Application of small molecule heat shock protein IbpA in improving environmental tolerance of host bacteria

Abstract

The present invention provides an application of a small molecule heat shock protein IbpA in improving the environmental tolerance of a host bacterium, belonging to the field of genetic engineering technology, wherein the small molecule heat shock protein encoding gene ibpA in Rhodobacter sphaeroides ATCC 17023 is cloned and an expression vector is constructed, and the expression vector is transferred into a host bacterium, so that the host bacterium overexpresses the small molecule heat shock protein IbpA, and the nucleotide sequence of the small molecule heat shock protein encoding gene ibpA is shown in SEQ ID NO. 1. The present invention greatly increases the resistance of the obtained recombinant bacteria under different stress conditions by overexpressing the small molecule heat shock protein IbpA in the host bacterium, and can improve the survival ability of the host bacterium under various adverse conditions.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, in particular to application of a small molecule heat shock protein IbpA in improving the environmental tolerance of host bacteria. Background Art

[0002] In the process of cultivating microorganisms, they can only grow normally under suitable external conditions and appropriate nutrients. If the external conditions are not suitable, the microorganisms will be affected by stress factors such as insufficient substrate, cold and heat, acidity and alkali, osmotic pressure, etc. These factors will inhibit the growth of microorganisms and even cause their death, and inhibit or reduce the production of their metabolites; acid stress, alkali stress, high temperature stress, oxygen stress, etc. are common stresses faced by microbial cells. Improving the stress resistance of microorganisms will be beneficial to the survival of microorganisms in adverse environments and maintain the normal life activities of cells; in addition, improving the high temperature stress resistance of industrial strains can effectively reduce the use of cooling water, reduce energy consumption and production costs, and effectively reduce the chance of infection; in order to improve the heat resistance of strains, traditional breeding techniques such as high temperature acclimation, natural selection, mutation breeding and other methods have played an important role, but have disadvantages such as low efficiency and lack of directionality.

[0003] The acquisition of biological heat resistance is closely positively correlated with the synthesis of heat shock proteins. Heat shock proteins are a superfamily found in all organisms such as archaea, bacteria and eukaryotes. They can act as molecular chaperones, bind to partially denatured proteins, assist in the degradation of denatured proteins, and prevent irreversible aggregation of proteins induced by various environmental pressures, helping cells adapt to external pressure environments. Overexpression of heat shock proteins can protect microorganisms from damage to cells caused by external high temperature pressure. At present, research on the function and application of microbial small molecule heat shock proteins has just started. These small molecule heat shock proteins are important biological resources and will have important application value in the food industry, environmental protection, medical health and other fields in the future.

[0004] Rhodobacter sphaeroides is a non-oxygenous photosynthetic bacterium that has applications in food, medicine, environmental protection, agriculture and other fields, and has high research and application value. In the field of food and medicine, Rhodobacter sphaeroides can be used to produce coenzyme Q10, synthesize lycopene, carotenoids, etc. In the field of environmental protection, Rhodobacter sphaeroides has strong nitrogen and phosphorus metabolism capabilities and can be used to degrade organophosphorus pesticides and enrich heavy metals; in the field of agriculture, Rhodobacter sphaeroides can be used as foliar fertilizer, and its synthesis of 5-aminolevulinic acid promotes plant maturity.

[0005] In order to enhance the tolerance of cells to damage, maintain normal metabolic functions of cells, and improve the survival ability of organisms under adverse conditions, the present invention intends to use transgenic technology to transfer the small molecule heat shock protein encoding gene ibpA in Rhodobacter sphaeroides ATCC 17023 into the strain for overexpression, so as to improve the tolerance of Rhodobacter sphaeroides and other host bacteria to adverse environments. Summary of the invention

[0006] In view of the above problems, the present invention provides an application of a small molecule heat shock protein IbpA in improving the environmental tolerance of host bacteria. The small molecule heat shock protein encoding gene ibpA is cloned and a recombinant expression vector is constructed. The recombinant expression vector is transferred into the host bacteria to overexpress the small molecule heat shock protein IbpA, so as to improve the resistance of the host bacteria under different stress conditions.

[0007] The technical solution of the present invention is as follows:

[0008] A small molecule heat shock protein IbpA is used in improving the environmental tolerance of a host bacteria. The small molecule heat shock protein IbpA is derived from Rhodobacter sphaeroides ATCC 17023, and its amino acid sequence is shown in SEQ ID NO.2.

[0009] Preferably, the nucleotide sequence of the gene ibpA encoding the small molecule heat shock protein IbpA is shown in SEQ ID NO.1.

[0010] Preferably, the host bacteria is Rhodobacter sphaeroides or Escherichia coli.

[0011] Preferably, a small molecule heat shock protein IbpA is used to improve the environmental tolerance of a host bacterium, and the application method is to clone the small molecule heat shock protein encoding gene ibpA in Rhodobacter sphaeroides ATCC 17023 and construct a recombinant expression vector, and transfer the recombinant expression vector into the host bacterium, so that the host bacterium overexpresses the small molecule heat shock protein IbpA.

[0012] Preferably, a small molecule heat shock protein IbpA is used to improve the environmental tolerance of host bacteria, and the application method specifically comprises the following steps:

[0013] S1, total DNA extracted from Rhodobacter sphaeroides ATCC 17023;

[0014] S2, using the extracted total DNA as a template, designing primers, and obtaining the small molecule heat shock protein coding gene ibpA, i.e., the cloned product, by PCR amplification; the primers are:

[0015] Upstream primer: 5'-CGGGGTACCATGCGTAGCTATGATTTCTCGCCGC-3', SEQ ID NO. 3;

[0016] Downstream primer: 5'-CCCAAGCTTTCAGGCCTCGACCGGCTCCTTCAC-3', SEQ ID NO. 4;

[0017] S3, digesting the cloned product and inserting it into the digested plasmid vector to construct a recombinant expression vector;

[0018] S4, the recombinant expression vector is transformed into the host bacteria by conjugation transfer, and the small molecule heat shock protein IbpA is overexpressed to improve the environmental tolerance of the host bacteria.

[0019] Preferably, the PCR amplification reaction conditions in S2 are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 70°C for 15 s, extension at 72°C for 2 min, repeated for 35 cycles; extension at 72°C for 5 min, and cooling to 4°C.

[0020] Preferably, the enzyme digestion method in S3 is digestion using restriction endonucleases Kpn I and Hind III.

[0021] Preferably, the plasmid vector in S3 is pBBR1MCS-2.

[0022] Preferably, the transformation method of the recombinant expression vector in S4 is:

[0023] (1) preparing a host bacterial solution and a donor bacterial solution containing a recombinant expression vector, washing the bacterial cells and then resuspending them to obtain a host bacterial resuspended solution and a donor bacterial resuspended solution;

[0024] (2) The host bacterial re-suspended liquid and the donor bacterial re-suspended liquid are mixed at a bacterial concentration ratio of 1:3 to 7, and the mixed bacterial liquid is spotted on a 0.22 μm sterile filter membrane placed on the corresponding solid culture medium, and statically cultured to allow conjugation transfer to occur, and the host bacteria containing the recombinant expression vector are screened.

[0025] A recombinant expression vector contains the above-mentioned coding gene ibpA.

[0026] A recombinant bacterium contains the above recombinant expression vector or the above coding gene ibpA.

[0027] Preferably, the host bacteria of the recombinant bacteria are Rhodobacter sphaeroides or Escherichia coli.

[0028] The present invention has found in the previous study that after the Rhodobacter sphaeroides ATCC 17023 was subjected to high temperature heat shock treatment (75°C heat shock for 10 minutes), the expression level of the small molecule heat shock protein encoding gene ibpA in the bacteria was significantly upregulated, so that the Rhodobacter sphaeroides ATCC 17023 bacteria had resistance to high temperature, and the higher the expression amount of the small molecule heat shock protein IbpA, the more obvious the differential expression, and the stronger the ability of the Rhodobacter sphaeroides ATCC 17023 to resist heat stress; further research found that the small molecule heat shock protein IbpA in the Rhodobacter sphaeroides ATCC 17023 may enhance the tolerance of cells to damage, maintain the normal metabolic function of cells, and is closely related to the survival ability of organisms under adversity. Therefore, the present invention uses transgenic technology to overexpress the encoding gene ibpA of the small molecule heat shock protein in the Rhodobacter sphaeroides ATCC 17023 in the host bacteria to improve the host bacteria's resistance to heat stress and tolerance to other adverse environments.

[0029] Beneficial effects of the present invention: By overexpressing the small molecule heat shock protein IbpA in the host bacteria, the resistance of the obtained recombinant bacteria under stress conditions such as high temperature, high acid, high alkali, high salt, high concentration of metal ions and high concentration of furfural is greatly increased, which can improve the survival ability of the host bacteria under various adverse conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the construction of IbpA recombinant expression vector;

[0031] Figure 2 The figure is a screening process of IbpA overexpression engineering bacteria, wherein: (a) is an electrophoresis diagram of ibpA gene amplification; (b) is an electrophoresis diagram of pBBR1MCS-2 plasmid restriction enzyme digestion; (c) is an electrophoresis diagram of recombinant Escherichia coli PCR amplification;

[0032] Figure 3 The survival rate of wild Rhodobacter sphaeroides and IbpA overexpressing engineered bacteria under high temperature conditions (85℃ heat shock for 10 minutes);

[0033] Figure 4 is the survival rate of wild-type Rhodobacter sphaeroides and IbpA overexpressing engineered bacteria under high acid conditions (pH 5.0);

[0034] Figure 5 is the survival rate of wild-type Rhodobacter sphaeroides and IbpA overexpressing engineered bacteria under high alkaline conditions (pH 11.0);

[0035] Figure 6 The survival rate of wild Rhodobacter sphaeroides and IbpA overexpressing engineered bacteria under high salt conditions (salinity 5%);

[0036] Figure 7The wild-type Rhodobacter sphaeroides and IbpA-overexpressing engineered bacteria were exposed to high concentrations of Cr 6+ Survival rate under conditions (350 mg / L);

[0037] Figure 8 The wild Rhodobacter sphaeroides and IbpA overexpressing engineered bacteria were exposed to high concentrations of Pb 2+ Survival rate under conditions (650 mg / L);

[0038] Fig. 9 The wild strains of Rhodobacter sphaeroides and the engineered strains overexpressing IbpA were exposed to high concentrations of Cd 2+ Survival rate under conditions (350 mg / L);

[0039] Fig.10 The survival rate of wild Rhodobacter sphaeroides and IbpA overexpressing engineered bacteria under high concentration furfural conditions (40 g / L);

[0040] Fig.11 is the survival rate of wild-type E. coli and IbpA-overexpressing engineered bacteria under high temperature conditions (85°C heat shock for 10 min);

[0041] Fig.12 is the survival rate of wild-type Escherichia coli and IbpA-overexpressing engineered bacteria under high acid conditions (pH 5.0);

[0042] Fig.13 is the survival rate of wild-type Escherichia coli and IbpA-overexpressing engineered bacteria under high alkaline conditions (pH 11.0);

[0043] Fig.14 The survival rate of wild Escherichia coli and IbpA overexpressing engineered bacteria under high salt conditions (salinity 5%);

[0044] Fig.15 The wild-type Escherichia coli and the engineered strains overexpressing IbpA were exposed to high concentrations of Cr 6+ Survival rate under conditions (450 mg / L);

[0045] Fig.16 The wild-type Escherichia coli and the engineered strains overexpressing IbpA were exposed to high concentrations of Pb 2+ Survival rate under conditions (350 mg / L);

[0046] Fig.17 The wild-type Escherichia coli and IbpA-overexpressing engineered bacteria were exposed to high concentrations of Cd 2+ Survival rate under the conditions (200 mg / L);

[0047] Fig.18 is the survival rate of wild Escherichia coli and IbpA overexpressing engineered bacteria under high concentration furfural conditions (6 g / L);

[0048] Fig.19 This is the survival rate of IbpA derived from Rhodobacter sphaeroides and IbpA derived from Escherichia coli after overexpression in Rhodobacter sphaeroides under high temperature conditions (heat shock at 85°C for 10 min). DETAILED DESCRIPTION

[0049] The following is explained in conjunction with specific embodiments:

[0050] Embodiment 1:

[0051] Cloning of the gene encoding small heat shock protein ibpA and construction of its recombinant expression vector

[0052] S1, total DNA was extracted from Rhodobacter sphaeroides ATCC 17023 using a genomic DNA extraction kit;

[0053] S2, using the extracted total DNA as a template, designed primers, and obtained the small molecule heat shock protein encoding gene ibpA, i.e., the cloned product, by PCR amplification. The agarose gel electrophoresis of the obtained cloned product is shown in Figure 2 As shown in Figure (a) in the figure; the PCR amplification reaction conditions are: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s, 70℃ annealing for 15s, 72℃ extension for 2min, repeated 35 cycles; 72℃ extension for 5min, cooling to 4℃;

[0054] The primers used were:

[0055] Upstream primer: 5'-CGGGGTACCATGCGTAGCTATGATTTCTCGCCGC-3', SEQ ID NO. 3;

[0056] Downstream primer: 5'-CCCAAGCTTTCAGGCCTCGACCGGCTCCTTCAC-3', SEQ ID NO. 4;

[0057] S3, the cloned product and pBBR1MCS-2 plasmid were double-digested with restriction endonucleases Kpn I and Hind III, respectively, and then ligated with T4 DNA ligase. The agarose gel electrophoresis of pBBR1MCS-2 plasmid after double-digestion is shown in FIG. Figure 2 As shown in (b) of FIG. 1 ; the molar ratio of the cloned product to the pBBR1MCS-2 plasmid during enzyme ligation is 4:1, and the total volume of the enzyme ligation reaction is 20 μL; the cloned product is inserted into the pBBR1MCS-2 plasmid by enzyme ligation reaction, and the enzyme ligation product is transferred into the competent cells of Escherichia coli S17-1, and the recombinant expression vector and the recombinant Escherichia coli S17-1 containing the recombinant expression vector are obtained by amplification and screening; wherein the colony PCR amplification agarose gel electrophoresis diagram of the recombinant Escherichia coli S17-1 is shown in Figure 2 As shown in Figure (c).

[0058] The nucleotide sequence of the small molecule heat shock protein encoding gene ibpA is shown in SEQ ID NO.1, and the amino acid sequence of the small molecule heat shock protein IbpA is shown in SEQ ID NO.2; the schematic diagram of constructing the recombinant expression vector is shown in Figure 1 shown.

[0059] Embodiment 2:

[0060] Transformation and expression of small molecule heat shock protein IbpA

[0061] (1) inoculating Rhodobacter sphaeroides ATCC 17023 into LB liquid culture medium, culturing at 32° C. and 200 rpm under light or without light for 2 to 3 days to obtain a Rhodobacter sphaeroides bacterial liquid;

[0062] The LB liquid culture medium comprises: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0;

[0063] (2) The recombinant Escherichia coli S17-1 (donor bacteria) containing the recombinant expression vector prepared in Example 1 was cultured overnight in LB liquid medium supplemented with 50 μg / mL kanamycin; the overnight cultured bacterial solution was transferred to fresh LB liquid medium and cultured until OD 600 is about 0.5, and the recombinant E. coli liquid is obtained;

[0064] (3) 1 mL of the Rhodobacter sphaeroides bacterial solution in step (1) and the recombinant Escherichia coli bacterial solution in step (2) were taken, centrifuged at 5000 rpm for 4 min, and the supernatant was discarded; the cells were very gently resuspended in 1 mL of fresh LB liquid medium, centrifuged at 5000 rpm for 4 min, and the supernatant was discarded. The process was repeated twice, and finally, the cells were very gently resuspended in 100 μL of fresh LB liquid medium to obtain a Rhodobacter sphaeroides resuspended bacterial solution and a recombinant Escherichia coli resuspended bacterial solution;

[0065] (4) mixing the resuspended bacterial solution of Rhodobacter sphaeroides and the resuspended bacterial solution of recombinant Escherichia coli in step (3) at a bacterial concentration ratio of 1:5, and spotting the mixed bacterial solution on a 0.22 μm sterile filter membrane placed on LB solid culture medium containing 25 μg / mL kanamycin and 50 μg / mL potassium tellurite; culturing at 32° C. for 1 day under light or no light conditions to allow conjugation transfer to occur;

[0066] (5) Scrape off the bacterial moss on the filter membrane in step (4), resuspend it in fresh LB liquid medium, and spread it on LB solid medium containing 25 μg / mL kanamycin and 50 μg / mL potassium tellurite, and culture it at 32°C for more than 3 days until small black colonies appear on the medium;

[0067] The components of the LB solid medium are: peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, pH 7.0;

[0068] (6) Pick out the black colonies in step (5), resuspend them in fresh LB liquid medium, and spread them on LB solid medium containing 25 μg / mL kanamycin and 50 μg / mL potassium tellurite after dilution. Incubate at 32°C for more than 3 days until separate colonies appear on the plate. Pick out the colonies to extract the plasmids and identify them to obtain the recombinant Rhodobacter sphaeroides.

[0069] The recombinant Rhodobacter sphaeroides obtained by the above screening was inoculated into LB liquid culture medium containing 25 μg / mL kanamycin to achieve overexpression of the small molecule heat shock protein IbpA; the pBBR1MCS-2 plasmid was constitutively expressed and did not require the addition of an inducer for induction.

[0070] Experimental Example 1:

[0071] Test of the temperature resistance of Rhodobacter sphaeroides overexpressing the small molecule heat shock protein IbpA

[0072] The recombinant Rhodobacter sphaeroides ibpA / op introduced with the recombinant expression vector and the Rhodobacter sphaeroides WT / op introduced with the empty vector in Example 2 were inoculated into LB liquid culture medium for activation, and cultured at 32° C. and 200 rpm for 48 h, wherein 25 μg / mL kanamycin was added to the culture medium containing ibpA / op; the activated bacterial solution was transferred to a new LB liquid culture medium at an inoculation amount of 2% of the volume of the new culture medium, and cultured at 32° C. and 200 rpm for 24 h; the OD of the bacterial solution was measured at a wavelength of 600 nm. 600 value, adjust the ibpA / op and WT / op bacterial solutions to the same concentration, OD 600 The value is 1.0.

[0073] The ibpA / op bacterial solution and WT / op bacterial solution adjusted to the same concentration were kept at 85°C for 10 min, and the survival rate of the strains at high temperature was tested. The results are shown in Table 1 and Figure 3 As shown;

[0074] Table 1. Average survival rate of Rhodobacter sphaeroides strains under high temperature conditions

[0075] strains WT / op ibpA / op Survival rate (%) 0.001 23

[0076] From Table 1 and Figure 3 It can be seen that the adaptability of the recombinant Rhodobacter sphaeroides ibpA / op to high temperature is significantly higher than that of the control strain; after 10 minutes of high temperature stress at 85°C, the average survival rate of ibpA / op can reach 23%, while the average survival rate of WT / op is only 0.001%, which indicates that the recombinant Rhodobacter sphaeroides overexpressing the small molecule heat shock protein IbpA has a higher resistance to high temperature.

[0077] Experimental Example 2:

[0078] Test of pH resistance of Rhodobacter sphaeroides overexpressing small heat shock protein IbpA

[0079] ① High acidity conditions

[0080] Different from Experimental Example 1, in this experimental example, the ibpA / op bacterial solution and WT / op bacterial solution adjusted to the same concentration were centrifuged at 8000 rpm for 5 min, the bacteria were collected, and then resuspended in LB liquid medium with a pH of 5.0, and cultured at 32°C for 2 h, 4 h, and 8 h, respectively, to detect the survival rate of the strains under high acidity conditions. The results are shown in Tables 2 and Figure 4 As shown;

[0081] Table 2. Average survival rate of Rhodobacter sphaeroides strains under high acidity conditions

[0082] Survival rate (%) 2h 4h 8h WT / op 18 21 3 ibpA / op 15 27 24

[0083] From Table 2 and Figure 4 It can be seen that the tolerance of the recombinant Rhodobacter sphaeroides ibpA / op to high acidity is generally higher than that of the control strain WT / op; after cultured under high acidity conditions of pH 5.0 for 4 hours, the average survival rate of ibpA / op can reach 27%, which is 6% higher than the 21% of WT / op; after cultured under high acidity conditions of pH 5.0 for 8 hours, the average survival rate of ibpA / op is 24%, while the average survival rate of WT / op is 3%, which indicates that the recombinant Rhodobacter sphaeroides overexpressing the small molecule heat shock protein IbpA has a higher resistance to high acidity.

[0084] ②High alkalinity conditions

[0085] Different from Experimental Example 1, in this experimental example, the ibpA / op bacterial solution and WT / op bacterial solution adjusted to the same concentration were centrifuged at 8000 rpm for 5 min, the bacteria were collected, and then resuspended in LB liquid culture medium with a pH of 11.0, and cultured at 32°C for 2 h, 4 h, and 8 h, respectively, to detect the survival rate of the strains under high alkalinity conditions. The results are shown in Tables 3 and Figure 5 As shown;

[0086] Table 3. Average survival rate of Rhodobacter sphaeroides strains under high alkalinity conditions

[0087] Survival rate (%) 2h 4h 8h WT / op 24 11 1 ibpA / op 33 37 24

[0088] From Table 3 and Figure 5 It can be seen that the tolerance of the recombinant Rhodobacter sphaeroides ibpA / op to high alkalinity is generally higher than that of the control strain WT / op; after cultivation under high alkalinity conditions of pH 11.0 for 2 to 8 hours, the average survival rate of ibpA / op was much higher than that of WT / op, especially after cultivation under high alkalinity conditions of pH 11.0 for 8 hours, the average survival rate of ibpA / op was 24%, while the average survival rate of WT / op was only 1%, which indicates that the recombinant Rhodobacter sphaeroides overexpressing the small molecule heat shock protein IbpA has a higher resistance to high alkalinity.

[0089] Experimental Example 3:

[0090] Resistance test of Rhodobacter sphaeroides overexpressing small heat shock protein IbpA to salt ions

[0091] Different from Experimental Example 1, in this experimental example, the ibpA / op bacterial solution and the WT / op bacterial solution adjusted to the same concentration were centrifuged at 8000 rpm for 5 min, the bacteria were collected, and then resuspended in LB liquid culture medium with a NaCl concentration of 5% (w / w), and cultured at 32°C for 2h, 4h, and 8h, respectively, to detect the survival rate of the strains under high salinity conditions. The results are shown in Tables 4 and Figure 6 As shown;

[0092] Table 4. Average survival rate of Rhodobacter sphaeroides strains under high salinity conditions

[0093] Survival rate (%) 2h 4h 8h WT / op 63 51 1 ibpA / op 42 24 45

[0094] From Table 4 and Figure 6 It can be seen that although the average survival rate of ibpA / op was lower than that of WT / op when cultured under high-salinity conditions for 2 to 4 hours, after cultured under high-salinity conditions for 4 hours, ibpA / op gradually adapted to the high-salinity environment and the strain further proliferated; thus, after cultured under high-salinity conditions for 8 hours, the average survival rate of ibpA / op was significantly increased to more than 45%, while the average survival rate of WT / op was only 1%, indicating that the recombinant Rhodobacter sphaeroides overexpressing the small molecule heat shock protein IbpA has a higher tolerance to high salinity.

[0095] Experimental Example 4:

[0096] Resistance test of Rhodobacter sphaeroides overexpressing small heat shock protein IbpA to heavy metal ions and furfural

[0097] Different from Experimental Example 1, in this experimental example, the ibpA / op bacterial solution and the WT / op bacterial solution adjusted to the same concentration were centrifuged at 8000 rpm for 5 min, the bacterial cells were collected, and then chromatographed with Cr-containing 6+ (350mg / L), Pb 2+ (650mg / L), Cd 2+ The strains were resuspended in LB liquid medium containing heavy metal ions (350 mg / L) and furfural (40 g / L) and cultured at 32°C for 4 h. The survival rate of the strains under heavy metal ions and the organic compound furfural was detected. The results are shown in Table 5 and Figures 7-10 As shown;

[0098] Table 5. Average survival rate of Rhodobacter sphaeroides strains under heavy metal ions and organic compound furfural

[0099] Survival rate (%) <![CDATA[Cr 6+ (350mg / L)]]> <![CDATA[Pb 2+ (650mg / L)]]> <![CDATA[Cd 2+ (350mg / L)]]> Furfural (40g / L) WT / op 7 8 10 51 ibpA / op 23 25 22 76

[0100] From Table 5 and Figures 7-10 It can be seen that under the influence of heavy metal ions and the organic compound furfural, the average survival rate of ibpA / op was higher than that of WT / op, which indicates that the recombinant Rhodobacter sphaeroides overexpressing the small molecule heat shock protein IbpA has a higher tolerance to heavy metal ions and the organic compound furfural.

[0101] Experimental Example 5:

[0102] Test of temperature resistance of Escherichia coli overexpressing small molecule heat shock protein IbpA

[0103] The recombinant Escherichia coli ibpA / op introduced with the recombinant expression vector and the Escherichia coli WT / op introduced with the empty vector in Example 1 were inoculated into LB liquid culture medium for activation, and cultured at 32°C and 200 rpm for 48 h, wherein 50 μg / mL kanamycin was added to the culture medium containing ibpA / op; the activated bacterial solution was transferred to a new LB liquid culture medium at an inoculation amount of 2% of the volume of the new culture medium, and cultured at 32°C and 200 rpm for 24 h; the OD of the bacterial solution was measured at a wavelength of 600 nm. 600 value, adjust the ibpA / op and WT / op bacterial solutions to the same concentration, OD 600 The value is 1.0.

[0104] The ibpA / op bacterial solution and WT / op bacterial solution adjusted to the same concentration were kept at 85℃ for 10 minutes, and the survival rate of the strains at high temperature was tested. The results are as follows Fig.11 As shown, the average survival rate of ibpA / op was 15%, and the survival rate of WT / op was 0. It can be seen that the recombinant Escherichia coli overexpressing the small molecule heat shock protein IbpA has a higher resistance to high temperature.

[0105] Experimental Example 6:

[0106] Test on the pH resistance of Escherichia coli overexpressing small heat shock protein IbpA

[0107] Different from Experimental Example 5, in this Experimental Example, the ibpA / op bacterial solution and the WT / op bacterial solution adjusted to the same concentration were centrifuged at 8000 rpm for 5 min, the bacteria were collected, and then resuspended in LB liquid culture medium with pH 5.0 and pH 11.0, respectively, and cultured at 32°C for 2 h, 4 h, and 8 h, respectively, to detect the survival rate of the strains under high acidity and high alkalinity conditions. The results are shown in Table 6, Fig.12 and Fig.13 As shown, it can be seen that the recombinant Escherichia coli overexpressing the small molecule heat shock protein IbpA has a higher resistance to high acidity and high alkalinity.

[0108] Table 6. Average survival rate of Escherichia coli strains under high acid and alkalinity conditions

[0109]

[0110] Experimental Example 7:

[0111] Resistance test of Escherichia coli overexpressing small molecule heat shock protein IbpA to salt ions

[0112] Different from Experimental Example 5, in this experimental example, the ibpA / op bacterial solution and the WT / op bacterial solution adjusted to the same concentration were centrifuged at 8000 rpm for 5 min, the bacteria were collected, and then resuspended in LB liquid culture medium with a NaCl concentration of 5% (w / w), and cultured at 32°C for 2h, 4h, and 8h, respectively, to detect the survival rate of the strain under high salinity conditions. The results are shown in Tables 7 and Fig.14 As shown, it can be seen that the recombinant Escherichia coli overexpressing the small molecule heat shock protein IbpA has a higher tolerance to high salinity.

[0113] Table 7. Average survival rate of E. coli strains under high salinity conditions

[0114] Survival rate (%) 2h 4h 8h WT / op 55 44 1 ibpA / op 62 57 42

[0115] Experimental Example 8:

[0116] Resistance test of Escherichia coli overexpressing small molecule heat shock protein IbpA to heavy metal ions and furfural

[0117] Different from Experimental Example 5, in this experimental example, the ibpA / op bacterial solution and the WT / op bacterial solution adjusted to the same concentration were centrifuged at 8000 rpm for 5 min, the bacterial cells were collected, and then chromatographed with Cr-containing 6+ (450mg / L), Pb 2+ (350mg / L), Cd2+ The strains were resuspended in LB liquid medium containing heavy metal ions (200 mg / L) and furfural (6 g / L) and cultured for 4 h. The survival rate of the strains under heavy metal ions and the organic compound furfural was detected. The results are shown in Table 8 and Figures 15 to 18 As shown, it can be seen that the recombinant Escherichia coli overexpressing the small molecule heat shock protein IbpA has a higher tolerance to heavy metal ions and the organic compound furfural.

[0118] Table 8. Average survival rate of Escherichia coli strains under heavy metal ions and organic compound furfural

[0119] Survival rate (%) <![CDATA[Cr 6+ (450mg / L)]]> <![CDATA[Pb 2+ (350mg / L)]]> <![CDATA[Cd 2+ (200mg / L)]]> Furfural (6g / L) WT / op 2 1 1 20 ibpA / op 17 19 12 42

[0120] Comparative Example 1:

[0121] The small heat shock protein IbpA from Escherichia coli S17-1 and the small heat shock protein IbpA from Rhodobacter sphaeroides ATCC 17023 were overexpressed in Rhodobacter sphaeroides ATCC 17023, and were named "ibpA (E.coli) / op" and "ibpA / op", respectively; wherein the nucleotide sequence of the small heat shock protein IbpA from Escherichia coli S17-1 is shown in SEQ ID NO.5;

[0122] The same concentration of ibpA (E. coli) / op and ibpA / op bacterial solutions were kept at 85 ° C for 10 minutes, and the survival rate of the recombinant strains after the small molecule heat shock protein IbpA from different sources was overexpressed in the host bacteria was detected at high temperature; the results are as follows Fig.19 As shown, the average survival rate of ibpA / op was 22%, while the average survival rate of ibpA(E.coli) / op was only 8%. This shows that the small molecule heat shock protein IbpA derived from Rhodobacter sphaeroides ATCC 17023 has higher resistance to high temperature.

[0123] In summary, the present invention provides a small molecule heat shock protein IbpA derived from Rhodobacter sphaeroides ATCC 17023 and capable of improving the environmental tolerance of host bacteria. After overexpressing it in the host bacteria, the resistance of the obtained recombinant bacteria to stress conditions such as high temperature, high acid, high alkali, high salt, high concentration of metal ions and high concentration of furfural is greatly increased, and the survival ability of the host bacteria under various adverse conditions can be significantly improved.

Claims

1. Application of a small molecule heat shock protein IbpA in improving the environmental tolerance of a host bacterium, wherein the small molecule heat shock protein IbpA is derived from Rhodobacter sphaeroides ATCC 17023, and its amino acid sequence is shown in SEQ ID NO.2; in, The host bacteria is Rhodobacter sphaeroides or Escherichia coli.

2. The use according to claim 1, characterized in that The nucleotide sequence of the gene ibpA encoding the small molecule heat shock protein IbpA is shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The method is to clone the small molecule heat shock protein encoding gene ibpA in Rhodobacter sphaeroides ATCC 17023 and construct a recombinant expression vector, and then transfer the recombinant expression vector into a host bacterium, so that the host bacterium overexpresses the small molecule heat shock protein IbpA.

4. The use according to claim 1, characterized in that The specific steps include: S1, total DNA extracted from Rhodobacter sphaeroides ATCC 17023; S2, using the extracted total DNA as a template, designing primers, and obtaining the small molecule heat shock protein encoding gene ibpA, i.e., the cloned product, by PCR amplification; the primers are: Upstream primer: 5'-CGGGGTACCATGCGTAGCTATGATTTCTCGCCGC-3', SEQ ID NO. 3; Downstream primer: 5'-CCCAAGCTTTCAGGCCTCGACCGGCTCCTTCAC-3', SEQ ID NO. 4; S3, digesting the cloned product and inserting it into the digested plasmid vector to construct a recombinant expression vector; S4, the recombinant expression vector is transformed into the host bacteria by conjugation transfer, and the small molecule heat shock protein IbpA is overexpressed to improve the environmental tolerance of the host bacteria.

5. The use according to claim 4, characterized in that The PCR amplification reaction conditions in S2 are: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 70°C for 15 s, extension at 72°C for 2 min, repeated for 35 cycles; extension at 72°C for 5 min, and cooling to 4°C.

6. The use according to claim 4, characterized in that The enzyme digestion method in S3 is to use restriction endonucleases Kpn I and Hind III for digestion.

7. The use according to claim 4, characterized in that The plasmid vector in S3 is pBBR1MCS-2.

8. The use according to claim 4, characterized in that The transformation method of the recombinant expression vector in S4 is: (1) preparing a host bacterial solution and a donor bacterial solution containing a recombinant expression vector, washing the bacterial cells and then resuspending them to obtain a host bacterial resuspended solution and a donor bacterial resuspended solution; (2) The host bacterial re-suspended liquid and the donor bacterial re-suspended liquid are mixed at a bacterial concentration ratio of 1:3 to 7, and the mixed bacterial liquid is spotted on a 0.22 μm sterile filter membrane placed on the corresponding solid culture medium, and statically cultured to allow conjugation transfer to occur, and the host bacteria containing the recombinant expression vector are screened.

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