcriptional reprogramming.

[0010] The present invention tested Corynebacterium glutamicum ATCC 13032, knockout strain ΔsigE and knockout strains Δcg2888 OD at pH 5.5 and neutral conditions 600 , it was found that the OD of ATCC 13032 was 600 is about 7.44, ΔsigE OD 600 is 0.59, Δcg2888 OD 600was 0.47, which was 92.05% and 93.68% lower than that of the wild type, indicating that pH 5.5 seriously affected ΔsigE and Δcg2888 The growth capacity of say and cg2888 After that, the OD value of ATCC13032 was 600 is about 7.44, ΔsigE::sigE OD 600 is 4.82, Δcg2888::cg2888 OD 600 It is 7.28, which shows that after covering, cg2888 It has basically returned to the growth level of wild mushrooms.

[0011] The present invention provides an acid-resistant protein, which is encoded by the acid-resistant element.

[0012] The present invention also provides a recombinant vector containing an acid-resistant element; specifically, the acid-resistant element is inserted into an expression vector (such as pXMJ19) to form a recombinant plasmid.

[0013] The present invention further provides a recombinant bacterium containing an acid-resistant element, which contains the acid-resistant element or the recombinant vector.

[0014] Specifically, the recombinant vector is transferred into a target host bacterium (eg, Corynebacterium glutamicum) for expression.

[0015] More specifically, the recombinant vector is introduced into Corynebacterium glutamicum by vector-mediated electrotransformation to achieve heterologous expression and impart acid resistance to the bacterium.

[0016] In a specific embodiment, the electroporation operation includes the following steps: mixing the constructed plasmid with pre-treated competent Corynebacterium glutamicum cells, and placing them in a special electroporation cup for treatment; then incubating them to allow the bacteria to repair; and then spreading them on a solid culture medium containing antibiotics, and performing subsequent screening and verification after a single colony appears.

[0017] Furthermore, the acid resistance of the obtained single colony strains was further verified by culturing and observing the growth under acidic conditions of pH 5.5 and neutral conditions of pH 7.0 for three treatment times of 20 minutes, 2 hours and 16 hours.

[0018] The present invention also provides the use of the acid-resistant element or its recombinant vector in enhancing the adaptability of Corynebacterium to acidic environments; specifically, the Corynebacterium is Corynebacterium glutamicum.

[0019] Specifically, the acid-resistant element is introduced into the target host bacteria so that the resulting recombinant bacteria has improved acid resistance. More specifically, this is achieved by constructing a recombinant expression vector with the acid-resistant element and then introducing it into the target host bacteria.

[0020] By applying the above technical solution, the present invention has the following advantages over the existing technologies: Based on transcriptome analysis, the present invention systematically screened and identified two key target genes closely related to acid stress response, clarifying their core role in the acid resistance mechanism of Corynebacterium glutamicum, providing a scientific basis for the rational design of acid-resistant strains. Knocking out these two key genes in Corynebacterium glutamicum significantly reduced the strain's growth ability, survival rate, and metabolic activity in acidic environments, demonstrating acid resistance far lower than that of existing engineered strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Corynebacterium glutamicum ATCC 13032, ΔsigE and Δcg2888 OD at pH 5.5 and neutral conditions 600 .

[0022] Figure 2 Corynebacterium glutamicum ATCC 13032, Δcg2888::cg2888 and ΔsigE::sigE OD at pH 5.5 and neutral conditions 600 . DETAILED DESCRIPTION

[0023] The present invention is described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0024] Example 1: Transcriptome-based screening of acid-resistance response elements in Corynebacterium glutamicum This example uses standard strains Corynebacterium glutamicum ATCC 13032 was used as the research object and cultured in BHI medium until the logarithmic growth phase. The initial OD 600 The inoculation was performed after the pH was set to 0.1. Three treatment durations (20 minutes, 2 hours, and 16 hours) and two pH conditions (acidic (pH 5.5) and neutral (pH 7.0) were used, resulting in a total of six sample treatments. Biological replicates were set for all treatments. After treatment, the bacteria were collected and high-throughput transcriptome sequencing was commissioned to Beijing Novogene Technology Co., Ltd. Sequencing data demonstrated high correlation between replicate samples and good expression consistency. The data have been uploaded to the NCBI SRA database under the accession number PRJNA1068289.

[0025] By systematically analyzing transcriptome data, we identified core response genes that were significantly upregulated under low pH stress conditions. The screening criteria included: significant upregulation of expression in short-term acid treatment (20 min). Based on the above criteria, we initially screened out two acid stress response genes whose expression patterns met the requirements, namely say and cg2888 , which will be subsequently used in functional verification experiments as a potential key acid-resistant component.

[0026] The potential functions of Sigma factors and response regulatory proteins in acid stimulation were further analyzed. The results showed that under 20-min acid stress conditions, the bacterial transcription factor SigE showed significant upregulation and was one of the most sensitive Sigma factors in response to acid stimulation, indicating that it plays an important regulatory role in the early stages of stress. However, in the subsequent 2-h adaptation and 16-h tolerance stages, SigE expression did not change significantly, suggesting that its main activity is concentrated in the early response stage. In addition, the response regulatory proteins in the two-component system cg2888 It also showed obvious activation in the early stage of acid stress, indicating that this signaling pathway may be involved in the early perception and transduction of low-acid stimulation signals and is one of the important regulatory mechanisms for initiating acid response reactions.

[0027] Example 2: Construction and verification of gene knockout and complementation strains of Corynebacterium glutamicum 1. Extraction of Corynebacterium glutamicum genome In this example, a genomic DNA extraction kit provided by Beijing Tiangen Biochemical Technology Co., Ltd. was used to extract genomic DNA from Corynebacterium glutamicum according to the instructions. The specific steps are as follows: Take 1-5 mL of bacterial culture and centrifuge at 12,000 rpm for 1 minute. Discard the supernatant. Add an appropriate amount of buffer to wash once. Repeat the centrifugation step, discard the remaining liquid, and retain the cell pellet.

[0028] Add 110 μL of lysis buffer (containing 20 mM Tris-HCl, pH 8.0; 2 mM EDTA; 1.2% Triton X-100) to the precipitate, followed by 70 μL of lysozyme solution (50 mg / mL). Mix well and place in a 37°C incubator for more than 30 min to fully lyse the cell wall.

[0029] Add 20 μL of Proteinase K enzyme solution and mix thoroughly, then add 220 μL of buffer GB, shake vigorously for 15 seconds, and incubate at 70°C for 10 minutes. The mixture will gradually turn from turbid to clear.

[0030] Then, 220 μL of anhydrous ethanol was added and mixed thoroughly. The mixture was transferred to a centrifugal adsorption column CA and centrifuged at 12,000 rpm for 1 min. The filtrate was discarded.

[0031] Add 500 μL of buffer GD in sequence, centrifuge for 1 min, and discard the effluent; then add 600 μL of buffer PW, centrifuge for 1 min, and discard the waste liquid; repeat once, add 600 μL of buffer PW, and centrifuge for 2 min to remove residual ethanol.

[0032] After washing, centrifuge again for 2 minutes without loading to ensure that there is no residual liquid in the column.

[0033] Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 50 μL of elution buffer, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute; repeat the elution process once, and collect the eluate twice to obtain the target DNA product.

[0034] The final genomic DNA should be stored at -20℃ for future use.

[0035] 2. Construction of knockout plasmid This example uses the temperature-controlled plasmid pCRD206 as the basic vector, combined with sacB The gene's lethal effect in high sucrose concentrations was knocked out at a specific site in Corynebacterium glutamicum through double crossover homologous recombination. The specific steps are as follows: The target genes were amplified by polymerase chain reaction using the extracted Corynebacterium glutamicum ATCC13032 genome as a template. cg2888 and say The upstream and downstream homologous fragments of the primers are shown in SEQ ID NO. 3 to SEQ ID NO. 10. The above homologous sequences were assembled into the pCRD206 backbone vector using seamless ligation technology. The recombinant plasmids constructed were transformed into Escherichia coli, and the plasmids contained in the positive clones were extracted after resistance screening.

[0036] Screen the recombinant plasmid, confirmed to be correct by sequencing, and introduce it into competent cells of Corynebacterium glutamicum by electroporation. Spread the plasmid onto the surface of LBHIS solid medium containing kanamycin and incubate at 25°C. Select a single colony from the plate and inoculate it into LBHIS liquid medium containing kanamycin. Cultivate the culture at 25°C with shaking.

[0037] The bacterial suspension obtained by culture at 25°C was inoculated into LBHIS liquid medium containing kanamycin and cultured at 37°C for another 24 h to enhance the selection pressure of plasmid inactivation.

[0038] The bacterial solution obtained in the previous step was streaked onto an LBHIS plate containing kanamycin and incubated at 37°C for 24 h to promote single exchange.

[0039] Samples were taken from the mixed colonies, inoculated into culture medium A without antibiotics, and cultured at 32°C for 24 h.

[0040] The above culture was transferred to liquid medium A containing 10% sucrose, cultured at 32°C for 24 h, then streaked onto solid medium A containing the same concentration of sucrose and continued to be cultured at 32°C to screen for strains that underwent a second recombination.

[0041] Select individual colonies from the screening plate and inoculate them onto solid LBHIS plates containing kanamycin and those without the antibiotic. Compare their growth patterns. Select colonies that grow on the antibiotic-free plates but not on the antibiotic-containing plates. Extract the colony template and perform PCR verification. If the amplified fragment size is inconsistent with the positive control and the sequencing results are consistent with expectations, the target gene can be determined to have been successfully knocked out or replaced.

[0042] cg2888 and say The knockout bacteria, named Δcg2888 and ΔsigE .

[0043] 3. Construction of complementing plasmids and acquisition of complementing strains This example constructs a complementation expression plasmid based on the temperature-controlled shuttle plasmid pCRD206, and reintroduces the target gene into the knockout background strain by electroporation, thereby obtaining a gene-complementing strain of Corynebacterium glutamicum. The specific steps are as follows: The target gene was amplified by polymerase chain reaction using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template. cg2888 and say The coding region of the primers is shown in SEQ ID NO.11 to SEQ ID NO.14.

[0044] The above-mentioned target fragment was connected to the pCRD206 vector backbone that had been treated with the same enzyme digestion, and seamless cloning assembly was used to construct the complementing expression plasmids pCRD206-cg2888 and pCRD206-sigE, which were transformed into Escherichia coli DH5α competent cells, respectively. The correctness of the inserted fragment was verified by chloramphenicol resistance screening and sequencing.

[0045] The correct recombinant plasmid was introduced into Δcg2888 and ΔsigE Among the knockout strains, screening was performed on LBHIS solid medium containing chloramphenicol. The positive clones were named as: cg2888 The strain is Δ cg2888::cg2888, Cover say The strain is ΔsigE::sigE .

[0046] Positive clones were selected and inoculated into LBHIS liquid medium containing chloramphenicol. The culture was shaken at 30°C, and genomic DNA was extracted. PCR was used to verify the expression and insertion of the complemented gene. Successful complementation was confirmed if the PCR amplification product was the correct size and the target gene was stably expressed in the complemented strain.

[0047] Example 3: Growth test of knockout bacteria and complementing bacteria under low acid conditions Corynebacterium glutamicum ATCC 13032, ΔsigE and Δcg2888 OD at pH 5.5 and neutral conditions 600 ,like Figure 1 As shown, it was found that the OD of ATCC 13032 was 600 is about 7.44, ΔsigE OD 600 is 0.59, Δcg2888 OD 600 was 0.47, which was 92.05% and 93.68% lower than that of the wild type, indicating that pH 5.5 seriously affected ΔsigE and Δcg2888 growth capacity.

[0048] Cover say and cg2888 Afterwards, Figure 2 It can be seen that the OD of ATCC 13032 at pH 5.5 600 is about 7.44, ΔsigE::sigE OD 600 is 4.82, Δcg2888::cg2888 OD 600 It is 7.28, which shows that after covering, cg2888 It has basically returned to the growth level of wild mushrooms.

[0049] In Corynebacterium glutamicum, SigE is closely associated with cell surface stress. It is hypothesized that the loss of sigE causes abnormal membrane permeability, preventing cells from maintaining homeostasis in acidic environments, leading to a decrease in intracellular pH and thus inhibiting growth. Sigma factors are important regulatory modules for bacterial responses to external stimuli. Further research into their mechanisms of action will help optimize strain stress tolerance and enhance the ability to industrially synthesize target products.

Claims

1. An acid-resistant component, characterized in that: The acid-resistant element is selected from Corynebacterium glutamicum sigE or cg2888, or a homologous gene thereof; preferably, the nucleotide sequence thereof is as shown in SEQ ID NO.1-SEQ ID NO.2 or a degenerate sequence thereof, or a homologous gene thereof having more than 99% identity therewith and derived from Lactobacillus brevis.

2. An acid-resistant protein, characterized in that It is obtained by coding the acid-resistant element as claimed in claim 1.

3. A recombinant vector containing an acid-resistant element; specifically, the acid-resistant element according to claim 1 is inserted into an expression vector (such as pXMJ19) to form a recombinant plasmid.

4. A recombinant bacterium containing an acid-resistant element, characterized in that: It contains the acid-resistant element according to claim 1 or the recombinant vector according to claim 3.

5. The recombinant bacterium according to claim 4, characterized in that The recombinant vector as described in claim 3 is transformed into a target host bacterium (eg, Corynebacterium glutamicum) for expression.

6. The recombinant bacterium according to claim 5, characterized in that The recombinant vector is introduced into Corynebacterium glutamicum through vector-mediated electrotransformation to achieve heterologous expression and impart acid resistance to the bacterium.

7. The recombinant bacterium according to claim 6, characterized in that The electroporation operation includes the following steps: mixing the constructed plasmid with pre-treated competent Corynebacterium glutamicum cells, placing them in a special electroporation cup for treatment; then incubating them to allow the bacteria to repair; and then spreading them on a solid culture medium containing antibiotics, and performing subsequent screening and verification after a single colony appears.

8. The recombinant bacterium according to claim 7, characterized in that Furthermore, the acid resistance of the obtained single colony strains was further verified by culturing and observing the growth under acidic conditions of pH 5.5 and neutral conditions of pH 7.0 for three treatment times of 20 minutes, 2 hours and 16 hours.

9. Use of the acid-resistant element or its recombinant vector according to claim 1 in enhancing the adaptability of Corynebacterium to acidic environment; specifically, the Corynebacterium is Corynebacterium glutamicum.

10. The use according to claim 9, characterized in that The acid-resistant element is introduced into the target host bacteria so that the resulting recombinant bacteria has improved acid resistance. More specifically, this is achieved by constructing a recombinant expression vector with the acid-resistant element and then introducing it into the target host bacteria.

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