Functional module SyOrRW for improving cell stress resistance, protein coded by functional module SyOrRW and application of functional module SyOrRW
Designing the composite anti-resistance functional module SyOrRW through synthetic biological methods significantly improves the anti-oxidation, high salt and anti-poration stress capabilities of microbial cell factories, solves the problem of insufficient stress resistance in the existing technology, and realizes the chassis strain Improved robustness.
Patent Information
- Application Number
- CN202411678388.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-01-17
AI Technical Summary
The prior art has bottlenecks in improving the robustness and cell activity in the fermentation adversarial environment of microbial cell factories. The individual resistance genes have limited effects in gene transcription and expression, and it is difficult to significantly without affecting the normal growth of the chassis strain. Improve stress resistance.
Through synthetic biological methods, protein functional elements are designed and optimized, and the complex anti-reflection functional module SyOrRW is constructed to significantly improve the anti-oxidation, high salt and anti-poration stress capabilities of the organisms, and transferred them into the chassis strain to improve stress resistance.
It significantly improves the antioxidant, high salt and anti-osmosis stress capabilities of cells, enhances the robustness of the chassis strain, increases oxidative resistance by 2 orders of magnitude, increases salt resistance by 100,000 times, and improves the anti-osmosis capability by more than 5 times.
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Figure CN119320433A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biosynthesis, and in particular relates to a functional module SyOrRW for improving cell stress resistance, a protein encoded by the module, and an application thereof. Background Art
[0002] Synthetic biology redesigns organisms in an engineering way or even synthesizes them from scratch, which will overcome the limitations of natural evolution and create synthetic organisms that surpass natural life capabilities. It not only has important scientific significance for exploring the basic laws of life activities, but also has great application prospects in industrial and agricultural production, environmental protection, health care and other fields.
[0003] The design and construction of microbial cell factories using synthetic biology technology has been widely used to produce a wide variety of chemicals, foods, medicines and energy, and is the core link of green biomanufacturing. The construction of strain cell factories is completed by introducing new enzymes into microbial chassis cells, constructing new pathways and adapting to chassis cells. However, increasing the chassis' tolerance to substrates and products, increasing growth rate, increasing substrate utilization and increasing product yield are the goals of improving chassis strain performance.
[0004] To address the bottleneck problem of stress resistance and production performance of low-version chassis strains, a separate resistance gene can be introduced. However, the effect of introducing a separate resistance gene in terms of gene transcription, expression and function needs to be improved. Compared with a single stress-resistant gene, the artificially designed high-efficiency stress-resistant gene module is assembled with a suitable promoter element, so that the stress-resistant gene is only expressed and functions in specific environments and periods, and does not affect the normal growth of the chassis strain.
[0005] At present, how to improve the robustness and cell activity of chassis cell factories under fermentation stress environments has become a hot topic in the research of synthetic biology cell factories. Summary of the invention
[0006] In view of this, the purpose of the present invention is to provide a functional module SyOrRW for improving cell stress resistance, and its encoded protein and application. The present invention uses synthetic biology methods to construct a composite stress resistance functional module SyOrRW through artificial design and optimization of protein functional elements. The stress resistance functional module SyOrRW can significantly improve the ability of organisms to resist oxidation, high salt and osmotic stress.
[0007] The present invention provides a functional module SyOrRW for improving cell stress resistance, and the nucleotide sequence of the functional module SyOrRW is shown in SEQ ID No.1.
[0008] The present invention provides a protein encoded by the functional module SyOrRW, and the amino acid sequence of the protein is shown in SEQ ID No.2.
[0009] The present invention provides a recombinant expression plasmid for expressing the functional module SyOrRW, comprising an initial expression plasmid and the functional module SyOrRW.
[0010] Preferably, the initial expression plasmid is pRADZ3 plasmid.
[0011] Preferably, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID No.3.
[0012] The present invention provides the application of the functional module SyOrRW, the protein or the recombinant expression plasmid in improving the stress resistance of organisms.
[0013] Preferably, the stress resistance includes resistance to oxidation, resistance to high salt and / or resistance to osmotic stress.
[0014] Preferably, the organism includes a microorganism or a plant.
[0015] The present invention provides a recombinant engineering strain expressing the functional module SyOrRW, which is obtained by transferring the recombinant expression plasmid into a chassis strain.
[0016] Preferably, the chassis strain is Escherichia coli.
[0017] Compared with the prior art, the present invention has the following beneficial effects: the functional module SyOrRW for improving cell stress resistance provided by the present invention is obtained by artificial design and optimization of protein functional elements using synthetic biology methods, and the functional module SyOrRW can significantly improve the ability of biological antioxidant, high salt and osmotic stress resistance. The module SyOrRW is transferred into the biosynthetic chassis strain, which can significantly improve the stress resistance of the chassis strain and can be used for synthetic biology chassis cell stress resistance improvement applications.
[0018] The embodiments of the present invention record that after oxidation, high salt and osmotic stress shocks, the engineered strain BW-SyOrRW containing the stress resistance module SyOrRW grew well, and the number of colonies was significantly more than that of the BW-Z3 strain containing only an empty plasmid. Compared with the control BW-Z3 strain, the oxidative resistance was increased by 2 orders of magnitude, about 100 times. Under the high salt stress conditions of 5M sodium chloride, the growth of the engineered strain BW-SyOrRW expressing the stress resistance module SyOrRW was almost unaffected, and the salt resistance was increased by about 100,000 times. Under the osmotic stress conditions simulated by 2M sorbitol, the survival rate of the engineered strain BW-SyOrRW expressing the stress resistance gene SyOrRW was 18.28%, and the anti-osmotic ability was more than 5 times higher than that of the control. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1The figure shows the comparison of the stress resistance and survival ability of the engineered strain BW-SyOrRW and the control strain after 20 mM hydrogen peroxide stress, 5 M sodium chloride high salt stress, and 2 M sorbitol osmotic stress in Example 2. The control group does not show the survival rate, and the * represents a survival rate lower than 0.01%. DETAILED DESCRIPTION
[0020] The present invention provides a functional module SyOrRW for improving cell stress resistance, wherein the nucleotide sequence of the functional module SyOrRW is shown in SEQ ID No. 1, and is specifically as follows:
[0021]
[0022] In the present invention, the functional module SyOrRW includes a promoter region (1-742 bp) and a coding region (743-1798 bp). The functional module SyOrRW is 1798 bp in length and encodes a complex protein composed of 351 amino acids.
[0023] The present invention provides a protein encoded by the functional module SyOrRW, the amino acid sequence of which is shown in SEQ ID No. 2, and is as follows:
[0024] MAGHPNTPPETLAALGAEFPAEVLGNPALPLLRLAQPGLLARWPVRTLEALAAAPAAPVWLLRLAAAHEKIDVQLAAVTRPDLPGDVLLKLSHSPFWTIREAVARKPQLPPGVLGQLGHDLDYGVRLSVANRSDLSRELRGALRRDPHPLVRAVVILGEPQVSELSLSATNFSLLK QAGDVEENPGPMKKMAFAAIALGLVACAPAQQQRLVYPPTVEVERIQLTSFSLPAPGRAAVAGVRLELNVDNPNPLPVRLVQLAGVLVLDGQEAGTVTVPDIRLPGQGRARQVAQLTLPVTFKTATTFLNIARGQEVSYRLDGTLTADLGPLGQPTFGPFTLTQGVWKQAPLSVF.
[0025] The present invention provides a recombinant expression plasmid expressing the functional module SyOrRW, comprising an initial expression plasmid and the functional module SyOrRW; in the present invention, the initial expression plasmid is preferably a pRADZ3 plasmid, and the functional module SyOrRW is preferably cloned between the Spe I and Nde I restriction sites of the pRADZ3 plasmid. The present invention does not specifically limit the preparation method of the recombinant expression plasmid, and conventional enzyme cutting and connection in the art can be used. After obtaining the nucleotide sequence of the functional module SyOrRW, the present invention preferably connects the functional module SyOrRW to a cloning plasmid to construct a recombinant cloning plasmid containing the functional module SyOrRW, and the cloning plasmid is sequenced and verified. Then the recombinant cloning plasmid is transferred to a host cell for propagation, and the expanded recombinant cloning plasmid is enzymatically cut to obtain the functional module SyOrRW, and finally the functional module SyOrRW is connected to the expression plasmid pRADZ3 to obtain a recombinant expression plasmid. In the present invention, the nucleotide sequence of the recombinant plasmid is preferably as shown in SEQ ID No.3, which is as follows:
[0026]
[0027] The present invention provides the use of the functional module SyOrRW, the protein or the recombinant expression plasmid in improving biological stress resistance. In the specific implementation of the present invention, the functional module SyOrRW is preferably expressed in an organism, and the organism preferably includes a microorganism or an animal or plant cell, and is further preferably an engineered strain. In the specific implementation of the present invention, the engineered strain is preferably Escherichia coli BW25113. In the present invention, the stress resistance preferably includes anti-oxidation, resistance to high salt and / or resistance to osmotic stress.
[0028] The present invention provides a recombinant engineering strain expressing the functional module SyOrRW, which is obtained by transferring the recombinant expression plasmid into the chassis strain. In the present invention, the chassis strain is Escherichia coli. The recombinant engineering strain provided by the present invention has excellent salt resistance, antioxidant and osmotic stress resistance, can adapt to different environments, and is more suitable for production applications.
[0029] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0030] Example 1
[0031] Construction of recombinant engineering strain containing the functional module SyOrRW
[0032] 1. Experimental Materials
[0033] Cloning vector pJET: a commercial product from ThermoFisher;
[0034] Shuttle plasmid pRADZ3: commercially available, see reference doi:10.1128 / aem.66.9.3856-3867.2000.
[0035] Escherichia coli BW25113: purchased from Wuhan Miaoling Biotechnology Co., Ltd., product number T0035.
[0036] 2. Experimental Methods
[0037] 1. Through the synthetic biology design module, the full-length nucleic acid sequence of the functional module SyOrRW was obtained by artificial chemical synthesis, with a length of 1798bp, including the promoter and the coding region. The coding region gene encodes a complex protein of 351 amino acids. It was cloned into the vector pJET, and the recombinant cloning plasmid pJET-SyOrRW containing the complete functional module SyOrRW was constructed and sequenced for verification; then the functional module SyOrRW and pRADZ3 vector fragments containing sticky ends were obtained by double digestion with Spe I / Nde I, and the functional module SyOrRW was connected to the pRADZ3 vector to construct the E. coli expression vector pRADZ-SyOrRW, and the expression vector was transformed into E. coli BW25113. The specific steps are as follows:
[0038] Enzyme digestion reaction: 5.0 μL of the sequence DNA of the artificially synthesized functional module SyOrRW, 0.5 μL of restriction endonucleases Spe I / Nde I, 2.0 μL of 10×CS Buffer, ddH 2 O 12μL. Digest at 37℃ for 30min.
[0039] The target DNA fragment was recovered and purified using the DNA recovery kit purchased from Beijing Juhemei Biotechnology Co., Ltd. (Cat. No. MF029-04).
[0040] Ligation reaction: vector DNA fragment 3.0 μL; target DNA fragment 4.0 μL; T4 DNA ligase 0.5 μL; 10× ligation buffer 1.0 μL; ddH 2 O 1.5 μL. Enzyme digestion at 16℃ for 4 h.
[0041] E. coli transformation: Add 10 μL of the ligation product to 50 μL of competent cells, mix the liquid by inverting, and place on ice for 30 minutes. Place the centrifuge tube containing the mixture in a 42°C heating block and heat shock for 60-90 seconds. Quickly place the centrifuge tube on ice and cool for 2 minutes. Add 800 μL of LB liquid culture medium to the clean bench and culture on a shaker at 37°C, 150-180 rpm for 45-60 minutes. Take 200 μL of the bacterial solution and spread it on an LB plate containing the corresponding antibiotics, and culture it in a 37°C incubator for 14 hours.
[0042] 3. Experimental Results
[0043] The full-length nucleic acid sequence of the functional module SyOrRW was obtained by artificial chemical synthesis, and a recombinant E. coli engineering strain expressing the functional module SyOrRW was successfully constructed. The insertion sequence was verified to be correct by PCR, enzyme digestion, and sequencing, and the strain was named BW-SyOrRW. BW25113 containing the pRADZ3 control empty plasmid was named BW-Z3.
[0044] Example 2
[0045] Analysis of resistance to oxidative, high-salt and osmotic stress of engineered strain BW-SyOrRW
[0046] 1. Experimental Materials
[0047] Recombinant engineering strain: the engineering strain BW-SyOrRW expressing the SyOrRW functional module obtained in Example 1
[0048] Control strain: strain BW-Z3 containing an empty plasmid as described in Example 1.
[0049] 2. Experimental Methods
[0050] 1. Activate the control strain and the recombinant engineered strain by streaking on LB solid medium plates;
[0051] 2. Pick a single colony and inoculate it into liquid LB medium supplemented with corresponding antibiotics, and culture it at 37°C until the mid-to-late exponential period;
[0052] 3. Collect the cells by centrifugation at 6,000 rpm for 5 min at room temperature, then wash the cells twice with the same volume of phosphate buffer (pH 7.0) and shake evenly;
[0053] Next, the oxidative resistance, high salt and dryness stress resistance experiments were carried out.
[0054] A. Hydrogen Peroxide Oxidation Resistance Analysis:
[0055] 1) Divide the bacterial solution into two equal volumes;
[0056] 2) One portion was centrifuged at 6,000 rpm for 5 min at room temperature to collect the bacterial cells as a control;
[0057] 3) The cells were resuspended in LB liquid medium containing hydrogen peroxide at a final concentration of 10 mM and cultured at 30°C and 220 rpm on a shaker for 10 min, 15 min, and 20 min;
[0058] 4) Take bacterial suspension from different time periods and dilute 10 times to 10 -5 , take 10 μL and spot it on LB solid medium plate, culture at 37℃ for 2 days, observe and record the growth of different strains;
[0059] 5) Take bacterial solution from different time periods and dilute it 10 times to 10 -5 , take 200 μL to spread on LB solid culture medium plate, culture at 37℃ for 2 days, record the number of colonies, calculate the survival rate of the strain, and repeat the experiment 3 times.
[0060] B. High salt resistance analysis:
[0061] 1) Divide the bacterial solution into two equal volumes;
[0062] 2) One portion was centrifuged at 6,000 rpm for 5 min at room temperature to collect the bacterial cells as a control;
[0063] 3) The cells were resuspended in LB liquid medium with a final concentration of 5 M sodium chloride and cultured at 30°C and 220 rpm for 2 h;
[0064] 4) Take bacterial suspension from different time periods and dilute 10 times to 10 -5 , take 10 μL and spot it on LB solid medium plate, culture at 37℃ for 2 days, observe and record the growth of different strains;
[0065] 5) Take bacterial solution from different time periods and dilute it 10 times to 10 -5 , take 200 μL to spread on LB solid culture medium plate, culture at 37℃ for 2 days, record the number of colonies, calculate the survival rate of the strain, and repeat the experiment 3 times.
[0066] C. Penetration resistance analysis:
[0067] 1) Divide the bacterial solution into two equal volumes;
[0068] 2) One portion was centrifuged at 6,000 rpm for 5 min at room temperature to collect the bacterial cells as a control;
[0069] 3) The cells were resuspended in LB liquid medium with a final concentration of 2 M sorbitol and cultured at 30°C and 220 rpm for 2 h.
[0070] 4) Take bacterial suspension at different time points and dilute 10-fold to 10 -5 , take 10 μL and spot it on LB solid culture medium plate, culture it at 37℃ for 2 days, observe and record the growth of different strains.
[0071] 5) Take bacterial solution from different time periods and dilute it 10 times to 10 -5 , take 200 μL to spread on LB solid culture medium plate, culture at 37℃ for 2 days, record the number of colonies, calculate the survival rate of the strain, and repeat the experiment 3 times.
[0072] 3. Experimental Results
[0073] like Figure 1 It was shown that before oxidative, high salt and osmotic stress shocks, the growth status of the engineered strain BW-SyOrRW containing the stress resistance module SyOrRW was basically the same as that of the BW-Z3 strain containing an empty plasmid.
[0074] After oxidative, high-salt and osmotic stress shocks, the engineered strain BW-SyOrRW containing the stress resistance module SyOrRW grew well, and the number of colonies was significantly higher than that of the BW-Z3 strain containing only an empty plasmid ( Figure 1 ). Under the oxidative stress condition of 20mM hydrogen peroxide shock for 20 minutes, the resistance of the engineered strain expressing the stress resistance module SyOrRW was significantly improved. Compared with the control BW-Z3 strain, the oxidative resistance was increased by 2 orders of magnitude, about 100 times. Under the high salt stress condition of 5M sodium chloride, the survival rate of the control strain BW-Z3 strain decreased by 5 orders of magnitude, while the growth of the engineered strain BW-SyOrRW expressing the stress resistance module SyOrRW was almost unaffected, and the salt resistance was increased by about 100,000 times. Under the osmotic stress condition simulated by 2M sorbitol, the survival rate of the control strain BW-Z3 strain was 2.69%, while the survival rate of the engineered strain BW-SyOrRW expressing the stress resistance gene SyOrRW was 18.28%, and the anti-osmotic ability was more than 5 times higher than the control.
[0075] Table 1 Number of strains surviving under different stress treatments (unit: individual)
[0076] Unprocessed Oxidative stress High salt stress Osmotic stress BW-Z3 <![CDATA[3.25×10 7 ]]> <![CDATA[6.4×10 3 ]]> <![CDATA[2.78×10 2 ]]> <![CDATA[8.7×10 5 ]]> BW-SyOrRW <![CDATA[4.55×10 7 ]]> <![CDATA[8.64×10 5 ]]> <![CDATA[1.46×10 7 ]]> <![CDATA[8.1×10 6 ]]>
[0077] From the above examples, it can be seen that the functional module SyOrRW can significantly enhance the resistance of cells to oxidation, high salt and osmotic stress. The engineered strain provided by the present invention has excellent antioxidant, salt resistance and osmotic stress resistance properties and is more suitable for production applications.
[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A functional module SyOrRW for improving cell stress resistance, characterized in that: The nucleotide sequence of the functional module SyOrRW is shown in SEQ ID No.
1.
2. The protein encoded by the functional module SyOrRW 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 expressing the functional module SyOrRW according to claim 1, characterized in that: It comprises an initial expression plasmid and the functional module SyOrRW according to claim 1.
4. The recombinant expression plasmid according to claim 3, characterized in that: The initial expression plasmid is pRADZ3 plasmid.
5. The recombinant expression plasmid according to claim 4, characterized in that: The nucleotide sequence of the recombinant plasmid is shown in SEQ ID No.
3.
6. Use of the functional module SyOrRW according to claim 1, the protein according to claim 2 or the recombinant expression plasmid according to any one of claims 3 to 5 in improving biological stress resistance.
7. The use according to claim 6, characterized in that: The stress resistance includes resistance to oxidation, resistance to high salt and / or resistance to osmotic stress.
8. The use according to claim 6 or 7, characterized in that: The organisms include microorganisms.
9. A recombinant engineering strain expressing the functional module SyOrRW according to claim 1, characterized in that: The method is obtained by transferring the recombinant expression plasmid described in any one of claims 3 to 5 into a chassis strain.
10. The recombinant engineering strain according to claim 9, characterized in that The chassis strain is Escherichia coli.