Acid-resistant element from escherichia coli and application of acid-resistant element

Abstract

The invention discloses an acid-resistant element derived from Escherichia coli and application of the acid-resistant element. The acid-resistant elements are respectively HdeB and GlsA, and the acid-resistant elements are constructed on an expression vector and are introduced into corynebacterium glutamicum to obtain an acid-resistant engineering strain. A research result shows that the acid-resistant element has a good effect on improving the acid resistance of corynebacterium glutamicum in an acid environment such as a condition that the pH value is 5.5, and the application potential of the acid-resistant element in fermentation strain modification is proved.

Description

Technical Field

[0001] The invention relates to an acid-resistant element derived from Escherichia coli and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] Corynebacterium glutamicum ( Corynebacterium glutamicum As a typical industrial microorganism, β-amino acid (AMIN) is widely used in the fermentation production of amino acids and their derivatives. However, in actual production, the pH of the culture system often decreases continuously with the accumulation of fermentation products, creating an acidic stress environment that severely affects the growth rate and metabolic activity of the bacteria, ultimately leading to reduced product yield and prolonged fermentation cycles.

[0003] To alleviate these issues and improve strain adaptability to acid stress, various strategies for acid-tolerance modification have been developed, including mutation breeding, gene overexpression, genome editing, and regulatory network reconstruction. However, these currently rely primarily on modifying the acid-tolerance genes of Corynebacterium glutamicum. This is due to technical bottlenecks such as incomplete intrinsic acid-tolerance mechanisms, limited targets, and instability and limited effectiveness of modification.

[0004] In-depth research into bacterial physiology has revealed that certain strains, such as lactic acid bacteria and Escherichia coli, contain multiple functional genes involved in acid stress response. These exogenous acid-resistant elements have demonstrated remarkable environmental adaptability in other hosts, but their application in Corynebacterium glutamicum remains limited. Therefore, developing and validating the expression and mechanism of action of exogenous acid-resistant elements in these host strains has become an important research direction for improving their acid resistance. Summary of the Invention

[0005] In order to obtain the key regulatory element that can enhance the viability of Corynebacterium glutamicum in a low pH environment, the present invention selects Escherichia coli MG1655 was used as the source of acid-resistant functional factors. Through functional screening and sequence analysis, two potential acid-resistant related genes were identified and obtained from this strain. hdeB and glsA The above genes were constructed for heterologous expression and introduced into the target host, Corynebacterium glutamicum, to further evaluate their effects on host growth performance under different acidic conditions.

[0006] The present invention provides an acid-resistant element, wherein the acid-resistant element is selected from HdeB and GlsA derived from Escherichia coli ， 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] in, hdeBEncodes an acid stress chaperone protein located in the bacterial periplasm that can prevent acid-induced denaturation of extracellular proteins under low pH conditions, thereby alleviating cell damage by maintaining their native conformation; glsA It is the structural gene encoding glutaminase A, which catalyzes the hydrolysis of glutamine to produce glutamate and ammonia. It can regulate the intracellular pH by releasing amino groups and achieve acid-resistant buffering effect.

[0008] The present invention also provides an acid-resistant protein, which is encoded by the acid-resistant element.

[0009] 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.

[0010] The present invention further provides a recombinant bacterium containing an acid-resistant element, which contains the acid-resistant element or the recombinant vector.

[0011] Specifically, the recombinant vector is transferred into a target host bacterium (eg, Corynebacterium glutamicum, Escherichia coli) for expression.

[0012] More specifically, the recombinant vector is introduced into the target host bacteria through vector-mediated electrotransformation to achieve heterologous expression and impart acid resistance.

[0013] In a specific embodiment, the electroporation operation includes the following steps: mixing the constructed plasmid with the target host bacterial cells pretreated to be competent, and placing them in a special electroporation cup for treatment; then incubating 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.

[0014] Furthermore, the acid resistance of the obtained single colony strain was further verified by culturing at pH 5.5, 6.0 and 7.0 for 18 hours and 24 hours to observe the growth.

[0015] The present invention also provides the use of the acid-resistant element or its recombinant vector in enhancing the ability of bacteria to adapt to acidic environments; specifically, the bacteria is Corynebacterium or Enterobacterium, more preferably Corynebacterium glutamicum.

[0016] Specifically, the acid-resistant element is introduced into the target host bacteria so that the resulting recombinant bacteria have 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.

[0017] The advantages of the present invention compared with the prior art are: Experimental data showed that under pH 5.5, the OD of the recombinant strain containing the acid-resistant element of the present invention was600 The values ​​were significantly higher than those in the empty vector control group, with the HdeB-expressing strain showing a significant increase of over 20-fold within 24 hours, confirming its strong adaptability to acid stress. The exogenous acid-resistant element not only exhibited a significant enhancement effect under strong acid (pH 5.5), but also exhibited a certain degree of tolerance improvement under moderate and weak acid (pH 6.0), demonstrating that the present invention has a wide range of acid adaptation.

[0018] The present invention introduces two acid-resistant genes, HdeB and GlsA, from the Escherichia coli MG1655 strain for the first time, avoiding the limitations of relying solely on native genes of Corynebacterium glutamicum for modification and expanding the available sources of acid-resistant elements. Furthermore, the acid-resistant elements of the present invention can be introduced into the host by electroporation using the pXMJ19 plasmid as an expression vector. This simple and mature method is widely applicable to industrial strain construction and high-throughput screening, and has promising engineering application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 , WT-pXMJ19, HdeB-pXMJ19 and GlsA-pXMJ19 bacterial growth under pH 5.5, 6.0 and 7.0, 18h and 24h respectively. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1: Mining and amplification of acid-resistant elements in Escherichia coli 1. Extraction of the genome of Escherichia coli MG1655 strain E. coli genome extraction was performed according to the instructions for the Beijing Tiangen Biochemical Technology Genome Extraction Kit. The specific steps were as follows: 1 to 5 mL of an overnight E. coli culture suspension was centrifuged at 10,000 rpm for 1 minute, and the supernatant was discarded. To the cell pellet, 110 μL of custom-made lysis buffer (20 mM Tris, pH 8.0, 2 mM EDTA, 1.2% Triton X-100) and 70 μL of 50 mg / mL lysozyme solution were added and incubated at 37°C for at least 30 minutes. Next, 20 μL of proteinase K and 220 μL of lysis enhancement buffer (GB) were added sequentially. The mixture was thoroughly mixed, shaken vigorously for 15 seconds, and incubated at 70°C for 10 minutes. The solution was allowed to gradually clear before proceeding. 220 μL of anhydrous ethanol was then added, mixed thoroughly, and transferred to a silica-based adsorption column. Binding was then performed by centrifugation at 12,000 rpm for 1 minute. Wash twice with 500 μL of Buffer GD and 600 μL of Wash Buffer PW, centrifuging after each wash and discarding the remaining liquid. Centrifuge the empty tube for another 2 minutes to remove any residual liquid. Finally, slowly add 50 μL of TE buffer to the center of the column in two separate drops. Allow to stand at room temperature for 2 minutes each time before centrifuging each time. A 100 μL genomic DNA sample is obtained and stored at low temperatures for later use.

[0022] 2. Screening of acid-resistant genes from Escherichia coli MG1655 In order to screen for elements that can improve the acid tolerance of Corynebacterium glutamicum, the present invention uses Escherichia coli MG1655 as the strain from which the acid-resistant gene originates, and screening is performed by the following steps: First, based on the public full genome information of Escherichia coli MG1655 and the acid stress response genes reported in the literature, MG1655 was cultured at pH 5.0 and pH 7.0, and transcriptome sequencing was used to analyze the differentially expressed genes under acid stress. The results showed that under pH 5.0, hdeB, glsA, gadA, yhiM The expression of multiple genes was up-regulated by more than 2 times (log2 fold change >1, p<0.01), among which hdeB and glsA The increase was the most significant.

[0023] Secondly, conservation analysis and functional structure prediction of the protein products of these differentially expressed genes were performed using BLAST and protein structure prediction software. The results showed that the acid-stimulated protein encoded by hdeB is highly evolutionarily conserved, with homologous proteins found in a variety of Gram-positive and Gram-negative bacteria, and its N-terminal region is involved in maintaining intracellular pH homeostasis. Furthermore, the glutamine synthetase encoded by glsA may increase intracellular pH by consuming protons under low pH conditions, potentially exerting a buffering effect.

[0024] Based on the above data, hdeB and glsA were finally determined to be the target acid-resistant elements of the present invention, which are used to enhance the acid resistance of Corynebacterium glutamicum.

[0025] 3. Construction method of recombinant plasmid All primers used in this example were designed with the aid of SnapGene software and synthesized by a third-party professional synthesis agency. The target gene fragments were amplified using high-fidelity DNA polymerase and FastPfu Fly DNA polymerase, respectively. The E. coli MG1655 genome extracted in the first part was used as a template. hdeB and glsA Forward primers and reverse primers were designed, and their primer sequences were shown as SEQ ID NOs. 3 and 4, and SEQ ID NOs. 5 and 6, and PCR amplification was performed.

[0026] PCR products were detected by agarose gel electrophoresis, excised, and recovered. Fragments were purified using a commercial DNA purification kit. The resulting target DNA sequence was then assembled into an expression vector using the pXMJ19 backbone using a seamless cloning system. The resulting recombinant product was used to transform competent host cells. Screening was performed using 2× Rapid Taq Master Mix. Positive clones were then processed using a plasmid extraction kit to successfully obtain the target recombinant vectors, designated HdeB-pXMJ19 and GlsA-pXMJ19, for subsequent analysis or expression experiments. The original plasmid pXMJ19 was designated WT-pXMJ19.

[0027] Example 2: Screening of acid-resistant functional elements 1. Electroporation of target plasmid A single colony of Corynebacterium glutamicum was selected and inoculated into 5 mL of LBHIS liquid medium. The culture was shaken at 32°C until the cell density OD 600 The cell density is about 0.6. After the cells are collected by centrifugation, they are washed three times in a row with 0.9% physiological saline, and then the bacteria are resuspended in cold saline at 1% of the initial volume to prepare competent cells. 10-100 ng of the recombinant plasmid constructed in Example 1 is mixed with the above-mentioned competent bacterial solution and placed in a pre-cooled electroporation cup for electric pulse treatment. Immediately after the electric shock, 1 mL of fresh LBHIS culture medium is added and the cells are placed at 32°C for 1 hour to repair. Finally, the culture is evenly spread on the surface of LBHIS solid culture medium containing kanamycin (concentration of 50 μg / mL), cultured at the same temperature for 24 hours, and positive clones are screened.

[0028] 2. Screening and evaluation methods for acid-resistant functional factors First, the recombinant strains carrying different exogenous acid-resistant genes were streaked and inoculated on LBHIS plates (containing an appropriate amount of selective antibiotics) in sequence and placed at 32°C for overnight culture. Secondly, a single colony was picked from each recombinant strain and inoculated into a liquid test tube, and the seed liquid was prepared by shaking culture at 32°C and 200rpm. Then, it was inoculated into 25mL BHI liquid culture medium at a volume ratio of 1%, and the shake flask culture was continued under the same conditions for about 16 hours until the bacterial growth entered the late logarithmic phase or the stable phase. The bacterial liquid was then centrifuged and washed using BHI medium with pH 7.0, and resuspended to OD 600 The standard concentration was 10. 100 μL of the standard bacterial solution was inoculated into BHI basal culture medium at pH 5.5, 6.0, and 7.0 (the culture medium contained 100 mM MES buffer and 0.4 mM IPTG inducer). The initial OD 600 The strain was cultured at 32°C and 200 rpm for 18 h and 24 h, and the OD at each time point was measured. 600 The growth performance of the strains under different pH conditions was evaluated, and the excellent recombinant strains with significant acid adaptability were screened out.

[0029] The results are shown in Table 1 and Figure 1 As shown in the figure, under neutral conditions, there was no significant difference in the growth state. After 18h stimulation at pH 5.5, the OD values ​​of HdeB-pXMJ19 and GlsA-pXMJ19 were significantly different. 600 Compared with the control WT-pXMJ19, the growth rate of HdeB-pXMJ19 increased by 5.62 and 14.16 times, respectively. After 24 hours, the growth effect of HdeB-pXMJ19 was more significant, increasing by 20.71 times, while GlsA-pXMJ19 only increased by 9.44 times. Under pH 6.0 stimulation, the effect was not as significant as pH 5.5. The OD values ​​of HdeB-pXMJ19 and GlsA-pXMJ19 at 18 hours were 600 The growth rates increased by 3.71 and 2.88 respectively after 24 hours, and by 3.85 and 2.96 times after 24 hours. It can be seen that the growth rate basically stabilized after 18 hours.

[0030] Table 1 .

Claims

1. An acid-resistant component, characterized in that: The acid-resistant element is selected from HdeB and GlsA derived from Escherichia coli ， 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, Escherichia coli) for expression.

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

7. The recombinant bacterium according to claim 6, characterized in that The electroporation operation includes the following steps: mixing the constructed plasmid with the target host bacterial cells that have been pretreated to be competent, 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.

8. The recombinant bacterium according to claim 7, characterized in that Furthermore, the acid resistance of the obtained single colony strain was further verified by culturing at pH 5.5, 6.0 and 7.0 for 18 hours and 24 hours to observe the growth.

9. Use of the acid-resistant element or its recombinant vector according to claim 1 in enhancing the adaptability of bacteria to acidic environments; specifically, the bacteria is Corynebacterium or Enterobacterium, more preferably 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 have 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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