RNA chaperonin, coding gene, high-stress-resistance recombinant bacterium for expressing RNA chaperonin and preparation method of high-stress-resistance recombinant bacterium

Abstract

The invention discloses an RNA chaperonin, a coding gene, a high-stress-resistance recombinant bacterium for expressing the RNA chaperonin and a preparation method of the high-stress-resistance recombinant bacterium, and relates to the field of bioengineering. Wherein the RNA chaperonin PyCsp has the activity of binding RNA. A recombinant plasmid for expressing an RNA molecular chaperone PyCsp is constructed and transferred into a target bacterium to prepare a recombinant bacterium, namely the recombinant bacterium with high stress resistance, and the stress resistance is low-temperature tolerance, high-pressure tolerance or high-temperature tolerance. According to the invention, a heat-resistant RNA partner is excavated from hyperthermophilic and barophilic microorganisms (the optimum growth temperature is 95 DEG C), and the heat-resistant RNA partner, as a stress-resistant element, has important application value in the aspect of improving the stress resistance of the microorganisms to environmental stress conditions such as high temperature, low temperature, high hydrostatic pressure and the like; in bioengineering application scenarios involving high temperature and high pressure, the strain has unique advantages in the aspect of improving the stress resistance of strains used for fermentation.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to an RNA chaperone protein, a coding gene, and a highly stress-resistant recombinant bacterium expressing the RNA chaperone protein and a preparation method thereof. Background Art

[0002] RNA chaperones are a class of proteins that can bind to RNA without consuming ATP. This class of proteins can protect RNA from degradation by ribonucleases, change the structure of RNA, and assist RNA in binding to its target. The characteristics of RNA chaperones allow it to play an important role in RNA-mediated regulation, especially in response to low temperature stress. The RNA chaperone CspA in bacteria not only plays an important role in response to low temperature stress, but is also a stress protein under high hydrostatic pressure; the RNA chaperones CspB and CspC in Clostridium botulinum ATCC 3502 play an important role in the strain's response to low pH, high salt and ethanol stress.

[0003] After the hyperthermophilic barophilic archaeon P.yayanosii A1 was cold-shocked, the gene PYCH_0765 was induced, and the protein PyCsp encoded by this gene is a new type of cold-shock protein. In addition, the online tool catRAID signature shows that PyCsp can bind to RNA. The RNA chaperone PyCsp found in P.yayanosii A1 can be used as a stress-resistant element, and has important application value in improving the stress resistance of microorganisms to environmental stress conditions such as high temperature, low temperature, and high hydrostatic pressure. For example, in the process of industrial fermentation, increasing the fermentation temperature requires strains that tolerate high temperatures. Inducing the expression of recombinant proteins at lower temperatures inhibits the growth of genetically engineered host bacteria. Bioengineering applications under high temperature and high pressure conditions require improving the tolerance of microorganisms to high pressure. Therefore, technicians in this field are committed to developing a strain and preparation method that has stress resistance to environmental stress conditions such as high temperature, low temperature, and high hydrostatic pressure. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to enhance the resistance of microorganisms to environmental stress conditions such as low temperature, high temperature, and high hydrostatic pressure.

[0005] To achieve the above object, the present invention provides an RNA chaperone protein, characterized in that the RNA chaperone protein has the activity of binding to RNA.

[0006] In a preferred embodiment of the present invention, the RNA chaperone protein is PyCsp, and the amino acid sequence is shown in SEQ ID NO.1. In a preferred embodiment of the present invention,

[0007] The present invention also provides a gene for the RNA chaperone protein, characterized in that the nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO.2.

[0008] The present invention also provides a highly stress-resistant recombinant bacterium expressing the above-mentioned RNA chaperone protein, characterized in that the recombinant bacterium has low temperature tolerance, high pressure tolerance or high temperature tolerance.

[0009] In a preferred embodiment of the present invention, the recombinant bacteria contains the gene as described above.

[0010] The present invention also provides a method for preparing the above-mentioned highly stress-resistant recombinant bacteria, characterized in that the method comprises the following steps:

[0011] Step 1, constructing a recombinant plasmid expressing RNA molecular chaperone PyCsp;

[0012] Step 2: The recombinant plasmid expressing the RNA molecular chaperone PyCsp is transferred into the target bacteria to prepare the recombinant bacteria, that is, the recombinant bacteria with high stress resistance.

[0013] In a preferred embodiment of the present invention, the step 1 specifically includes:

[0014] The nucleotide sequence shown in SEQ ID NO.2 was connected to a vector to construct a recombinant plasmid expressing the RNA chaperone protein PyCsp.

[0015] In another preferred embodiment of the present invention, the step 1 specifically includes:

[0016] Using the genomic DNA of the hyperthermophilic barophilic archaeon Pyrococcusyayanosii CH1 as a template, the full-length fragment of the PyCsp protein gene was amplified by PCR using primers 1 and 2. The base sequences of primers 1 and 2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0017] Using plasmid pET28a as a template, primers 3 and 4 were used to amplify the linearized expression vector pET28a by PCR. The base sequences of primers 3 and 4 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0018] In a 20 μL reaction system, the full-length fragment of the PyCsp protein gene and the linearized vector pET28a fragment were added in a molar ratio of 2:1 between the gene fragment and the linearized vector fragment, and then 2 μL of ExnaseⅡ ligase and 4 μL of 5×CEⅡ buffer were added. After mixing, the mixture was incubated at 37°C for 30 min to obtain the recombinant plasmid.

[0019] In another preferred embodiment of the present invention, the step 2 specifically includes:

[0020] The recombinant plasmid was transferred into competent Escherichia coli BL21 cells by heat shock to obtain recombinant bacteria.

[0021] In another preferred embodiment of the present invention, the target bacteria is one or more of cold-sensitive Escherichia coli BX04, Escherichia coli DH5α, Pyrococcus yayanosii, Thermococcus kodakarensis and Streptomyces xiamenensis.

[0022] Technical Effects

[0023] 1. Introducing genetic elements from thermophilic microorganisms into mesophilic microorganisms can improve the host cell's tolerance to high temperatures. RNA chaperones have a wide range of regulatory effects and can function under a variety of stress conditions. Overexpression of the RNA chaperone PyCsp from Pyrococcus yayanosii in Escherichia coli significantly increased the biomass at 45°C, reaching 1.72 times that of the control strain.

[0024] 2. The RNA molecular chaperone PyCsp has anti-transcription termination activity and can also improve the host bacteria's tolerance to low temperatures. In Escherichia coli RL211, PyCsp exhibits anti-transcription termination activity. Overexpression of PyCsp can increase the biomass of cold-sensitive Escherichia coli BX04 at 22°C and 18°C.

[0025] 3. Stress-resistant gene elements were discovered from the barophilic microorganism P.yayanosiiA1 from deep-sea hydrothermal vents. RNA chaperones have a wide range of regulatory effects and can function under a variety of stress conditions. Overexpression of the RNA chaperone PyCsp can significantly increase the biomass of the hyperthermophilic archaeon P.yayanosii A1 under high hydrostatic pressure.

[0026] The present invention excavates a heat-resistant RNA chaperone from a hyperthermophilic barophilic microorganism (optimum growth temperature 95°C) originating from a deep-sea hydrothermal vent. Compared with the existing RNA molecular chaperones derived from thermophilic bacteria (generally growing at a temperature below 60°C), PyCsp has great advantages in thermal stability. This type of high-temperature resistant RNA chaperone, as a stress-resistant element, has important application value in improving the resistance of microorganisms to environmental stress conditions such as high temperature, low temperature, and high hydrostatic pressure. In bioengineering application scenarios involving high temperature and high pressure, it has unique advantages in improving the stress resistance of strains used in fermentation.

[0027] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the Tricine-SDS PAGE result of PyCsp of a preferred embodiment of the present invention;

[0029] Figure 2 is a binding and dissociation curve of PyCsp and RNA in a preferred embodiment of the present invention;

[0030] Figure 3 is the result of the PyCsp RNA melting activity of a preferred embodiment of the present invention;

[0031] Figure 4 It is the detection result of PyCsp anti-transcription termination activity of a preferred embodiment of the present invention;

[0032] Figure 5 This is the result of PyCsp complementation of cold-sensitive Escherichia coli BX04 in a preferred embodiment of the present invention;

[0033] Figure 6 It is a preferred embodiment of the present invention that the overexpression of PyCsp affects the biomass of Pyrococcus yayanosii at 85°C;

[0034] Figure 7 It is a preferred embodiment of the present invention that the overexpression of PyCsp affects the biomass of Pyrococcus yayanosii at 52MPa;

[0035] Figure 8 It is a preferred embodiment of the present invention that the overexpression of PyCsp affects the biomass of Thermococcus kodakarensis at 65°C;

[0036] Fig. 9It is a preferred embodiment of the present invention that the overexpression of PyCsp affects the biomass of Thermococcus kodakarensis at 20 MPa;

[0037] Fig.10 This is a growth curve of Escherichia coli overexpressing PyCsp at 45°C in a preferred embodiment of the present invention;

[0038] Fig.11 It is the effect of overexpression of PyCsp on the biomass of Escherichia coli at 45°C in a preferred embodiment of the present invention;

[0039] Fig.12 The present invention is a preferred embodiment of the effect of overexpression of PyCsp on the biomass of Streptomyces xiamenensis at 37°C. DETAILED DESCRIPTION

[0040] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0041] Example 1: Cloning, expression and purification of the thermostable RNA chaperone PyCsp

[0042] Construction of PyCsp expression plasmid: The amino acid sequence of PyCsp is shown in SEQ ID NO.1, and the full-length sequence encoding the PyCsp gene PYCH_0765 is shown in SEQ ID NO.2. Using Pyrococcus yayanosii CH1 genomic DNA as a template, primer 1 (base sequence as shown in SEQ ID NO.3) and primer 2 (base sequence as shown in SEQ ID NO.4) were used to amplify the full-length fragment of gene PYCH_0765 by PCR. Using plasmid pET28a as a template, primer 3 (base sequence as shown in SEQ ID NO.5) and primer 4 (base sequence as shown in SEQ ID NO.6) were used to amplify the linearized expression vector pET28a by PCR. In a 20 μL reaction system, add the PYCH_0765 fragment and the linearized vector pET28a fragment in a molar ratio of 2:1 between the gene fragment and the linearized vector fragment, then add 2 μL ExnaseⅡ (ligase) and 4 μL 5×CEⅡ buffer, mix well and incubate at 37°C for 30 minutes. Then, the ligation product was transferred into the competent cell Escherichia coli BL21 (DE3) by heat shock to obtain the recombinant bacteria BL21 / pET28a-PYCH_0765.

[0043] Expression of PyCsp: The recombinant bacteria BL21 / pET28a-PYCH_0765 were cultured in LB medium at 37°C and 200 rpm until OD600 reached 0.6, and IPTG was added at a final concentration of 0.5 mM for induction at 20°C for 16 h. After induction, the cells were collected by centrifugation at 4°C and 12,000 rpm for 5 min.

[0044] Purification of PyCsp: Resuspend the cells with lysis buffer (containing 0.5M NaCl, 50mM Tris-HCl, pH 8.0), break the cells with an ultrasonic disruptor on ice, centrifuge at 12000rpm, 4°C for 30min after lysis, and retain the supernatant. Pass the supernatant through a Ni-NTA affinity chromatography column, wash the column with a washing buffer containing 20mM imidazole (containing 0.5mol / LNaCl, 50mmol / LTris-HCl, pH 8.0), and then elute the target protein with an elution buffer containing 250mM imidazole (containing 0.5mol / LNaCl, 50mmol / LTris-HCl, pH 8.0). Use a 3kDa ultrafiltration tube to remove the high concentration of imidazole in the target protein, and store the target protein in a buffer containing 0.5M NaCl, 50mM Tris-HCl (pH8.0). The purified protein was identified by Tricine-SDS PAGE electrophoresis, as shown in Figure 1 As shown in (column 1: standard protein molecular weight Marker; column 2: purified PyCsp), the relative molecular masses of the purified PyCsp are about 9.5 kDa and 7.86 kDa, respectively, which are close to PyCsp 7.86 kDa. Therefore, PyCsp was successfully expressed in E. coli BL21 (DE3) in a soluble manner.

[0045] Example 2: RNA Binding Activity of RNA Chaperone PyCsp

[0046] A 20 nt long RNA with a biotin label at the 5' end was synthesized (the base sequence is shown in SEQ ID NO.7), and the synthesized RNA was loaded onto a streptavidin sensor in a PBS solution. The binding and dissociation curves of PyCsp and RNA were measured using a Fortebio Octet RED96 macromolecular interaction instrument in a PBS solution containing 0.02% Tween-20 (v / v). The results are shown in Figure 2 As shown, PyCsp can bind to RNA and is an RNA chaperone.

[0047] Example 3: RNA melting activity of RNA chaperone PyCsp

[0048] Synthesize two partially complementary molecular probes MB with a length of 40 nt 40-FITC (base sequence as shown in SEQ ID NO.8) and MB 40 -BHQ1 (base sequence as shown in SEQ ID NO.9), wherein MB 40 -FITC has a fluorescein isothiocyanate isomer (FITC) at the 5' end. MB 40 -BHQ1 has a fluorescence quencher group BHQ1 at the 3' end. 40 -FITC and MB 40 -BHQ1 are mixed at a molecular ratio of 1:1 and then annealed to form double-stranded RNA (dsRNA). Since the fluorescent group FITC is close to the fluorescent quenching group BHQ1, the fluorescence intensity of the double-stranded RNA formed by annealing is higher than that of MB. 40 -FITC dropped significantly. Under light-proof conditions, the following reaction system was prepared: 20μM purified PyCsp, 0.1μM annealed dsRNA, 200mM Tris-HCl (pH 7.4), 10mM MgCl2, and finally the reaction system was supplemented to 100μL with DEPC water. The prepared reaction system was incubated at 37°C for 30 minutes. After the incubation, 50μL of reaction solution was added to the black ELISA plate, and the fluorescence intensity was measured using a multifunctional ELISA reader. The measurement parameters are: excitation light is 460nm, and emission light is 515nm. The results are as follows: Figure 3 As shown, PyCsp has RNA depolymerization activity.

[0049] Example 4. Anti-transcription termination activity of RNA chaperone PyCsp

[0050] The chloramphenicol resistance gene cat in the model bacteria RL211 contains a strong terminator trpL upstream. If the protein has anti-transcription termination activity, the gene cat can be transcribed normally, and the strain also shows chloramphenicol resistance. The gene PYCH_0765 was connected to the plasmid pINⅢ to obtain the recombinant plasmid pIN-0765, and then the recombinant plasmid was transferred into the model bacteria RL211 by heat shock to obtain the recombinant bacteria RL211 / pIN-0765. The transferred genes cspA and cspL were connected to the plasmid pINⅢ to obtain the recombinant plasmids pIN-cspA and pIN-cspL. The recombinant bacteria RL211 / pIN-cspA and RL211 / pIN-cspL transferred with the recombinant plasmids pIN-cspA and pIN-cspL were used as positive controls, and the recombinant bacteria RL211 / pIN transferred with the empty plasmid pINⅢ was used as a negative control. The recombinant bacteria RL211 / pIN-0765, RL211 / pIN-cspA, RL211 / pIN-cspL and RL211 / pIN were cultured in LB medium until OD 600 1.0, 6 μL of the culture was spotted on LB plates containing 100 μg / mL ampicillin, with or without 30 μg / mL chloramphenicol, and with or without 0.2 mM IPTG, and cultured at 37°C and 45°C for 2 days. Figure 4 As shown, PyCsp has anti-transcription termination activity at both 37°C and 45°C.

[0051] Example 5: RNA chaperone PyCsp complements cold-sensitive Escherichia coli BX04

[0052] The recombinant plasmids pIN-0765, pIN-cspA, pIN-cspL and the empty plasmid pINⅢ were transformed into cold-sensitive Escherichia coli BX04 by heat shock transformation to obtain recombinant bacteria BX04 / pIN-0765, BX04 / pIN-cspA, BX04 / pIN-cspL, and BX04 / pIN. The recombinant bacteria BX04 / pIN-cspA and BX04 / pIN-cspL were used as positive controls, and the recombinant bacteria BX04 / pIN was used as a negative control. The recombinant bacteria were cultured in LB medium at 37°C until OD 600 After the pH was 1.0, 6 μL of the culture was diluted with LB medium and spotted on LB plates containing 100 μg / mL ampicillin and with or without 1 mM IPTG, and cultured at 37°C, 22°C, and 18°C ​​for 3 days. Figure 5 As shown, PyCsp was able to restore the growth of cold-sensitive Escherichia coli BX04 at 22°C and 18°C.

[0053] Example 6: RNA chaperone PyCsp improves the low temperature tolerance of Pyrococcus yayanosiiA1

[0054] The gene PYCH_0765 was connected to the plasmid pLMOS to obtain the recombinant plasmid pLMOS-PYCH_0765, and then the recombinant plasmid was transferred into Pyrococcus yayanosii A1 by heat shock to obtain the recombinant bacteria A1 / pLMOS-PYCH_0765. The recombinant bacteria A1 / pLMOS-PYCH_0765 was transferred into the recombinant bacteria A1 / pLMOS containing the empty plasmid pLMOS and cultured in TRM medium at 85°C and 0.1MPa for 16 hours. After the culture was completed, the cells in the culture were counted using a hemocytometer. The results are shown in Figure 2. Figure 6 As shown, overexpression of PyCsp could significantly increase the biomass of Pyrococcus yayanosiiA1 at 85°C.

[0055] Example 7: RNA chaperone PyCsp improves the high pressure tolerance of Pyrococcus yayanosii

[0056] The recombinant bacteria A1 / pLMOS-PYCH_0765 and the recombinant bacteria A1 / pLMOS transformed with the empty plasmid pLMOS were cultured in TRM medium at 95°C and 52MPa for 16h. After the culture was completed, the cells in the culture were counted using a hemocytometer. The results are shown in Figure 7 As shown, overexpression of PyCsp could increase the biomass of Pyrococcus yayanosii A1 under 52 MPa.

[0057] Example 8: RNA chaperone PyCsp improves the low temperature tolerance of Thermococcus kodakarensis

[0058] The gene PYCH_0765 was connected to the plasmid pTE to obtain the recombinant plasmid pTE-0765, and then the recombinant plasmid was transferred into the agmatine auxotrophic strain Thermococcus kodakarensis TS559 by heat shock to obtain the recombinant bacteria TS559 / pTE-0765. The recombinant bacteria TS559 / pTE-0765 was transferred into the recombinant bacteria TS559 / pTE containing the empty plasmid pTE and cultured in ASW-YT medium at 65°C and 0.1MPa for 20h. After the culture was completed, the OD of the culture was measured. 600 The results are as follows Figure 8 As shown, overexpression of PyCsp could increase the biomass of Thermococcus kodakarensis at 65°C.

[0059] Example 9: PyCsp improves the growth of Thermococcus kodakarensis under high pressure

[0060] The recombinant bacteria TS559 / pTE-0765 was transformed with the empty plasmid pTE and cultured in ASW-YT medium at 85°C and 20 MPa for 20 h. 600 The results are as follows Fig. 9 As shown, overexpression of PyCsp could increase the biomass of Thermococcus kodakarensis under 20 MPa.

[0061] Example 10: RNA chaperone PyCsp improves the high temperature tolerance of Escherichia coli

[0062] The gene PYCH_0765 was connected to the plasmid pUC19 to obtain the recombinant plasmid pUC19-PYCH_0765, and then the recombinant plasmid was transferred into Escherichia coli DH5α by heat shock to obtain the recombinant bacteria DH5α / pUC19-PYCH_0765. The growth curve of the recombinant bacteria DH5α / pUC19-PYCH_0765 transferred into the empty plasmid pUC19 at 45°C was measured, and the cell dry weight at the stationary phase was measured. The growth curve of the overexpression of PyCsp is shown in Fig.10 As shown, the cell dry weight at the plateau phase is Fig.11 As shown in Figure 3, overexpression of PyCsp can increase the biomass of Escherichia coli at 45°C.

[0063] Example 11: RNA chaperone PyCsp improves the high temperature tolerance of Streptomyces xiamenensis

[0064] The gene PYCH_0765 was connected to the plasmid pIB139 to obtain the recombinant plasmid pIB139-0765, and then the recombinant plasmid was transformed into Xiamen Streptomyces. The cell dry weight of the wild-type strain of the recombinant bacteria transformed with the recombinant plasmid pIB139-0765 at 37°C was as follows: Fig.12 As shown in Figure 3, overexpression of PyCsp can increase the biomass of Streptomyces xiamenensis at 37°C.

[0065] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. An RNA chaperone protein, characterized in that The RNA chaperone protein has an activity of binding to RNA.

2. The RNA chaperone protein according to claim 1, characterized in that The RNA chaperone protein is PyCsp, and the amino acid sequence is shown in SEQ ID NO.

1.

3. A gene encoding the RNA chaperone protein according to any one of claims 1 to 2, characterized in that: The nucleotide sequence of the gene encoding the protein is shown in SEQ ID NO.

2.

4. A highly stress-resistant recombinant bacterium expressing the RNA chaperone protein according to claim 1, characterized in that: The recombinant bacteria have low temperature tolerance, high pressure tolerance or high temperature tolerance.

5. The highly stress-resistant recombinant bacterium according to claim 4, characterized in that: The recombinant bacterium contains the gene as claimed in claim 3.

6. A method for preparing a highly stress-resistant recombinant bacterium according to any one of claims 4 to 5, characterized in that: The method comprises the following steps: Step 1, constructing a recombinant plasmid expressing RNA molecular chaperone PyCsp; Step 2: The recombinant plasmid expressing the RNA molecular chaperone PyCsp is transferred into the target bacteria to prepare the recombinant bacteria, that is, the recombinant bacteria with high stress resistance.

7. The method according to claim 6, characterized in that The step 1 specifically includes: The nucleotide sequence shown in SEQ ID NO.2 was connected to a vector to construct a recombinant plasmid expressing the RNA chaperone protein PyCsp.

8. The method according to claim 6, characterized in that The step 1 specifically includes: The full-length fragment of the PyCsp protein gene was obtained by PCR amplification using the genomic DNA of the hyperthermophilic barophilic archaeon Pyrococcusyayanosii CH1 as a template using primers 1 and 2. The base sequences of primers 1 and 2 are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. Using plasmid pET28a as a template, primers 3 and 4 were used to amplify the linearized expression vector pET28a by PCR. The base sequences of primers 3 and 4 are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. In a 20 μL reaction system, the full-length fragment of the PyCsp protein gene and the linearized vector pET28a fragment were added in a molar ratio of 2:1 between the gene fragment and the linearized vector fragment, and then 2 μL of ExnaseⅡ ligase and 4 μL of 5×CEⅡ buffer were added. After mixing, the mixture was incubated at 37°C for 30 min to obtain the recombinant plasmid.

9. The method according to claim 8, characterized in that The step 2 specifically includes: The recombinant plasmid was transferred into competent cells of Escherichia coli BL21 by heat shock to obtain recombinant bacteria.

10. The method according to claim 6, characterized in that The target bacteria are one or more of cold-sensitive Escherichia coli BX04, Escherichia coli DH5α, hyperthermophilic barophilic archaeon Pyrococcusyayanosii, hyperthermophilic barophilic archaeon Thermococcuskodakarensis and Streptomyces xiamenensis.

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