Deinococcus radiodurans douV gene, protein and application of deinococcus radiodurans douV gene in improving stress resistance of chassis strain

Abstract

The invention provides a deinococcus radiodurans douV gene, a protein and an application of the deinococcus radiodurans douV gene in improving the stress resistance of a chassis strain, and belongs to the technical field of synthetic biology, the nucleotide sequence of the douV gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein is as shown in SEQ ID NO.2. According to the invention, the douV gene is found from the microbe deinococcus radiodurans in an extreme environment, and the douV gene is expressed in a prokaryotic microbe chassis strain escherichia coli by constructing an expression vector, so that the functional gene douV is determined to be capable of improving the biological stress resistance. The constructed engineering bacterium for expressing the douV gene has excellent salt resistance, oxidation resistance and osmotic stress resistance, can adapt to different environments, and is more suitable for production and application.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, and in particular relates to a radiation-resistant abnormal cocci douV gene, protein and its application in improving the stress resistance of chassis strains. Background Technology

[0002] Synthetic biology, which redesigns or even synthesizes organisms from scratch in an engineered manner, will overcome the limitations of natural evolution and create synthetic organisms that surpass the capabilities of natural life. This is not only of great scientific significance for exploring the basic laws of life activities, but also has great application prospects in fields such as industrial and agricultural production, environmental protection, and health care.

[0003] The design and construction of microbial cell factories using synthetic biology techniques has been widely used in the production of a wide variety of chemicals, foods, pharmaceuticals, and energy, and is a core component of green biomanufacturing. The construction of microbial cell factories is achieved by introducing novel enzymes and constructing new pathways into microbial chassis cells and allowing them to adapt to the chassis cells. However, improving the performance of chassis strains aims to increase the chassis's tolerance to substrates and products, enhance growth rates, increase substrate utilization, and improve product yield.

[0004] Addressing the bottlenecks in stress resistance and production performance of low-version chassis strains, improving the robustness and cell activity of chassis cell factories under fermentation stress environments by discovering stress-resistant functional genes of microorganisms in extreme environments and designing artificially efficient stress-resistant modules has become a hot topic in synthetic biology cell factory research.

[0005] Currently, there is limited research on genes that can enhance the stress resistance of chassis strains, and further development of stress-resistance functional genes is needed. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a radiation-resistant abnormal cocci douV gene, protein and its application in improving the stress resistance of chassis strains, wherein the douV gene can significantly improve the salt tolerance, antioxidant and osmotic stress resistance of chassis strains.

[0007] This invention provides a douV gene for radiation-resistant abnormal cocci, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The present invention provides a protein encoded by the douV gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] This invention provides a recombinant expression plasmid for improving biological stress resistance, wherein the recombinant expression plasmid includes the douV gene and the initial plasmid.

[0010] Preferably, the initial plasmid is the pRADZ3 plasmid.

[0011] This invention provides the application of the douV gene, the protein, or the recombinant expression plasmid in improving biological stress resistance.

[0012] Preferably, the organism is an engineered bacterium.

[0013] Preferably, the engineered bacteria include Escherichia coli.

[0014] Preferably, the stress resistance includes salt resistance, oxidation resistance, or resistance to osmotic stress.

[0015] This invention provides a highly resistant engineered bacterium, obtained by transferring the recombinant expression plasmid into a chassis strain.

[0016] This invention provides the application of the engineered bacteria in biosynthesis.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The present invention discovered the douV gene in the extreme environment microorganism *Radiation Abnormal Cocci*, and expressed the douV gene in the prokaryotic microorganism chassis strain *Escherichia coli* by constructing an expression vector, and determined that the functional gene douV can improve the stress resistance of organisms.

[0018] This invention constructed a recombinant Escherichia coli engineered strain, BW-douV, expressing a functional gene. Experiments showed that BW-douV exhibited significantly better stress resistance than the control strain. Under oxidative stress conditions of 20 mM hydrogen peroxide for 20 minutes, the survival rate of engineered strain BW-douV was 3 times higher than that of the control strain BW-Z3. Under high-salt stress conditions of 5 M sodium chloride, the survival rate of the control strain BW-Z3 decreased by 5 orders of magnitude, while the engineered strain BW-douV expressing the stress resistance gene douV showed 2 orders of magnitude higher resistance than the control, approximately 100 times. Under osmotic stress conditions simulated by 2 M sorbitol, the survival rate of the control strain BW-Z3 was 2.69%, while the survival rate of the engineered strain BW-douV expressing the stress resistance gene douV was 12.03%, demonstrating approximately 5 times better osmotic resistance than the control. Attached Figure Description

[0019] Figure 1 This is a comparison of the stress resistance and survival ability of engineered strain BW-douV and control strain after 20mM hydrogen peroxide stress, 5M sodium chloride high salt stress, and 2M sorbitol osmotic stress in Example 2; the control group does not show survival rate, and is represented by * to indicate a survival rate of less than 0.01%. Detailed Implementation

[0020] This invention provides a douV gene for radiation-resistant abnormal cocci, the nucleotide sequence of which is shown in SEQ ID NO.1, and is as follows:

[0021] ATGGTGGGGAATATCCTCCCGCCGACCTCTCATTGCGGGCCGCTGGAG

[0022] CAGTTCTCCGAATTACGCGTGCGTCGGAACAGCACCGCCACCCGCTCC

[0023] ATTCTCCGTCCTGCTCTGTGTTTTGCACTCGCTCCGCTCGCCAAAAAGC

[0024] TGTGCCACCTTTTTGTCAAATGCTCTAGCCTCGGCGGTATGCGTTTTCTT

[0025] CCCCTTTTCGCCGGCCTGCTGCTCGTTTCTGCCCTGCCTCAAGCTCGGG

[0026] CGCAGACCATCATTCCACTCGATTCGCGCCCGGCGACCCGCGTGCTGC

[0027] CCGCGCAAATCGCGGGGCTGACGGGGGGCACGCCGCATGTTCCCGCC

[0028] GCCGCGCAACTCGGCAACGCGGCGCAGGGGGCCGACGCCGCCGCGCT

[0029] GCTCGCGTGGCTGGAGGCGCAGCCCAAAGACGGCCCGCTCGTCGTCT

[0030] CGCTCGACGCGCTCGCTTATGGCGGGCTGGTGGAGTCGCGGAGGAGTC

[0031] CGCTGACAGCGGAGCAGGCCCTCGCCCACCTCGCGCCGCTGAAGGAC

[0032] TGGGCAGCGCGCAGCGGGCAGCCGGTCTACGCCTTCATCACCCTGCCG

[0033] CGTGAACCCGACGCCACTGACCGGGCGCGCAATCTGGCGGTGGTGCG

[0034] CGAGGTGATGGCGTGGGCGGGGAGCGGCTCATTCAAAGAACTGCACG

[0035] TCACCTGGGACGACGCGCTGAAGGGCAGCCCCGCTCCGGCAGAGGGC

[0036] GCCGAACTGGCAAAAACGGCCCCCGCCAACGTGCGCGTCTACCCCGG

[0037] CGCCGACGAAGTTGCCTCGCTGCTGGTGGCGCGGGCGCTCGCTCCGGT

[0038] GGCGCAGACGGTGCGGTTCGAATACAGCGACCCCAAAAAGGCTTCCC

[0039] AGGTCATCAAATACGAAGGTATTCCCCTCACCCAGAGCGCCCAGAACC

[0040] ACGCCGAGGCGAGCGGGTTCACCGTGACGGAGAATGGCCCCGCCGAC

[0041] CTCACCGTCTACGTCTACAACGGCGGCGACCCCCGGCAGGCGGCCCTG

[0042] CGAGTCAGTGCCCTGCTGCGGCGCGGGCCGGTGGCGGTGGCGGACGT

[0043] GAACGCCGTCAATCTCGGCAACGCCCGGCTGTGGCGCGACCTGCAAA

[0044] CCCTGCGCCAGAGCGCGAACCTGAAAGCCCTCGCCGCCTGGGGCACG

[0045] CCCGGCAACAACCTCGGCACGGCGCTGGCCCACGCCAAACTCGCGCT

[0046] CGCGGGCGCCGACCCGGTGAGGCAAGATGCCCTGCTCGCCCGCGAGT

[0047] ACGCCAACGACGTGATTTACAGCGGCGAACTGCGCGCGGCCCTGCGA

[0048] AAAGCCCTTCCCGAAAAGGAACTAAACACGCCCGCCGCCCAGGACGC

[0049] CCTCTTCAAGCTCGCGCAGGACTCGTTTCCACTGCGGCTCGGCAACAC

[0050] CTACACGCTGGAAGACGCCGCCTTGCCCTGGGGCCGCTCGTTCGAGTGGGACTTCGACCTCAAGGCGAACTGA.

[0051] In this invention, the douV gene is derived from *Radiatae abnormalis*, and its length is 1452 bp, encoding 483 amino acids. In this invention, the douV gene is obtained by designing primers using the *Radiatae abnormalis* genome as a template and amplifying it using PCR. In this invention, the primers for amplifying the douV gene include VF and VR; the specific sequences are as follows:

[0052] VF: CCCACTAGTATGGCCGGGCACCCCAACACA (SEQ ID NO. 3); VR: CCCCATATGTCAAGAGAGACTGAGTTCGCT (SEQ ID NO. 4).

[0053] This invention provides a protein encoded by the douV gene, the amino acid sequence of which is shown in SEQ ID NO.2, and is as follows:

[0054] MVGNILPPTSHCGPLEQFSELRVRRNSTATRSILRPALCFALAPLAKKLCHLFVKCSSLGGMRFLPLFAGLLLVSALPQARAQTIIPLDSRPATRVLPAQIAGLTGGTPHVPAAAQLGNAA QGADAAALLAWLEAQPKDGPLVVSLDALAYGGLVESRRSPLTAEQALAHLAPLKDWAARSGQPVYAFITLPREPDATDRARNLAVVREMAWAGSGSFKELHVTWDDALKGSPAPAEGAEL AKTAPANVRVYPGADEVASLLVARALAPVAQTVRFEYSDPKKASQVIKYEGIPLTQSAQNHAEASGFTVTENGPADLTVYVYNGGDPRQAALRVSALLRRGPVAVADVNAVNLGNARLWRD LQTLRQSANLKALAAWGTPGNNLGTALAHAKLALAGADPVRQDALLAREYANDVIYSGELRAALRKALPEKELNTPAAQDALFKLAQDSFPLRLGNTYTLEDAALPWGRSFEWDFDLKAN.

[0055] This invention provides a recombinant expression plasmid for enhancing biological stress resistance. The recombinant expression plasmid includes the douV gene and an initial expression plasmid. In this invention, the initial expression plasmid is preferably the pRADZ3 plasmid. After obtaining the nucleotide sequence of the douV gene, this invention preferably ligates the douV gene to a cloning plasmid to construct a recombinant cloning plasmid containing the complete douV gene, and verifies the cloning plasmid by sequencing. Then, the recombinant cloning plasmid is transferred to host cells for propagation. The propagated recombinant cloning plasmid is digested with enzymes to obtain the douV gene. Finally, the douV gene is ligated to the expression plasmid pRADZ3 to obtain the recombinant expression plasmid. In this invention, the nucleotide sequence of the recombinant expression plasmid is shown in SEQ ID No. 5, specifically as follows:

[0056]

[0057] gcccttccctgagcagggtttcaccgtgtggtgtcccttcgtccttgaaggcgaactggtaggccaggccgtaatgct

[0058] cctgcaccatcttttgctcatccaccgcgacaaagaaacggcgggtgttcgccgggctgctgagggtctgccactcg

[0059] ccggacgcgcccaccgcataggcgtctccggctccacattcctcggcctgctgatactgatacttcgtccacagcat

[0060] caccgccttatcgggcacctgcgcgaagctcaccagcgccccgaaccgcccggcgctcgtggtgaaggtggacg

[0061] actgcaccatgtcggacagttccagcgtggcaggcgcgggcgtgttcgtcggcgcgtacaccgtccccgcctcgc

[0062] acacgaccagcggggcggcccccgtgccgaactcggcggcgctgccgtttgccagcgtgagcgccgaagacgc

[0063] caccgcgcaggtacccatgggtgccgaccaccagcaggtccacgccgtgcgccgcctcggtgagcacctcgatt

[0064] ggacggcccctcagggtctgaaccctcccctgcacgcccgcctccccgatgagtgtctgcgccgtctgctgcgcct

[0065] gccgggcattctgctccagcatctcccggacgtagtcgggattcagtcccgcccactgaacgaagccgctgaccg

[0066] gcggctcctcgatcacagtcacgacctccagctcggcgcctgccgccttcccgagcgttatggcctgcttcaaggc

[0067] gcgctggctgggctcacttccgtcaaaggccaccacgaatcgcatccgtcctcctccctgaccagtcaggttcagca

[0068] agcagggtaagcctctcccagcactccggggcttcacacggccttgggtcttttctgcatacccatgagcctcagtac

[0069] ccgacagtttccggcagggcttgaaaaagttcggctgacacgcgagaactggacgcgtctcccgatgtcctggcca

[0070] agttgacccgctgcttcaccgcgcacttcccggagttccgcaagaactaggtcgagctgctctccctcatggtcctcg

[0071] ccctccttaggggcaaggacgtccggcatgctaaactcgccgcgcgcttccccggaagcgcgcacaccgcctcc

[0072] gtcatccggcgggtggaacgcttcttcgaccgtcatcctcttcggccagctgatgtcgcccgggtcgttctgacgctc

[0073] cttcccgccgcgcagccacgcgaatttatccttgaccggaccaactggaagtatgggcagacggacgtgaacgtct

[0074] tgttgctggccgtcatttggcgggacgtcgccatccccctgctctacgagttgctgccccatgggggcagcagcgac

[0075] accgagattcggcacaccctgatggacgatgccctgtgcctgctgtccgccgctgacatccgggtgctgtattagcc

[0076] gaccgcgaattcccagcttgagtaggacaaatccgccgagcttcgacgagattttcaggagctaaggaagctaaaa

[0077] tggagaaaaaaatcactggatataccaccgttgatatatcccaatcgcatcgtaaagaacattttgaggcatttcagtc

[0078] agttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcac

[0079] aagttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggaattccgtatggcaatgaaagacggt

[0080] gagctggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagt

[0081] gaataccacgacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctattt

[0082] ccctaaagggtttattgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggcc

[0083] aatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctg

[0084] gcgattcaggttcatcatgccgtctgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatg

[0085] agtggcagggcggggcgtaatttttttaaggcagttattggtgcccttaaacgcctggtgctacgcctgaataagtgat

[0086] aataagcggatgaatggcagaaattcgtcgaggcggcacctcgctaacggattcaccactccaagaattggagcca

[0087] atcaattcttgcggagaactgtgaatgcatcccgcaagaggcccggcagt.

[0088] This invention provides the application of the douV gene, the protein, or the recombinant plasmid in improving biological stress resistance.

[0089] In this invention, the organism is preferably an engineered bacterium, and the engineered bacterium preferably includes Escherichia coli. In the specific implementation of this invention, the Escherichia coli is preferably strain BW25113.

[0090] In this invention, the stress resistance preferably includes salt tolerance, antioxidant properties, or resistance to osmotic stress. In this invention, the salt tolerance is preferably determined by sodium chloride stress, the antioxidant properties are preferably determined by hydrogen peroxide stress, and the resistance to osmotic stress is preferably determined by sorbitol stress.

[0091] This invention also provides a highly resistant engineered bacterium, obtained by transferring the recombinant expression plasmid into a chassis strain. In this invention, the chassis strain is preferably *Escherichia coli*, and more preferably *Escherichia coli* strain BW25113.

[0092] This invention provides the application of the engineered bacteria in biosynthesis. The engineered bacteria provided by this invention have excellent salt tolerance, antioxidant properties, and resistance to osmotic stress, enabling them to adapt to different environments and making them more suitable for production applications.

[0093] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0094] Example 1

[0095] Construction of recombinant engineered strains expressing the functional gene douV

[0096] I. Experimental Materials

[0097] Cloning vector pJET: a commercially available product from ThermoFisher.

[0098] The shuttle plasmid pRADZ3, reference doi:10.1128 / aem.66.9.3856-3867.2000.

[0099] Escherichia coli BW25113: Purchased from Wuhan Miaoling Biotechnology Co., Ltd., product code T0035.

[0100] II. Experimental Methods

[0101] 1. Primers were designed using the genome of *Radiation-resistant Abnormal Cocci* as a template.

[0102] VF:CCCACTAGTATGGCCGGGCACCCCAACACA(SEQ ID NO.3);

[0103] VR:CCCCATATGTCAAGAGAGACTGAGTTCGCT (SEQ ID NO. 4).

[0104] The full-length nucleic acid sequence of the functional gene douV, 1452 bp in size, encoding 483 amino acids, was obtained by PCR. It was cloned into the vector pJET to construct the recombinant cloning plasmid pJET-douV containing the complete functional gene douV, and sequenced for verification. Then, the functional gene douV with sticky ends and the pRADZ3 vector fragment were obtained by Spe I / Nde I double digestion. The functional gene douV was ligated into the pRADZ3 vector to construct the *E. coli* expression vector pRADZ-douV, which was then transformed into *E. coli* BW25113. Specific procedures are as follows:

[0105] PCR amplification: The PCR amplification system is 20 μL, including 10 μL 2×Phanta Max Master Mix, 1 μL DNA template, 1 μL upstream primer VF with an initial concentration of 10 μmol / L, 1 μL downstream primer VR with an initial concentration of 10 μmol / L and 7 μL ddH2O;

[0106] The PCR amplification program was 35 cycles of denaturation at 95℃ for 3 min → (denaturation at 95℃ for 30 s → annealing at 60℃ for 30 s → extension at 72℃ for 1 min) → extension at 72℃ for 5 min → storage at 4℃.

[0107] Enzyme digestion reaction: 5.0 μL target DNA, 0.5 μL each of restriction endonucleases Spe I and Nde I, 2.0 μL 10×CS Buffer, and 12 μL ddH2O. Digest at 37℃ for 30 min.

[0108] DNA fragments were recovered and purified using a DNA recovery kit purchased from Beijing Polymer Biotechnology Co., Ltd. (catalog number MF029-04).

[0109] Ligation reaction: 3.0 μL vector DNA fragment; 4.0 μL target DNA fragment; 0.5 μL T4 DNA ligase; 1.0 μL 10× ligation buffer; 1.5 μL ddH2O. Digestion at 16℃ for 4 h.

[0110] Transformation of *E. coli*: Add 10 μL of the ligation product to 50 μL of competent cells, invert to mix, and incubate on ice for 30 min. Place the centrifuge tube containing the mixture in a 42°C heating block for heat shock for 60-90 sec. Immediately place the centrifuge tube on ice to cool for 2 min. Add 800 μL of LB liquid medium to a clean bench and incubate at 37°C and 150-180 rpm for 45-60 min on a shaker. Spread 200 μL of the bacterial culture onto an LB agar plate containing the appropriate antibiotic and incubate at 37°C for 14 h.

[0111] III. Experimental Results

[0112] The full-length nucleic acid sequence of the functional gene douV was obtained using PCR, and a recombinant E. coli strain expressing the douV gene was successfully constructed. The inserted sequence was verified to be correct by PCR, enzyme digestion, and sequencing; this strain was named BW-douV. BW25113, containing the pRADZ3 control empty plasmid, was named BW-Z3.

[0113] Example 2

[0114] Stress resistance analysis of recombinant engineered strain BW-douV

[0115] I. Experimental Materials

[0116] Recombinant engineered strain: BW-douV, an engineered strain expressing the douV functional gene obtained in Example 1.

[0117] Control strain: BW-Z3, the strain containing the empty plasmid described in Example 1.

[0118] II. Experimental Methods

[0119] 1. Activate the control strain and the recombinant engineered strain by streaking them onto LB agar plates;

[0120] 2. Pick a single colony and inoculate it into liquid LB medium supplemented with the corresponding antibiotic, and incubate at 37°C until the mid-to-late stage of the exponential growth.

[0121] 3. Collect bacterial cells by centrifugation at 6,000 rpm for 5 min at room temperature, then wash the bacterial cells twice with the same volume of phosphate buffer (pH 7.0) and shake well.

[0122] Next, experiments were conducted on oxidation resistance, high salt resistance, and resistance to drying stress.

[0123] A. Hydrogen peroxide resistance analysis:

[0124] 1) Divide the bacterial solution into two equal volumes;

[0125] 2) One control was collected by centrifuging at 6,000 rpm for 5 min at room temperature;

[0126] 3) The bacterial body was suspended in LB liquid medium with a final concentration of 20 mM hydrogen peroxide and cultured at 30°C and 220 rpm for 10 min, 15 min, and 20 min.

[0127] 4) Take bacterial suspensions from different time periods and dilute them 10-fold to 10⁻⁶. -5 Take 10 μL and spot it on an LB solid medium plate. Incubate at 37°C for 2 days and observe and record the growth of different strains.

[0128] 5) Take bacterial suspensions from different time periods and dilute them 10-fold to 10⁻⁶. -5 Spread 200 μL of the culture medium onto an LB agar plate and incubate at 37°C for 2 days. Record the number of colonies and calculate the survival rate of the strains. Repeat the experiment 3 times.

[0129] B. High salt resistance analysis:

[0130] 1) Divide the bacterial solution into two equal volumes;

[0131] 2) One control was collected by centrifuging at 6,000 rpm for 5 min at room temperature;

[0132] 3) The bacterial body was suspended in LB liquid medium with a final concentration of 5M sodium chloride and cultured at 30°C and 220 rpm for 2 hours.

[0133] 4) Take bacterial suspensions from different time periods and dilute them 10-fold to 10⁻⁶. -5 Take 10 μL and spot it on an LB solid medium plate. Incubate at 37°C for 2 days and observe and record the growth of different strains.

[0134] 5) Take bacterial suspensions from different time periods and dilute them 10-fold to 10⁻⁶. -5 Spread 200 μL of the culture medium onto an LB agar plate and incubate at 37°C for 2 days. Record the number of colonies and calculate the survival rate of the strains. Repeat the experiment 3 times.

[0135] C. Permeability resistance analysis:

[0136] 1) Divide the bacterial solution into two equal volumes;

[0137] 2) One control was collected by centrifuging at 6,000 rpm for 5 min at room temperature;

[0138] 3) The bacterial cells were suspended in LB liquid medium with a final concentration of 2M sorbitol and cultured at 30°C and 220 rpm for 2 hours.

[0139] 4) Take bacterial suspensions from different time points and dilute them 10-fold to 10⁻⁶. -5 Take 10 μL and spot it on an LB solid medium plate. Incubate at 37°C for 2 days, observe and record the growth of different strains.

[0140] 5) Take bacterial suspensions from different time periods and dilute them 10-fold to 10⁻⁶. -5 Spread 200 μL of the culture medium onto an LB agar plate and incubate at 37°C for 2 days. Record the number of colonies and calculate the survival rate of the strains. Repeat the experiment 3 times.

[0141] III. Experimental Results

[0142] like Figure 1 The results showed that before oxidation, high salt, and osmotic stress, the growth status of the engineered strain BW-douV containing the functional gene douV was basically the same as that of the BW-Z3 strain containing the empty plasmid; the growth status of the engineered strain BW-douV containing the stress-resistant functional gene douV was no different from that of the control.

[0143] After stress shock, strain BW-douV showed significantly better resistance than the control strain. Figure 1 Under oxidative stress conditions of 20 mM hydrogen peroxide for 20 minutes, the survival rate of the engineered strain BW-douV was 3 times higher than that of the control strain BW-Z3. Under high-salt stress conditions of 5 M sodium chloride, the survival rate of the control strain BW-Z3 decreased by 5 orders of magnitude, while the engineered strain BW-douV expressing the stress resistance gene douV showed 2 orders of magnitude higher resistance than the control, approximately 100 times. Under osmotic stress conditions simulated by 2 M sorbitol, the survival rate of the control strain BW-Z3 was 2.69%, while the survival rate of the engineered strain BW-douV expressing the stress resistance gene douV was 12.03%, showing approximately 5 times higher osmotic resistance than the control.

[0144] Table 1. Number of bacterial strains surviving under different stress treatments (unit: strains)

[0145]

[0146] As demonstrated by the above embodiments, the functional gene douV significantly enhances the cell's resistance to high salt and osmotic stress. The engineered bacteria provided by this invention exhibit excellent salt tolerance, antioxidant properties, and resistance to osmotic stress, making them more suitable for production applications.

[0147] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A douV gene of Deinococcus radiodurans, characterized in that: The nucleotide sequence of the douV gene is shown as SEQ ID NO.

1.

2. The protein encoded by the douV gene according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A recombinant expression plasmid for improving biological stress resistance, characterized in that: The recombinant expression plasmid comprises the douV gene according to claim 1 and an initial plasmid.

4. The recombinant expression plasmid according to claim 3, characterized in that: The initial plasmid is pRADZ3 plasmid.

5. Use of the douV gene according to claim 1, the protein according to claim 2 or the recombinant expression plasmid according to claim 3 or 4 in improving biological stress resistance.

6. The use according to claim 5, characterized in that: The organism is an engineered bacterium.

7. The use according to claim 6, characterized in that: The engineered bacteria include Escherichia coli.

8. The use according to any one of claims 5 to 7, characterized in that: The stress resistance is salt resistance, oxidation resistance and / or osmotic stress resistance.

9. An engineered bacterium with high stress resistance, characterized in that: The method is obtained by transferring the recombinant expression plasmid described in claim 3 or 4 into a chassis strain.

10. Use of the engineered bacteria according to claim 9 in biosynthesis.

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