An RNA chaperone, a coding gene and a high-stress-resistance recombinant bacterium expressing the RNA chaperone and a preparation method thereof

Abstract

The application discloses a kind of RNA chaperone, coding gene and high stress resistance recombinant bacteria expressing the RNA chaperone and its preparation method, it is related to the field of bioengineering.RNA chaperone PyCsp has the activity of binding RNA.By constructing the recombinant plasmid of expression RNA molecule PyCsp chaperone, the recombinant plasmid is transferred into target bacteria to prepare recombinant bacteria, i.e., the recombinant bacteria with high stress resistance, the stress resistance is low-temperature tolerance, high-pressure tolerance or high-temperature tolerance.The application digs out a heat-resistant RNA chaperone from super-thermophilic pressure-loving microorganism (optimum growth temperature 95 DEG C).Such high-temperature-resistant RNA chaperone as stress resistance element has important application value in improving the stress resistance of microorganism to high temperature, low temperature and high hydrostatic pressure and other environmental stress conditions, and has unique advantages in improving the stress resistance of strain used in fermentation in the application scene of bioengineering related to high temperature and high pressure.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to an RNA chaperone protein, its encoding gene, a highly stress-resistant recombinant bacterium expressing the RNA chaperone protein, and a method for preparing the same. Background Technology

[0002] RNA chaperones are a class of proteins that can bind to RNA without consuming ATP. These proteins protect RNA from degradation by ribonucleases, alter RNA structure, and assist RNA in binding to its targets. The properties of RNA chaperones make them play an important role in RNA-mediated regulation, especially in response to low-temperature stress. In bacteria, RNA chaperone CspA plays a crucial role not only in responding to low-temperature stress but also as a stress protein under high hydrostatic pressure; in Clostridium botulinum ATCC 3502, RNA chaperones CspB and CspC play important roles in the strain's response to low pH, high salt, and ethanol stress.

[0003] Cold shock treatment of the hyperthermophilic and barophilic archaea *P. yayanosii* A1 induced the gene PYCH_0765, which encodes the novel cold shock protein PyCsp. Furthermore, online tool catRAID signature showed that PyCsp can bind to RNA. The RNA chaperone PyCsp discovered in *P. yayanosii* A1 can serve as a stress-resistance element, possessing significant application value in enhancing the resistance of microorganisms to environmental stresses such as high temperature, low temperature, and high hydrostatic pressure. For example, increasing fermentation temperature in industrial fermentation requires heat-tolerant strains; inducing recombinant protein expression at lower temperatures inhibits the growth of genetically engineered host bacteria; and bioengineering applications under high temperature and high pressure conditions require improved microbial tolerance to high pressure. Therefore, those skilled in the art are dedicated to developing strains and preparation methods that possess resistance to environmental stresses such as high temperature, low temperature, and high hydrostatic pressure. Summary of the Invention

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

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

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

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

[0008] The present invention also provides a highly 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 genes as described above.

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

[0011] Step 1: Construct a recombinant plasmid expressing the RNA molecular chaperone PyCsp;

[0012] Step 2: Transform the recombinant plasmid expressing the RNA molecular chaperone PyCsp into the target bacteria to prepare recombinant bacteria, which are recombinant bacteria with high stress resistance.

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

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

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

[0016] Using the genomic DNA of the hyperthermophilic and barophilic archaea Pyrococcus yayanosii 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, the linearized expression vector pET28a was obtained by PCR amplification using primers 3 and 4. 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 at a molar ratio of 2:1 to the gene fragment and the linearized vector fragment, respectively. Then, 2 μL of Exnase II ligase and 4 μL of 5×CE II buffer were added, mixed well, and incubated at 37°C for 30 min to obtain the recombinant plasmid.

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

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

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

[0022] Technical effect

[0023] 1. Introducing gene 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 functions and can function under various 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 showed anti-transcription termination activity, and overexpression of PyCsp can increase the biomass of cold-sensitive Escherichia coli BX04 at 22℃ and 18℃.

[0025] 3. Discover stress-resistance gene elements from the barophilic microorganism P. yayanosii A1 from deep-sea hydrothermal vents. RNA chaperones have a wide range of regulatory functions and can play a role under various stress conditions. Overexpression of RNA chaperone PyCsp can significantly increase the biomass of the hyperthermophilic archaea P. yayanosii A1 under high hydrostatic pressure.

[0026] This invention identifies a thermostable RNA chaperone from hyperthermophilic and barotropic microorganisms (optimal growth temperature 95°C) originating from deep-sea hydrothermal vents. Compared to existing RNA chaperones derived from thermophilic bacteria (generally growing at temperatures below 60°C), PyCsp exhibits significant advantages in thermal stability. This type of thermostable RNA chaperone, as a stress-resistance element, has important application value in enhancing the resistance of microorganisms to environmental stresses such as high temperature, low temperature, and high hydrostatic pressure. It also offers unique advantages in improving the stress resistance of strains used in fermentation within bioengineering applications involving high temperature and high pressure.

[0027] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

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

[0029] Figure 2 This is a preferred embodiment of the PyCsp binding and dissociation curve of RNA.

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

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

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

[0033] Figure 6 This is a preferred embodiment of the present invention regarding the effect of PyCsp overexpression on the biomass of Pyrococcus yayanosii at 85°C;

[0034] Figure 7 This is a preferred embodiment of the present invention regarding the effect of PyCsp overexpression on the biomass of Pyrococcus yayanosii at 52 MPa;

[0035] Figure 8 This is a preferred embodiment of the present invention regarding the effect of PyCsp overexpression on the biomass of Thermococcus kodakarensis at 65°C;

[0036] Figure 9This is a preferred embodiment of the present invention regarding the effect of PyCsp overexpression on the biomass of Thermococcus kodakarensis at 20 MPa;

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

[0038] Figure 11 This is a preferred embodiment of the present invention regarding the effect of PyCsp overexpression on Escherichia coli biomass at 45°C;

[0039] Figure 12 This is a preferred embodiment of the present invention, showing the effect of PyCsp overexpression on the biomass of *Streptomyces xiaensis* at 37°C. Detailed Implementation

[0040] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0041] Example 1: Cloning, expression, and purification of the thermostable RNA molecular 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, the full-length fragment of the gene PYCH_0765 was amplified by PCR using primer 1 (base sequence shown in SEQ ID NO.3) and primer 2 (base sequence shown in SEQ ID NO.4). Using plasmid pET28a as a template, the linearized expression vector pET28a was amplified by PCR using primer 3 (base sequence shown in SEQ ID NO.5) and primer 4 (base sequence shown in SEQ ID NO.6). In a 20 μL reaction system, the PYCH_0765 fragment and the linearized vector pET28a fragment were added at a molar ratio of 2:1 (gene fragment to linearized vector fragment). Then, 2 μL of Exnase II (ligase) and 4 μL of 5×CEII buffer were added, mixed thoroughly, and incubated at 37°C for 30 min. The ligation product was then transformed into competent Escherichia coli BL21(DE3) cells via heat shock to obtain the recombinant strain BL21 / pET28a-PYCH_0765.

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

[0044] Purification of PyCsp: The bacterial cells were resuspended in lysis buffer (containing 0.5 M NaCl, 50 mM Tris-HCl, pH 8.0), and the cells were lysed on ice using an ultrasonic homogenizer. After lysis, the cells were centrifuged at 12,000 rpm for 30 min at 4°C, and the supernatant was retained. The supernatant was passed through a Ni-NTA affinity chromatography column, and the column was washed with wash buffer containing 20 mM imidazole (containing 0.5 mol / L NaCl, 50 mmol / L Tris-HCl, pH 8.0), followed by elution with 250 mM imidazole elution buffer (containing 0.5 mol / L NaCl, 50 mmol / L Tris-HCl, pH 8.0). The high concentration of imidazole in the target protein was removed using a 3 kDa ultrafiltration tube, and the target protein was stored in buffer containing 0.5 M NaCl and 50 mM Tris-HCl (pH 8.0). The purified protein was identified by Tricine-SDSPAGE electrophoresis. Figure 1 As shown in the figure (Column 1: Standard protein molecular weight marker; Column 2: Purified PyCsp), the relative molecular mass of purified PyCsp is approximately 9.5 kDa, which is close to that of PyCsp (7.86 kDa). Therefore, PyCsp was successfully expressed in soluble form in E. coli BL21(DE3).

[0045] Example 2: RNA-binding activity of the RNA molecular chaperone PyCsp

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

[0047] Example 3: RNA unwinding activity of the RNA molecular 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), where MB 40 -FITC has a fluorescein isothiocyanate isomer (FITC) at its 5' end, MB 40 -BHQ1 has a fluorescence quenching group BHQ1 at its 3' end. MB 40 -FITC and MB 40 BHQ1 molecules are mixed in a 1:1 molecular ratio and then annealed to form double-stranded RNA (dsRNA). Because the FITC fluorescent group is close to the BHQ1 fluorescence quencher, the fluorescence intensity of the annealed double-stranded RNA is higher than that of MB. 40 -FITC levels decreased significantly. Under light-protected conditions, the following reaction mixture was prepared: 20 μM purified PyCsp, 0.1 μM annealed dsRNA, 200 mM Tris-HCl (pH 7.4), and 10 mM MgCl2. The final volume was increased to 100 μL with DEPC water. The reaction mixture was incubated at 37°C for 30 min. After incubation, 50 μL of the reaction solution was added to a black ELISA plate, and the fluorescence intensity was measured using a multi-functional microplate reader. The measurement parameters were: excitation light 460 nm, emission light 515 nm. The results are as follows: Figure 3 As shown, PyCsp possesses RNA unwinding activity.

[0049] Example 4: Anti-transcription termination activity of the RNA molecular chaperone PyCsp

[0050] The chloramphenicol resistance gene *cat* in the model strain RL211 contains a strong terminator *trpL* upstream. If the protein has anti-transcriptional termination activity, the *cat* gene can be transcribed normally, and the strain will exhibit chloramphenicol resistance. The gene *PYCH_0765* was ligated into plasmid *pINⅢ* to obtain the recombinant plasmid *pIN-0765*. This recombinant plasmid was then transformed into the model strain RL211 via heat shock, resulting in the recombinant strain RL211 / pIN-0765. The transformed genes *cspA* and *cspL* were ligated into plasmid *pINⅢ* to obtain the recombinant plasmids *pIN-cspA* and *pIN-cspL*. The recombinant strains RL211 / pIN-cspA and RL211 / pIN-cspL transformed into recombinant plasmids *pIN-cspA* and *pIN-cspL* served as positive controls, while the recombinant strain RL211 / pIN transformed into the empty vector plasmid *pINⅢ* served as a negative control. Recombinant bacteria RL211 / pIN-0765, RL211 / pIN-cspA, RL211 / pIN-cspL, and RL211 / pIN were cultured in LB medium until OD500. 600 The concentration was 1.0. 6 μL of culture was spotted onto LB agar plates containing 100 μg / mL ampicillin, with or without 30 μg / mL chloramphenicol, and with or without 0.2 mM IPTG, and incubated for 2 days at 37℃ and 45℃, respectively. Results are as follows: Figure 4 As shown, PyCsp exhibits anti-transcription termination activity at both 37℃ and 45℃.

[0051] Example 5: PyCsp RNA molecular chaperone reintroduction of cold-sensitive E. coli BX04

[0052] Recombinant plasmids pIN-0765, pIN-cspA, pIN-cspL, and the empty vector pINⅢ were transformed into cold-sensitive *E. coli* BX04 via heat shock transformation to obtain recombinant bacteria BX04 / pIN-0765, BX04 / pIN-cspA, BX04 / pIN-cspL, and BX04 / pIN, respectively. Recombinant bacteria BX04 / pIN-cspA and BX04 / pIN-cspL served as positive controls, and recombinant bacteria BX04 / pIN served as a negative control. The recombinant bacteria were cultured in LB medium at 37°C until OD2000. 600 After reaching a concentration of 1.0, the culture was diluted with LB medium, and 6 μL of the culture was spotted onto LB plates containing 100 μg / mL ampicillin and with or without 1 mM IPTG. The plates were then incubated at 37°C, 22°C, and 18°C ​​for 3 days, respectively. Results are as follows: Figure 5 As shown, PyCsp can restore the growth of cold-sensitive Escherichia coli BX04 at 22°C and 18°C.

[0053] Example 6: RNA molecular chaperone PyCsp enhances the low-temperature tolerance of Pyrococcus yayanosii A1

[0054] The gene PYCH_0765 was ligated into the plasmid pLMOS to obtain the recombinant plasmid pLMOS-PYCH_0765. This recombinant plasmid was then transformed into *Pyrococcus yayanosii* A1 via heat shock to obtain the recombinant bacterium A1 / pLMOS-PYCH_0765. The recombinant bacterium A1 / pLMOS-PYCH_0765 was then transformed into the empty vector pLMOS and cultured in TRM medium at 85°C and 0.1 MPa for 16 h. After culture, the cells in the culture were counted using a hemocytometer. The results are as follows: Figure 6 As shown, overexpression of PyCsp significantly increases the biomass of Pyrococcus yayanosii A1 at 85℃.

[0055] Example 7: RNA molecular chaperone PyCsp enhances the high stress tolerance of Pyrococcus yayanosii

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

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

[0058] The gene PYCH_0765 was ligated into plasmid pTE to obtain recombinant plasmid pTE-0765. This recombinant plasmid was then transformed into the guanidine auxotrophic strain *Thermococcus kodakarensis* TS559 via heat shock to obtain recombinant bacteria TS559 / pTE-0765. The recombinant bacteria TS559 / pTE-0765 were then transformed into the recombinant bacteria TS559 / pTE containing the empty plasmid pTE and cultured in ASW-YT medium at 65°C and 0.1 MPa for 20 h. After culture, the OD of the culture was measured. 600 The measurement was performed. The results are as follows: Figure 8 As shown, overexpression of PyCsp can increase the biomass of Thermococcus kodakarensis at 65°C.

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

[0060] The recombinant strain TS559 / pTE-0765 was transformed into the recombinant strain TS559 / pTE with the empty vector pTE and cultured in ASW-YT medium at 85℃ and 20MPa for 20 h. After culture, the OD of the culture was measured. 600 The measurement was performed. The results are as follows: Figure 9 As shown, overexpression of PyCsp can increase the biomass of Thermococcus kodakarensis at 20 MPa.

[0061] Example 10: RNA molecular chaperone PyCsp enhances the heat tolerance of Escherichia coli

[0062] The gene PYCH_0765 was ligated into plasmid pUC19 to obtain the recombinant plasmid pUC19-PYCH_0765. This recombinant plasmid was then transformed into *E. coli* DH5α via heat shock, resulting in the recombinant bacterium DH5α / pUC19-PYCH_0765. The growth curve of the recombinant bacterium DH5α / pUC19 transformed into the empty vector pUC19 was measured at 45°C, and the cell dry weight at the plateau phase was also determined. The growth curve of PyCsp overexpression is shown below. Figure 10 As shown, the cell stem weight during the plateau phase is as follows: Figure 11 As shown, overexpression of PyCsp can increase the biomass of Escherichia coli at 45°C.

[0063] Example 11: RNA molecular chaperone PyCsp enhances the heat tolerance of Streptomyces xiaensis.

[0064] The gene PYCH_0765 was ligated into plasmid pIB139 to obtain the recombinant plasmid pIB139-0765, which was then transformed into *Streptomyces xiaensis*. The dry weight of the wild-type strain of recombinant bacteria transformed with recombinant plasmid pIB139-0765 at 37°C was as follows. Figure 12 As shown, overexpression of PyCsp can increase the biomass of *Streptomyces xiaensis* at 37°C.

[0065] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. Use of overexpression of RNA chaperone proteins for increasing the cold tolerance of microorganisms, characterized in that, The RNA chaperone is PyCsp, the amino acid sequence of which is shown as SEQ ID NO. 1, and the microorganism is cold-sensitive Escherichia coli BX04 or Pyrococcus yayanosii A1.

2. Use of overexpression of RNA chaperone proteins for increasing thermotolerance of microorganisms, characterized in that, The RNA chaperone is PyCsp, the amino acid sequence of which is shown as SEQ ID NO. 1, and the microorganism is Escherichia coli DH5α or Streptomyces xiamenensis.

3. Use of overexpression of RNA chaperone proteins for increasing high pressure tolerance of microorganisms, characterized in that, The RNA chaperone is PyCsp, the amino acid sequence of which is shown as SEQ ID NO. 1, and the microorganism is Pyrococcus yayanosii or Thermococcus kodakarensis.

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