CN116925196A - Application of small molecule heat shock protein RSP_1572 in improving environmental tolerance of host bacteria - Google Patents

Application of small molecule heat shock protein RSP_1572 in improving environmental tolerance of host bacteria Download PDF

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CN116925196A
CN116925196A CN202310729626.5A CN202310729626A CN116925196A CN 116925196 A CN116925196 A CN 116925196A CN 202310729626 A CN202310729626 A CN 202310729626A CN 116925196 A CN116925196 A CN 116925196A
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rsp
heat shock
shock protein
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杨飞越
亓正良
韩郁杰
叶亦珂
刘新利
董蝶
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Qilu University of Technology
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Abstract

The invention provides an application of a small molecular heat shock protein RSP_1572 in improving environmental tolerance of host bacteria, belongs to the technical field of genetic engineering, and is characterized in that a small molecular heat shock protein coding gene in rhodobacter sphaeroides ATCC 17023 is cloned and an expression vector is constructed, the expression vector is transferred into host bacteria, so that the host bacteria overexpress the small molecular heat shock protein RSP_1572, and the nucleotide sequence of the coding gene of the small molecular heat shock protein RSP_1572 is shown as SEQ ID NO. 1. According to the invention, through over-expressing the small molecular heat shock protein RSP_1572 in the host bacteria, the resistance of the obtained recombinant bacteria under different stress conditions is greatly increased, and the survivability of the host bacteria under various adverse conditions can be improved.

Description

Application of small molecule heat shock protein RSP_1572 in improving environmental tolerance of host bacteria
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of a small molecule heat shock protein RSP_1572 in improving environmental tolerance of host bacteria.
Background
During the culture process of the microorganism, the microorganism can normally grow only under proper external conditions and proper nutrient substances, and if the external conditions are not proper, the microorganism can be influenced by stress factors such as insufficient substrates, cold and hot, acid and alkali, osmotic pressure and the like, and the factors can inhibit the growth of the microorganism and even cause the death of the microorganism and inhibit or reduce the generation of metabolites of the microorganism; acid stress, alkali stress, high temperature stress, oxygen stress and the like are stresses faced by common microbial cells, so that the stress resistance of the microorganisms is improved, the survival of the microorganisms in an adverse environment is facilitated, and the normal vital activities of the cells are maintained; in addition, the high temperature stress resistance of the industrial strain is improved, the use of cooling water can be effectively reduced, the energy consumption and the production cost are reduced, and meanwhile, the probability of bacteria contamination can be effectively reduced; in order to improve the heat resistance of the strain, the traditional breeding technology such as high-temperature domestication, natural breeding, mutation breeding and other methods play an important role, but have the defects of low efficiency, nondirectionality and the like.
The temperature has profound effects on the microbial evolution, and is one of important external factors for generating microbial diversity; after long-term evolution, the microbiota produced thermophilic, mesophilic and psychrophilic microorganisms, respectively; mesophilic microorganisms are also called mesophilic microorganisms, most common microorganisms belong to the category, and although the mesophilic microorganisms can only survive normally at a conventional temperature, when the temperature of the surrounding environment changes, the mesophilic microorganisms can activate some heat shock proteins in thalli and the expression of transcription factors for regulating and controlling the stress reaction of the thalli so as to ensure the normal growth and metabolism of thalli cells at a higher temperature; the heat shock proteins are important biological resources, and have important application values in the fields of food industry, environmental protection, medical health and the like in the future.
Rhodobacter sphaeroides is a photosynthetic bacterium which does not produce oxygen, has application in the fields of food, medicine, environmental protection, agriculture and the like, and has higher research and application values. In the field of food medicine, rhodobacter sphaeroides can be used for producing coenzyme Q10, synthesizing lycopene, carotenoid and the like; in the field of environmental protection, rhodobacter sphaeroides has stronger nitrogen and phosphorus metabolism capability, and can be used for degrading organophosphorus pesticides and enriching heavy metals; in the agricultural field, rhodobacter sphaeroides can be used as foliar fertilizer, and 5-aminolevulinic acid is synthesized by using rhodobacter sphaeroides to promote plant maturation.
In order to enhance the tolerance of cells to damage, maintain the normal metabolic function of cells and improve the viability of organisms in adverse circumstances, the invention aims to utilize a transgenic technology to transfer the coding gene of the small molecule heat shock protein RSP_1572 in rhodobacter sphaeroides ATCC 17023 into a strain for over-expression so as to improve the tolerance of rhodobacter sphaeroides and other host bacteria to adverse environments.
Disclosure of Invention
Aiming at the problems, the invention provides application of a small molecular heat shock protein RSP_1572 in improving environmental tolerance of host bacteria, cloning and constructing a recombinant expression vector of a coding gene of the small molecular heat shock protein RSP_1572, and transferring the recombinant expression vector into the host bacteria to overexpress the small molecular heat shock protein RSP_1572 so as to improve the resistance of the host bacteria under different stress conditions.
The technical scheme of the invention is as follows:
an application of a small molecular heat shock protein RSP_1572 in improving environmental tolerance of host bacteria, wherein the small molecular heat shock protein RSP_1572 is derived from rhodobacter sphaeroides ATCC 17023, and the amino acid sequence of the small molecular heat shock protein RSP_1572 is shown as SEQ ID NO. 2.
Preferably, the nucleotide sequence of the encoding gene of the small molecule heat shock protein RSP_1572 is shown as SEQ ID NO. 1.
Preferably, the host bacterium is rhodobacter sphaeroides or escherichia coli.
Preferably, the application of the small molecule heat shock protein RSP_1572 in improving the environmental tolerance of host bacteria is to clone the coding gene of the small molecule heat shock protein RSP_1572 in rhodobacter sphaeroides ATCC 17023 and construct a recombinant expression vector, and transfer the recombinant expression vector into host bacteria to enable the host bacteria to overexpress the small molecule heat shock protein RSP_1572.
Preferably, the application of the small molecule heat shock protein RSP_1572 in improving the environmental tolerance of host bacteria comprises the following steps:
s1, extracting total DNA from rhodobacter sphaeroides ATCC 17023;
s2, designing a primer by taking the extracted total DNA as a template, and obtaining a coding gene of the small molecule heat shock protein RSP_1572, namely a cloning product, through PCR amplification; the primer is as follows:
an upstream primer: 5'-CCCAAGCTTATGACCAAACTGACTTTCGGGGG-3', SEQ ID No.3;
a downstream primer: 5'-GGACTAGTTCATTGCTCGACTCCTTCCTTTATC-3', SEQ ID No.4;
s3, inserting the cloned product after enzyme digestion into the plasmid vector after enzyme digestion to construct a recombinant expression vector;
s4, transforming the recombinant expression vector into host bacteria by adopting a joint transfer mode, and overexpressing the small molecule heat shock protein RSP_1572 to improve the environmental tolerance of the host bacteria.
Preferably, the PCR amplification reaction conditions in S2 are: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 70℃for 15s, extension at 72℃for 2min, 35 cycles were repeated; extending at 72 ℃ for 5min, and cooling to 4 ℃.
Preferably, the cleavage method in S3 is cleavage with restriction enzymes HindIII and SpeI.
Preferably, the plasmid vector in S3 is pBBR1MCS-2.
Preferably, the transformation method of the recombinant expression vector in S4 is as follows:
(1) Preparing a host bacterium liquid and a donor bacterium liquid containing a recombinant expression vector, washing thalli, and then re-suspending to obtain a host bacterium re-suspension bacterium liquid and a donor bacterium re-suspension bacterium liquid;
(2) Mixing the host bacteria re-suspension and donor bacteria re-suspension according to a bacterial concentration ratio of 1:3-7, dibbling the mixed bacterial solution on a 0.22 mu m sterile filter membrane placed on a corresponding solid culture medium, standing for culture to enable the bacterial solution to be subjected to joint transfer, and screening to obtain the host bacteria containing the recombinant expression vector.
A recombinant expression vector, which contains the coding gene of the small molecule heat shock protein RSP_1572.
A recombinant bacterium comprising the recombinant expression vector or the encoding gene of the small molecule heat shock protein RSP_1572.
Preferably, the host bacteria of the recombinant bacteria are rhodobacter sphaeroides or escherichia coli.
In the early research, the invention discovers that after rhodobacter sphaeroides ATCC 17023 is subjected to high-temperature heat shock treatment (75 ℃ heat shock for 10 min), the expression level of the encoding gene of the small molecule heat shock protein RSP_1572 in the bacterium body is obviously up-regulated, so that rhodobacter sphaeroides ATCC 17023 bacterium body has high temperature resistance, and the higher the expression quantity of the small molecule heat shock protein RSP_1572 is, the more obvious the differential expression is, and the stronger the capacity of rhodobacter sphaeroides ATCC 17023 against heat stress is; further research shows that the small molecule heat shock protein RSP_1572 in rhodobacter sphaeroides ATCC 17023 may enhance the tolerance degree of cells to damage, maintain the normal metabolic function of the cells and have close relation with the viability of organisms in adverse conditions. Therefore, the coding gene of the small molecular heat shock protein RSP_1572 in rhodobacter sphaeroides ATCC 17023 is over-expressed in host bacteria by utilizing a transgenic technology, so that the heat stress resistance and the tolerance to other adverse environments of the host bacteria are improved.
The invention has the beneficial effects that: according to the invention, through over-expressing the small molecular heat shock protein RSP_1572 in the host bacteria, the resistance of the obtained recombinant bacteria under the stress conditions of high temperature, high acid, high alkali, high salt, high concentration metal ions, high concentration furfural and the like is greatly increased, and the survivability of the host bacteria under various adverse conditions can be improved.
Drawings
FIG. 1 is a schematic diagram of the construction of a RSP_1572 recombinant expression vector;
FIG. 2 shows a screening process of RSP_1572 over-expressed engineering bacteria, wherein: (a) an amplified electrophoresis pattern of the gene encoding RSP_1572; (b) FIG. is a pBBR1MCS-2 plasmid enzyme digestion electrophoresis; (c) performing enzyme digestion verification on the recombinant expression vector to obtain an electrophoresis chart; FIG. d shows PCR amplification electrophoresis of recombinant E.coli.
FIG. 3 shows the survival rates of rhodobacter sphaeroides wild-type bacteria and RSP_1572 overexpressing engineering bacteria under high temperature conditions (heat shock at 85 ℃ for 10 min);
FIG. 4 shows the survival rates of rhodobacter sphaeroides wild-type bacteria and RSP_1572 overexpressing engineering bacteria under high acid conditions (pH 5.0);
FIG. 5 shows the survival rates of rhodobacter sphaeroides wild-type bacteria and RSP_1572 overexpressing engineering bacteria under high alkaline conditions (pH 11.0);
FIG. 6 shows the survival rate of rhodobacter sphaeroides wild-type bacteria and RSP_1572 overexpressing engineering bacteria under high salt conditions (salinity 5%);
FIG. 7 shows the high concentration Cr of rhodobacter sphaeroides wild strain and RSP_1572 overexpressing engineering strain 6+ Survival under conditions (350 mg/L);
FIG. 8 shows the presence of rhodobacter sphaeroides wild strain and RSP_1572 over-expressed engineering bacteria at high Pb concentration 2+ Survival under conditions (650 mg/L);
FIG. 9 shows the high concentration Cd of rhodobacter sphaeroides wild strain and RSP_1572 over-expressed engineering strain 2+ Survival under conditions (350 mg/L);
FIG. 10 shows the survival rate of rhodobacter sphaeroides wild-type bacteria and RSP_1572 over-expressed engineering bacteria under high concentration furfural conditions (40 g/L);
FIG. 11 shows the survival rates of E.coli wild-type bacteria and RSP_1572 overexpressing engineering bacteria under high temperature conditions (heat shock at 85 ℃ for 10 min);
FIG. 12 shows the survival rates of E.coli wild-type bacteria and RSP_1572 overexpressing engineering bacteria under high acid conditions (pH 5.0);
FIG. 13 shows the survival rates of E.coli wild-type bacteria and RSP_1572 overexpressing engineering bacteria under high alkaline conditions (pH 11.0);
FIG. 14 shows the survival rate of E.coli wild-type bacteria and RSP_1572 overexpressing engineering bacteria under high salt conditions (salinity 5%);
FIG. 15 shows the high concentration Cr of E.coli wild strain and RSP_1572 overexpressing engineering bacteria 6+ Survival under conditions (450 mg/L);
FIG. 16 shows that E.coli wild strain and RSP_1572 overexpressing engineering strain are in high Pb concentration 2+ Survival under conditions (350 mg/L);
FIG. 17 shows the presence of E.coli wild strain and RSP_1572 over-expressed engineering bacteria at high Cd concentration 2+ Survival rate under conditions (200 mg/L);
FIG. 18 shows survival rates of E.coli wild bacteria and RSP_1572 overexpressing engineering bacteria under high concentration furfural conditions (6 g/L);
FIG. 19 shows the survival rates of R.sphaeroides-derived RSP_1572 and E.coli-derived IbpB under high temperature conditions (heat shock at 85℃for 10 min) after overexpression in R.sphaeroides.
Detailed Description
The following description is made in connection with specific embodiments:
example 1:
cloning of small molecule heat shock protein RSP_1572 encoding gene and construction of recombinant expression vector
S1, extracting total DNA from rhodobacter sphaeroides ATCC 17023 by using a genome DNA extraction kit;
s2, designing a primer by taking the extracted total DNA as a template, and amplifying by PCR to obtain a coding gene of a small molecule heat shock protein RSP_1572, namely a clone product, wherein an agarose gel electrophoresis diagram of the clone product is shown in a diagram (a) in FIG. 2; the PCR amplification reaction conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 70℃for 15s, extension at 72℃for 2min, 35 cycles were repeated; extending at 72 ℃ for 5min, and cooling to 4 ℃;
the primers used were:
an upstream primer: 5'-CCCAAGCTTATGACCAAACTGACTTTCGGGGG-3', SEQ ID No.3;
a downstream primer: 5'-GGACTAGTTCATTGCTCGACTCCTTCCTTTATC-3', SEQ ID No.4;
s3, carrying out double enzyme digestion on the clone product and the pBBR1MCS-2 plasmid by using restriction enzymes Hind III and Spe I respectively, and then carrying out enzyme ligation by using T4 DNA ligase, wherein an agarose gel electrophoresis diagram of the pBBR1MCS-2 plasmid after double enzyme digestion is shown in a diagram (b) in FIG. 2; the molar ratio of the cloning product to the pBBR1MCS-2 plasmid during enzyme ligation is 4:1, and the total volume of the enzyme ligation reaction is 20 mu L; inserting the cloned product into pBBR1MCS-2 plasmid by enzyme ligation, transferring the enzyme ligation product into competent cells of the escherichia coli S17-1, and obtaining a recombinant expression vector and recombinant escherichia coli S17-1 containing the recombinant expression vector through amplification and screening; wherein, agarose gel electrophoresis diagram of recombinant expression vectors HindIII and SpeI after double digestion is shown in (c) diagram of FIG. 2, colony PCR amplified agarose gel electrophoresis diagram of recombinant E.coli S17-1 is shown in (d) diagram of FIG. 2.
The nucleotide sequence of the coding gene of the small molecule heat shock protein RSP_1572 is shown as SEQ ID NO.1, and the amino acid sequence of the small molecule heat shock protein RSP_1572 is shown as SEQ ID NO. 2; a schematic diagram of the construction of the recombinant expression vector is shown in FIG. 1.
Example 2:
transformation and expression of small molecule heat shock protein RSP_1572
(1) Inoculating rhodobacter sphaeroides ATCC 17023 into an LB liquid culture medium, and culturing for 2-3 days at 32 ℃ with 200rpm under illumination or without illumination to obtain rhodobacter sphaeroides bacterial liquid;
wherein, the LB liquid medium comprises the following components: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, pH7.0;
(2) Recombinant E.coli S17-1 (donor strain) containing the recombinant expression vector prepared in example 1 was addedCulturing overnight in LB liquid medium with 50. Mu.g/mL kanamycin; transferring the bacterial liquid cultured overnight into fresh LB liquid culture medium, culturing to OD 600 About 0.5 to obtain recombinant escherichia coli bacterial liquid;
(3) Respectively taking 1mL of rhodobacter sphaeroides bacterial liquid in the step (1) and recombinant escherichia coli bacterial liquid in the step (2), centrifuging at 5000rpm for 4min, and discarding the supernatant; re-suspending the thallus with 1mL fresh LB liquid culture medium, centrifuging at 5000rpm for 4min, discarding supernatant, repeating the process twice, and re-suspending the thallus with 100 μl fresh LB liquid culture medium to obtain rhodobacter sphaeroides re-suspension and recombinant Escherichia coli re-suspension;
(4) Mixing the rhodobacter sphaeroides resuspension bacteria liquid and the recombinant escherichia coli resuspension bacteria liquid in the step (3) according to the ratio of the bacteria concentration of 1:4, and spotting the mixed bacteria liquid on a 0.22 mu m sterile filter membrane placed on an LB solid medium containing 25 mu g/mL kanamycin and 50 mu g/mL potassium tellurite; culturing at 32deg.C for 1 day under light or no light to allow joint transfer;
(5) Scraping the lawn on the filter membrane in the step (4), re-suspending the lawn by using fresh LB liquid culture medium, coating the re-suspension on LB solid culture medium containing 25 mug/mL kanamycin and 50 mug/mL potassium tellurite, and standing and culturing the re-suspension at 32 ℃ for more than 3 days until small black colonies appear on the culture medium;
wherein, the LB solid medium comprises the following components: 10g/L of peptone, 5g/L of yeast extract powder, 10g/L of sodium chloride, 15g/L of agar powder and pH7.0;
(6) Picking out black colonies in the step (5), re-suspending with fresh LB liquid medium, diluting, coating onto LB solid medium containing 25 mug/mL kanamycin and 50 mug/mL potassium tellurite, standing at 32 ℃ for 3 days or more until single colonies appear on a flat plate, picking out colony to extract plasmids, and identifying to obtain recombinant rhodobacter sphaeroides;
inoculating the recombinant rhodobacter sphaeroides obtained by screening into LB liquid medium containing 25 mug/mL kanamycin for culture, and then implementing overexpression of the small molecule heat shock protein RSP_1572; the pBBR1MCS-2 plasmid is expressed constitutively, and is not required to be induced by adding an inducer.
Experimental example 1:
test of temperature resistance of rhodobacter sphaeroides over-expressed small molecule heat shock protein RSP_1572
Taking recombinant rhodobacter sphaeroides RSP_1572/op introduced with the recombinant expression vector and rhodobacter sphaeroides WT/op introduced with the empty vector in the example 2, respectively inoculating the recombinant rhodobacter sphaeroides RSP_1572/op and the rhodobacter sphaeroides WT/op into LB liquid culture medium for activation, and carrying out shaking culture at 32 ℃ and 200rpm for 48 hours, wherein 25 mug/mL kanamycin is added into the culture medium containing the RSP_1572/op; transferring the activated bacterial liquid into a new LB liquid culture medium according to the inoculum size of 2% of the volume of the new culture medium, and carrying out shaking culture for 24 hours at the temperature of 32 ℃ and at the speed of 200 rpm; measuring OD of the bacterial liquid under the condition of 600nm wavelength 600 The value is adjusted to the same concentration, OD, of the RSP_1572/op bacterial liquid and the WT/op bacterial liquid 600 The value was 1.0.
The RSP_1572/op bacterial liquid and the WT/op bacterial liquid which are adjusted to the same concentration are respectively kept at the high temperature of 85 ℃ for 10min, and the survival rate of the strain at the high temperature is detected, and the results are shown in Table 1 and FIG. 3;
TABLE 1 average survival rate of rhodobacter sphaeroides strains under high temperature conditions
Strain WT/op RSP_1572/op
Survival (%) 0.5 24
As can be seen from Table 1 and FIG. 3, the recombinant rhodobacter sphaeroides RSP_1572/op has significantly higher adaptability to high temperatures than the control strain; after 10min of high temperature stress at 85 ℃, the average survival rate of RSP_1572/op can reach 24%, and the average survival rate of WT/op is less than 1%, which indicates that the recombinant rhodobacter sphaeroides overexpressing the small molecule heat shock protein RSP_1572 has higher resistance to high temperature.
Experimental example 2:
resistance test of rhodobacter sphaeroides over-expressed small molecule heat shock protein RSP_1572 to pH
(1) High acidity condition
In this experimental example, the rsp_1572/op bacterial liquid and the WT/op bacterial liquid, which were adjusted to have the same concentration, were centrifuged at 8000rpm for 5min, respectively, and the bacterial cells were collected, resuspended in LB liquid medium having pH of 5.0, and cultured at 32 ℃ for 2h, 4h, 8h, respectively, and the viability of the strain under high acidity conditions was examined, and the results are shown in table 2 and fig. 4;
TABLE 2 average survival of rhodobacter sphaeroides strains under high acidity conditions
Survival (%) 2h 4h 8h
WT/op 19 21 4
RSP_1572/op 19 17 14
As can be seen from Table 2 and FIG. 4, although the survival rate of RSP_1572/op was smaller than that of WT/op when cultured for 4 hours under high acidity conditions, RSP_1572/op gradually became adaptive to the high acidity environment after a certain period of culture under high acidity conditions, and the strain further proliferated, so that the average survival rate of RSP_1572/op remained at 14% after 8 hours of culture under high acidity conditions, which was 10% higher than that of 4% of WT/op, indicating that recombinant rhodobacter sphaeroids overexpressing small molecule heat shock protein RSP_1572 had higher resistance to high acidity.
(2) High alkalinity condition
In this experimental example, the rsp_1572/op bacterial liquid and the WT/op bacterial liquid, which were adjusted to have the same concentration, were centrifuged at 8000rpm for 5min, respectively, and the bacterial cells were collected, resuspended in LB liquid medium having pH of 11.0, and cultured at 32 ℃ for 2h, 4h, 8h, respectively, and the viability of the strain under high alkalinity condition was examined, and the results are shown in table 3 and fig. 5;
TABLE 3 average survival of rhodobacter sphaeroides strains under high alkalinity conditions
As can be seen from table 3 and fig. 5, the recombinant rhodobacter sphaeroides rsp_1572/op had overall higher tolerance to high alkalinity than the control strain WT/op; the survival rate of RSP_1572/op is far higher than that of WT/op when cultured for 2-8 h under the high alkalinity condition with pH of 11.0, and especially after being cultured for 8h under the high alkalinity condition with pH of 11.0, the average survival rate of RSP_1572/op is 25 percent, and the average survival rate of WT/op is only 2 percent, which indicates that the recombinant rhodobacter sphaeroides over-expressing the small molecular heat shock protein RSP_1572 has higher resistance to high alkalinity.
Experimental example 3:
resistance test of rhodobacter sphaeroides over-expressed small molecule heat shock protein RSP_1572 on salt ions
In this experimental example, the rsp_1572/op bacterial liquid and the WT/op bacterial liquid, which were adjusted to have the same concentration, were centrifuged at 8000rpm for 5min, and the bacterial cells were collected, resuspended in LB liquid medium having 5% (w/w) NaCl concentration, and cultured at 32 ℃ for 2 hours, 4 hours, and 8 hours, respectively, and the viability of the strain under high salinity was examined, and the results are shown in table 4 and fig. 6;
TABLE 4 average survival of rhodobacter sphaeroides strains under high salinity conditions
Survival (%) 2h 4h 8h
WT/op 67 51 2
RSP_1572/op 30 50 140
As can be seen from Table 4 and FIG. 6, although the survival rate of RSP_1572/op is smaller than that of WT/op when cultured for 2-4 h under high salinity conditions, after 8h of culture under high salinity conditions, RSP_1572/op gradually adapts to the high salinity environment, the average survival rate of RSP_1572/op is obviously improved to 140%, and the average survival rate of WT/op is only 2%, which indicates that the recombinant rhodobacter sphaeroides over-expressing small molecule heat shock protein RSP_1572 has higher tolerance to high salinity.
Experimental example 4:
test of resistance of rhodobacter sphaeroides over-expressed small molecule heat shock protein RSP_1572 to heavy metal ions and furfural
In the present experimental example, the RSP_1572/op bacterial liquid and the WT/op bacterial liquid, which were adjusted to have the same concentration, were centrifuged at 8000rpm for 5min, respectively, and the bacterial cells were collected and then treated with Cr-containing bacteria, respectively 6+ (350mg/L)、Pb 2+ (650mg/L)、Cd 2+ (350 mg/L) and furfural (40 g/L) and culturing for 4 hours at 32 ℃, and detecting the survival rate of the strain under heavy metal ions and organic compound furfural, wherein the results are shown in Table 5 and FIGS. 7-10;
TABLE 5 average survival of rhodobacter sphaeroides strains under heavy metal ions and organic Furfural
As can be seen from table 5 and fig. 7 to 10, the survival rate of rsp_1572/op is higher than WT/op under the influence of heavy metal ions and organic compound furfural, which indicates that recombinant rhodobacter sphaeroides overexpressing small molecule heat shock protein rsp_1572 has higher tolerance to heavy metal ions and organic compound furfural.
Experimental example 5:
test of temperature resistance of E.coli over-expressed small molecule heat shock protein RSP_1572
Taking recombinant escherichia coli RSP_1572/op introduced with a recombinant expression vector and escherichia coli WT/op introduced with an empty vector in the example 1, respectively inoculating the recombinant escherichia coli RSP_1572/op and the escherichia coli WT/op into an LB liquid culture medium for activation, and carrying out shaking culture at 32 ℃ and 200rpm for 48 hours, wherein 50 mug/mL kanamycin is added into the culture medium containing the RSP_1572/op; transferring the activated bacterial liquid with an inoculum size of 2% of the volume of the new culture mediumCulturing in new LB liquid medium at 32deg.C and 200rpm with shaking for 24 hr; measuring OD of the bacterial liquid under the condition of 600nm wavelength 600 The value is adjusted to the same concentration, OD, of the RSP_1572/op bacterial liquid and the WT/op bacterial liquid 600 The value was 1.0.
The rsp_1572/op bacterial liquid and the WT/op bacterial liquid which are adjusted to the same concentration are respectively kept at the high temperature of 85 ℃ for 10min, and the survival rate of the strain at the high temperature is detected, and the result is shown in fig. 11, wherein the average survival rate of the rsp_1572/op is 34%, the average survival rate of the WT/op is 0.4%, and the recombinant escherichia coli which overexpresses the small molecule heat shock protein rsp_1572 is higher in resistance to the high temperature.
Experimental example 6:
resistance test of E.coli over-expressed small molecule heat shock protein RSP_1572 to pH
In contrast to experimental example 5, the experimental example was carried out by centrifuging RSP_1572/op bacterial liquid and WT/op bacterial liquid adjusted to the same concentration at 8000rpm for 5min, collecting bacterial cells, then re-suspending with LB liquid medium having pH of 5.0 and pH of 11.0, respectively, culturing at 32deg.C for 2h, 4h, 8h, and detecting survival rate of strain under high acidity and high alkalinity conditions, and the results are shown in Table 6, FIG. 12 and FIG. 13, wherein recombinant E.coli overexpressing small molecule heat shock protein RSP_1572 has high resistance to high acidity and high alkalinity.
TABLE 6 average survival of E.coli strains under high acid and alkalinity conditions
Experimental example 7:
resistance test of E.coli over-expressed small molecule heat shock protein RSP_1572 to salt ions
In contrast to experimental example 5, the recombinant E.coli over-expressing the small molecule heat shock protein RSP_1572 was found to have high tolerance to high salinity as shown in Table 7 and FIG. 14, in which the RSP_1572/op bacterial solution and the WT/op bacterial solution, which were adjusted to have the same concentration, were centrifuged at 8000rpm for 5min, respectively, and the bacterial cells were collected and resuspended in LB liquid medium having NaCl concentration of 5% (w/w), and cultured at 32℃for 2h, 4h, and 8h, respectively.
TABLE 7 average survival of E.coli strains under high salinity conditions
Survival (%) 2h 4h 8h
WT/op 65 50 1
RSP_1572/op 30 42 92
Experimental example 8:
resistance test of escherichia coli over-expressed small molecule heat shock protein RSP_1572 on heavy metal ions and furfural
In the present example, the RSP_1572/op solution and the WT/op solution, which were adjusted to the same concentration, were centrifuged at 8000rpm for 5min, respectively, and the cells were collected and then treated with Cr-containing bacteria 6+ (450mg/L)、Pb 2+ (350mg/L)、Cd 2+ LB liquid medium (200 mg/L) and furfural (6 g/L) were resuspended and cultured for 4 hours, and the survival rate of the strain under heavy metal ions and organic compound furfural was detected, and the results are shown in Table 8 and Table 2FIGS. 15-18 show that recombinant E.coli over-expressing the small molecule heat shock protein RSP_1572 has higher tolerance to heavy metal ions and organic compound furfural.
TABLE 8 average survival of E.coli strains under heavy metal ions and organic Furfural Compounds
Survival (%) Cr 6+ (450mg/L) Pb 2+ (350mg/L) Cd 2+ (200mg/L) Furfural (6 g/L)
WT/op 2 1 1 20
RSP_1572/op 16 24 16 52
Comparative example 1:
the small molecule heat shock protein IbpB from the escherichia coli S17-1 and the small molecule heat shock protein RSP_1572 from the rhodobacter sphaeroides ATCC 17023 are respectively over-expressed in the rhodobacter sphaeroides ATCC 17023 and are respectively named as ibpB (E.coll)/op and RSP_1572/op; wherein the amino acid sequence of the small molecule heat shock protein IbpB of Escherichia coli S17-1 is shown as SEQ ID NO. 5.
The RSP_1572/op bacterial liquid and ibpB (E.coli)/op bacterial liquid with the same concentration are respectively kept at a high temperature of 85 ℃ for 10min, and the survival rate of the recombinant strain after the overexpression of the micromolecule heat shock proteins of different sources on host bacteria at a high temperature is detected; the results are shown in FIG. 19, where the average survival rate of RSP_1572/op is 24% and the average survival rate of ibpB (E.coli)/op is 11%, showing that the small molecule heat shock protein RSP1572 derived from rhodobacter sphaeroides ATCC 17023 is more resistant to high temperatures.
In summary, the invention provides a small molecular heat shock protein RSP_1572 which is derived from rhodobacter sphaeroides ATCC 17023 and can improve the environmental tolerance of host bacteria, after the small molecular heat shock protein RSP_1572 is overexpressed 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 metal ions, high concentration furfural and the like is greatly increased, and the survivability of the host bacteria under various adverse conditions can be obviously improved.

Claims (10)

1. An application of a small molecular heat shock protein RSP_1572 in improving environmental tolerance of host bacteria, wherein the small molecular heat shock protein RSP_1572 is derived from rhodobacter sphaeroides ATCC 17023, and the amino acid sequence of the small molecular heat shock protein RSP_1572 is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the nucleotide sequence of the gene encoding the small molecule heat shock protein rsp_1572 is shown in SEQ ID No. 1.
3. The use according to claim 1, wherein the host bacterium is rhodobacter sphaeroides or escherichia coli.
4. The use according to claim 1, wherein the method of use is to clone the gene encoding the small molecule heat shock protein rsp_1572 in rhodobacter sphaeroides ATCC 17023 and construct a recombinant expression vector, and transfer the recombinant expression vector into a host bacterium, so that the host bacterium overexpresses the small molecule heat shock protein rsp_1572.
5. The application according to claim 1, wherein the application method comprises the steps of:
s1, extracting total DNA from rhodobacter sphaeroides ATCC 17023;
s2, designing a primer by taking the extracted total DNA as a template, and obtaining a small molecule heat shock protein RSP_1572 coding gene, namely a cloning product, through PCR amplification; the primer is as follows:
an upstream primer: 5'-CCCAAGCTTATGACCAAACTGACTTTCGGGGG-3', SEQ ID No.3;
a downstream primer: 5'-GGACTAGTTCATTGCTCGACTCCTTCCTTTATC-3', SEQ ID No.4;
s3, inserting the cloned product after enzyme digestion into the plasmid vector after enzyme digestion to construct a recombinant expression vector; preferably, the plasmid vector is pBBR1MCS-2;
s4, transforming the recombinant expression vector into host bacteria by adopting a joint transfer mode, and overexpressing the small molecule heat shock protein RSP_1572 to improve the environmental tolerance of the host bacteria.
6. The use according to claim 5, wherein the PCR amplification reaction conditions in S2 are: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 70℃for 15s, extension at 72℃for 2min, 35 cycles were repeated; extending at 72 ℃ for 5min, and cooling to 4 ℃;
preferably, the cleavage method in S3 is cleavage with restriction enzymes HindIII and SpeI.
7. The use according to claim 5, wherein the method of transforming the recombinant expression vector in S4 is:
(1) Preparing a host bacterium liquid and a donor bacterium liquid containing a recombinant expression vector, washing thalli, and then re-suspending to obtain a host bacterium re-suspension bacterium liquid and a donor bacterium re-suspension bacterium liquid;
(2) Mixing the host bacteria re-suspension and donor bacteria re-suspension according to a bacterial concentration ratio of 1:3-7, dibbling the mixed bacterial solution on a 0.22 mu m sterile filter membrane placed on a corresponding solid culture medium, standing for culture to enable the bacterial solution to be subjected to joint transfer, and screening to obtain the host bacteria containing the recombinant expression vector.
8. A recombinant expression vector comprising the gene encoding the small molecule heat shock protein rsp_1572 of claim 2.
9. A recombinant bacterium comprising the recombinant expression vector of claim 8 or the gene encoding the small molecule heat shock protein rsp_1572 of claim 2.
10. A recombinant bacterium according to claim 9, wherein the host bacterium of the recombinant bacterium is rhodobacter sphaeroides or escherichia coli.
CN202310729626.5A 2023-06-08 2023-06-19 Application of small molecule heat shock protein RSP_1572 in improving environmental tolerance of host bacteria Pending CN116925196A (en)

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