Application of heat shock protein gene derived from geobacillus stearothermophilus and heat-resistant engineering bacteria

Abstract

The invention belongs to the technical field of microbial engineering, and particularly relates to application of a heat shock protein gene derived from geobacillus stearothermophilus and heat-resistant engineering bacteria. According to the invention, through screening, the heat shock protein gene which is derived from over-expressed geobacillus stearothermophilus and is used for improving the heat resistance of microorganisms is obtained. The obtained heat shock protein gene with heat resistance is expressed in the same microorganism, so that the heat resistance of the microorganism is improved, the biomass of the microorganism is also improved, more importantly, the hydrocarbon decomposition capability of the microorganism is improved, and the application of the microorganism in crude oil degradation and oil displacement is promoted.

Description

Technical Field

[0001] The invention belongs to the field of microbial engineering technology, and particularly relates to the application of heat shock protein genes derived from Geobacillus stearothermophilus and heat-resistant engineering bacteria. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] Oil is a non-renewable energy source. Oil recovery technologies include primary, secondary and tertiary recovery. After primary and secondary recovery, 60%-70% of the oil remains in the formation. This is because the low reservoir permeability, high crude oil viscosity and high oil-water interfacial tension lead to high capillary forces that retain the oil in the reservoir rock. These factors are the main obstacles to effective oil recovery. Due to the inherent properties of heavy oil and oil sands, these residual oils in the formation respond poorly to traditional primary and secondary recovery methods. For this reason, tertiary oil recovery technology came into being.

[0004] Tertiary oil recovery technology, namely enhanced oil recovery (EOR) technology, is mainly divided into thermal recovery, chemical drive, gas drive and microbial drive. Microbial drive oil recovery, also known as microbial enhanced oil recovery (MEOR), uses the movement of microorganisms themselves and their metabolites to change the chemical and physical properties of reservoir rocks and crude oil, achieving the effect of displacing and stripping oil from depleted and high-water-content reservoirs, thereby extending the life of oil wells and effectively coping with the various obstacles currently faced by effective oil recovery.

[0005] MEOR technology can be divided into two types: endogenous microbial oil recovery technology, which involves injecting biological products into oil wells to activate native microorganisms, and exogenous microbial oil recovery technology, which involves introducing exogenous microorganisms to produce biosurfactants to increase oil recovery. Due to the influence of environmental pH, temperature, permeability, mineralization and other factors, it is difficult to find endogenous microorganisms suitable for microbial oil recovery technology in some oil reservoirs. Exogenous microbial oil recovery technology, which involves obtaining microorganisms with oil recovery potential through screening methods, performing modifications such as improving their oil recovery capacity and enhancing their tolerance, and then supplementing them into the formation after large-scale fermentation, is an important direction for solving the current bottleneck problem of MEOR. The inventor’s team screened a strain of bacteria that can emulsify and disperse crude oil, which was identified as Geobacillus stearothermophilus Geobacillus stearothermophilus SL-1, this bacterium can tolerate high temperatures above 60°C, and its culture fluid, metabolites after culture and bacteria after culture can all improve displacement efficiency, of which the culture fluid can improve displacement efficiency by 11.8%, and the other two displacement fluids can improve displacement efficiency by 4.5% and 3%, respectively. However, the microorganisms screened for oil recovery potential often have poor heat resistance and are difficult to withstand high temperatures in the formation, so their number is small, resulting in low oil recovery effect.

[0006] Obtaining engineered bacteria based on genetic engineering is an effective way to expand the oil-producing microbial population and accelerate shale oil extraction. Summary of the invention

[0007] In order to overcome the deficiencies of the above-mentioned prior art, the present invention provides a group of heat shock protein genes derived from Bacillus stearothermophilus and their applications. In the present invention, through screening, overexpressed heat shock protein genes derived from Bacillus stearothermophilus that improve the heat resistance of microorganisms are obtained. The obtained heat shock protein genes with heat resistance are expressed in the same microorganism, which not only improves the heat resistance of the microorganism, but also increases the biomass of the microorganism, and more importantly, improves the hydrocarbon-degrading ability of the microorganism, promoting the application of microorganisms in crude oil degradation and oil recovery. The problem of poor heat resistance of microorganisms in the prior art is solved, filling the gap of crude oil degradation microorganisms.

[0008] To achieve the above objectives, one or more embodiments of the present invention provide the following technical solutions: One of the technical solutions provided by the present invention is: application of a heat shock protein gene derived from Geobacillus stearothermophilus in any of the following: (1) Application in improving the heat resistance of microorganisms; (2) Applications to increase microbial biomass; (3) Application of improving the hydrocarbon decomposition capacity of microorganisms; (4) Application of thermotolerant engineered bacteria; The heat shock protein includes any one or more of DnaK, DnaJ and HtpX; The gene sequences of DnaK, DnaJ and HtpX are shown in SEQ ID NO.1-3 respectively.

[0009] The present invention also provides a heat-resistant engineered bacterium, which comprises the heat shock protein gene or the expression vector; two or more heat shock protein genes or their expression vectors are connected in series and then introduced into a host cell.

[0010] Preferably, the expression vector further comprises a promoter; the nucleotide sequence of the promoter is shown in SEQ ID NO.4.

[0011] Preferably, the host is selected from any one of bacteria, fungi and archaea.

[0012] The present invention also provides the use of the heat-resistant engineering bacteria in the degradation of crude oil or microbial oil recovery.

[0013] The present invention also provides a method for degrading crude oil or microbial oil recovery, which utilizes the bacterial body, culture fluid or fermented metabolites of the heat-resistant engineering bacteria to replace the displacement fluid to degrade crude oil or perform microbial oil recovery.

[0014] One or more of the above technical solutions have the following beneficial effects: Geobacillus stearothermophilus Geobacillus stearothermophilus The heat shock protein gene from SL-1 was introduced into the thermophilic Bacillus stearothermophilus in series, which significantly increased the fermentation temperature of the strain, reduced the use of cooling water, and improved economic benefits. On the other hand, it improved the tolerance of the strain, significantly increased the heat resistance temperature of the strain, promoted the renaturation and folding of the protein in the strain, and improved the stability of the protein in the strain, thereby increasing the biomass of the strain itself and developing new applications. The details are as follows: (1) The engineered bacteria SL-1-H1 provided by the present invention has a maximum tolerance temperature increased from 70°C to 85°C, which is equivalent to the temperature of shale oil reservoirs; it can survive for 120 hours at an extreme temperature of 85°C.

[0015] (2) The engineered bacteria SL-1-H1 can effectively grow using crude oil as the sole carbon source and is a dominant strain for microbial oil recovery, providing a harmless and economically feasible strategy for oil extraction. The crude oil degradation rate reached 50% after 20 days of cultivation, effectively degrading crude oil and having great application prospects.

[0016] Advantages of additional aspects of the present invention will be given in part in the following description, and in part will become obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the source plasmid map of the vector used to express stress-related proteins in Example 1 of the present invention; Figure 2 The heat shock protein transformants of Example 2 of the present invention are grown at 80°C, and from left to right are DnaK, DnaJ, HtpX transformants and transformants expressing three heat shock proteins in combination; Figure 3 This is the heat shock protein combination expression plasmid map of Example 2 of the present invention; Figure 4 This is a comparison chart of the survival rates of the wild type and the heat-resistant engineered bacteria SL-1-H1 at 85°C in Example 2 of the present invention; Figure 5 This is a growth curve of the heat-resistant engineering bacteria SL-1-H1 in Example 3 of the present invention in a crude oil degradation medium with crude oil as the sole carbon source; Figure 6 This is the degradation of crude oil by the heat-resistant engineering bacteria SL-1-H1 in Example 3 of the present invention; Figure 7 This is the change in biomass of the heat-resistant engineering bacteria SL-1-H1 in Example 3 of the present invention at different culture temperatures. DETAILED DESCRIPTION

[0018] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001, or the conditions recommended by the manufacturer's instructions.

[0019] It should be noted that the terms used herein are for describing specific embodiments only and are not intended to be limiting of exemplary embodiments according to the present invention.

[0020] Reagents and instruments: The reagents in this embodiment are mainly molecular biology experimental reagents, single-stranded nucleotides are synthesized from Shanghai Shenggong Biotechnology Co., Ltd., DNA polymerase is from Hunan Aikerui Bioengineering Co., Ltd., Gibson assembly kit is from Wuhan Abotek Biotechnology Co., Ltd., restriction endonucleases are from New England Biolabs, and antibiotics are purchased from Invitrogen. The starting strain involved in the invention is Bacillus stearothermophilus ( Geobacillus stearothermophilus SL-1 is an oil reservoir native microorganism isolated by the inventor in a block of Shengli Oilfield. The isolation and identification of the starting strain can be found in the non-patent literature DOI: 10.3724 / SP.J.1145.2015.04053 (https: / / journals.sagepub.com / doi / 10.3233 / JCM-237072?icid=int.sj-full-text.similar-articles.7). The involved Escherichia coli DH10B electroporation competent cells were purchased from Shanghai Angyu Biotechnology Co., Ltd.

[0021] The culture medium involved in the following examples is as follows: LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 1 g / L.

[0022] Recovery medium: 2×LB+0.5% glucose+0.01% BSA.

[0023] Inorganic salt medium: Na 2 HPO 4 0.6 g / L, KH 2 PO4 0.2 g / L, NaNO 3 4 g / L, CaCl 2 0.01 g / L, FeSO 4 0.01 g / L, MgSO 4 0.3 g / L, yeast extract 0.5 g / L, pH 7.2.

[0024] Seed culture medium: inorganic salt medium + 1% sucrose.

[0025] Crude Oil Degraded Medium: 1% crude oil replaces yeast extract in mineral salts medium.

[0026] Solid culture medium was prepared by adding 1.2% agar powder to the corresponding liquid culture medium.

[0027] The applied concentration of kanamycin is 10 μg / mL for Escherichia coli and 20 μg / mL for Bacillus stearothermophilus in liquid culture medium; 20 μg / mL for Escherichia coli and 100 μg / mL for Bacillus stearothermophilus in solid culture medium.

[0028] In the present invention, the primers were set using Geneious Primer software (https: / / help.geneious.com / hc / en-us), and the primer information provided by the software can be used to determine the conditions required for amplifying the fragment.

[0029] Table 1 Primer sequences involved in the present invention .

[0030] Example 1: Determination of heat resistance-related temperature stress effector proteins 1. Construction of heat stress effector protein expression plasmid The genome sequence of Bacillus stearothermophilus SL-1 was analyzed using geneious primer software (https: / / help.geneious.com / hc / en-us), and it was found that it encodes a variety of temperature stress effector proteins, including DnaK, DnaJ, HtpX, GroES, GroEL, HslO, GrpE, Hsp20, Hsp33, HemW, ClpB and ClpX.

[0031] In this embodiment, the pBBR1-Km vector (from non-patent literature doi:10.1016 / j.isci.2019.03.007) was used as the backbone for constructing the above protein overexpression plasmid. The plasmid backbone was derived from the pBBR1-kan-ccdB-hyg plasmid (Baosai Biotechnology). Figure 1As shown, the pBBR1 replicon was identified as a multi-copy replicon that stably replicates in Geobacillus stearothermophilus SL-1. km It is a constitutive promoter of the kanamycin resistance gene, which can function to initiate transcription of downstream genes under high temperature conditions. Using pBBR1-kan-ccdB-hyg plasmid (Baosai Biotechnology) as a template, pBBR1-Km-1 / pBBR1-Km-3 primers were used to amplify the pBBR1-Km linearized vector, and pkm-1 / pBBR1-Km-2 primers were used to amplify P km .

[0032] The PCR amplification system is shown in Table 2, and the amplification program is shown in Table 3: Table 2 PCR amplification system 2×PCR Mix 12.5μL 10 μM Primer-1 0.25μL 10 μM Primer-2 0.25μL Template (20ng / μL) 1μL <![CDATA[ddH 2 The]]> 11μL ; Table 3 PCR amplification program Pre-denaturation 98℃ 10 min transsexual 94℃ 15 s annealing X℃ 15 s extend 72℃ Y min Further extension 72℃ 5 min Insulation 4℃ Forever Number of cycles 35 ; For the pBBR1-Km linearized vector, X is 59 and Y is 2,4284 bp; for P km , X is 57.3, Y is 0.2, 257 bp.

[0033] The genome of the starting bacteria was used as a template and amplified by PCR. dnaK , dnaJ , xtP , groES , groEL , oeLh , gfrE , hsp20 , hsp33 , hem as well as clpB , clpX In this process, primers were designed to introduce a pair of primers into the 5' end of each gene for binding to P km Each gene was overlapped with the P promoter by overlap PCR. km The promoter was fused into a fragment, and 50 bp homology arms for homologous recombination with the vector were introduced at both ends. km Gene with promoter homology arms dnaK , amplified using P-DnaK-1 / 2 primers, X is 50.2, Y is 0.2, 1818 bp; for the DNA fragment with P km Gene with promoter homology arms dnaJ , amplified using P-DnaJ-1 / 2 primers, X is 54.5, Y is 0.2, 1146 bp; for the DNA sequence with P km Gene with promoter homology arms xtP , amplified using P-HtpX-1 / 2 primers, X is 54.9, Y is 0.2, 924 bp. km - dnaK The expression module was expressed by using primers pBBR1-Pkm-AB-1 / pBBR1-DnaK-AB-2 with P km The promoter and km Gene with promoter homology arms dnaK The template was amplified, X is 54.5, Y is 0.2, 2175bp; P with vector homology arms km - dnaJ The expression module was expressed using primers pBBR1-Pkm-AB-1 / pBBR1-DnaJ-AB-2 with P km The promoter and km Gene with promoter homology arms dnaJ The template was amplified, X is 54.5, Y is 0.2, 1502bp; P with vector homology arms km - xtP The expression module was expressed by using primers pBBR1-Pkm-AB-1 / pBBR1-HtpX-AB-2 with P km The promoter and km Gene with promoter homology arms xtP It was obtained by template amplification, X was 54.9, 0.2, and 1281 bp.

[0034] By P km The expression modules controlled were respectively cyclized with the pBBR1-Km vector by Gibson assembly and then transformed into E. coli DH10B electroporation competent cells to propagate the plasmid. The Gibson assembly system is shown in Table 4:

[0035] Table 4 Gibson assembly system ABclonal 2X MultiF Seamless Assembly Mix 10 μL pBBR1-Km vector 0.06 pmol = (0.04 × number of base pairs) ng Expression module 0.12 pmol = (0.04 × number of base pairs) ng <![CDATA[ddH 2 The]]> Make up to 20 μL ; Reaction procedure: Assemble at 50°C for 60 min; Keep at 4°C Forever; For the pBBR1-Km vector, the number of base pairs is 4284, so the usage amount is 171.36 ng, P km - dnaK Expression module, the number of base pairs is 2175, the usage is 87 ng, P km - dnaJ Expression module, base pair number is 1502, then use 60 ng, P km - xtPFor the expression module with a base pair number of 1281, 51.24 ng was used.

[0036] The specific method is as follows: (1) The Gibson assembly reaction product was transferred to a Millipore desalting membrane and desalted for 40 min before use; (2) Take the DH10B electroporated competent cells stored at -80℃ and put them in an ice bath to thaw, add the desalted Gibson assembly reaction product and gently mix by tapping the bottom of the EP tube and immediately put it in ice; (3) Use a 200 μl pipette tip to quickly transfer the competent cell-reaction product mixture to a dry, sterile 1 mm electroporation cup to avoid bubbles. Cover the cup and electroporate at 1350 V using an Eppendorf electroporator. (4) Immediately after electroporation, resuspend the cells in 1 mL of antibiotic-free LB medium and incubate for 1 h, then spread on resistant plates for culture; (5) Pick a single clone from the culture plate and inoculate it into LB medium containing kanamycin. After culturing for 16 h, extract the plasmid and confirm the correct plasmid by restriction endonuclease digestion and DNA second-generation sequencing.

[0037] 2. Transformation of temperature stress effector protein expression plasmid Each expression plasmid constructed in step 1 was electroporated into the starting strain Bacillus stearothermophilus SL-1 to verify the protein function. The specific transformation method is as follows: (1) Culture the thermophilic Bacillus SL-1 at 65°C until the OD 600 Value is about 1.0; (2) Collect the cells by centrifugation at 8000 rpm for 1 min at 4°C, wash them three times with sterile ice water as buffer at 4°C, and then concentrate them in 50 μl sterile water to prepare competent cells; (3) Add 1 μg of plasmid to 50 μl of competent cells and mix well. Transfer to a dry and sterile 2 mm electroporation cuvette and place in a Bio-Rad electroporator for electroporation at 2500 V, 200 Ω, and 25 μF. (4) Resuspend the bacterial solution in 1 mL of recovery medium, incubate at 48°C for 2 h, spread on a resistance plate, incubate at 65°C for 16 h, pick out a single colony on the resistance plate, and use PCR to determine the transformants carrying different plasmids.

[0038] 3. Screening of Geobacillus stearothermophilus transformants with improved heat resistance In order to evaluate the effect of temperature stress-related proteins on the heat resistance of the strain, the transformants of the 12 temperature stress-related protein expression plasmids obtained in step 2 were inoculated into 1.8 mL of resistant LB medium and cultured at 80°C and 960 rpm. After 18 h of culture, the results were as follows: Figure 2 As shown, three transformants carrying the expression plasmids of heat shock proteins DnaK, DnaJ or HtpX have the ability to grow at 80°C.

[0039] Example 2: Combination expression of heat shock proteins to construct the heat-resistant engineering strain SL-1-H1 of Bacillus stearothermophilus In order to further improve the heat resistance of Bacillus stearothermophilus, the three heat shock proteins DnaK, DnaJ and HtpX whose expression levels were positively correlated with the heat resistance of Bacillus stearothermophilus SL-1 determined in Example 1 were cloned into the same expression vector for co-expression to construct the combined expression plasmid pBBR1-Km-P km -DnaK-DnaJ-HtpX, map as Figure 3 As shown. km -DnaK-DnaJ-HtpX plasmid uses pBBR1-Km as the backbone vector. km R The gene expression cassettes of DnaK, DnaJ and HtpX were connected between the 3' end of pBBR1 ori, and the promoters of the three genes were P km pBBR1-Km-P km -DnaK-DnaJ-HtpX, the connection sequence of the three genes DnaK, DnaJ, and HtpX is: DnaK-DnaJ-HtpX. The expression vector, promoter, and plasmid construction method used in this example, and the method of transformation and initiation of bacteria are all consistent with those in Example 1.

[0040] The transformants carrying the combined expression plasmids of DnaK, DnaJ and HtpX were inoculated into 1.8 mL of resistant LB medium and cultured at 80°C and 960 rpm. Figure 2 As shown in the figure, the strain also has the ability to grow at 80°C and is named SL-1-H1. In addition, under the same inoculum size, transformants expressing three heat shock proteins and SL-1-H1 were cultured at 80°C. The OD of SL-1-H1 after 18 h was 600 The value is higher than the 0.3 level of DnaK and DnaJ and the 0.5 level of HtpX, reaching 0.6, which indicates that the engineered bacteria has a higher level of heat resistance.

[0041] The heat resistance of the engineered bacteria SL-1-H1 was further tested by a high temperature heat shock survival test. SL-1-H1 and the starting strain were cultured at the optimal culture temperature of 65°C and 960 rpm until OD 600 Value about 1.0, dilution 10 5 The plate was used to calculate the initial viable count t 0 Then, the two cultures with the same initial bacterial count were placed at 85°C for 24 h, 48 h, 72 h, 96 h, and 120 h of heat shock treatment, and the 10 3 Spread the plate and calculate the number of viable bacteria remaining after heat shock treatment 24 ,t 48 ,t 72 ,t 96 ,t 120 , survival rate = t x / t 0 The results are as follows Figure 4 As shown in the figure, the survival rate of the starting bacteria dropped to less than 1% at 24 h, while the survival rate of the engineered bacteria SL-1-H1 was still above this level after heat shock at 85℃ for 120 h.

[0042] Example 3: Use of heat-resistant engineering bacteria of Geobacillus stearothermophilus for microbial oil recovery 1. SL-1-H1 grows with crude oil as the only carbon source Inoculate the thermotolerant engineered bacteria SL-1-H1 of Geobacillus stearothermophilus into 50 mL of seed culture medium, culture at 60°C overnight, centrifuge at 5000 rpm for 5 min to collect the cells, wash and resuspend the cells with an equal volume of physiological saline as inoculum, inoculate 200 mL of crude oil degradation culture medium at a 1% inoculum volume, culture at 60°C, 120 rpm, sample and dilute appropriately every 3 hours, apply gradient agar plates to record the number of viable cells, and finally draw a growth curve with time as the horizontal axis and colons / mL as the vertical axis as shown in the figure. Figure 5 As shown, this indicates that SL-1-H1 is able to decompose crude oil as a carbon source for growth and is a thermophilic hydrocarbon-decomposing bacterium, and its ability to utilize crude oil is improved compared to the wild-type strain.

[0043] 2. Degradation of crude oil by SL-1-H1 The strain culture cultured overnight with seed culture medium was inoculated into 100 mL crude oil degradation medium at a 1% inoculation rate, and the same crude oil degradation medium without bacteria was used as a control, and cultured at 60°C and 200 rpm. With 5 days as one cycle, samples were taken to determine the crude oil degradation rate, and a total of 4 cycles were tested. After the fermentation liquid was filtered, all undegraded crude oil was collected with n-hexane for determining the crude oil degradation rate. The specific crude oil degradation rate test plan is as follows: (1) The initial crude oil mass contained in the culture medium is 1 g. The quantitative filter paper and the round-bottom flask are placed in a 60°C oven for 24 h and the mass of the round-bottom flask plus the filter paper is recorded as M. 1 ; (2) After the fermentation cycle is completed, the fermentation liquid is filtered using a constant weight quantitative filter paper to collect the residual oil in the culture medium onto the filter paper; (3) Use 80 mL of 50-60°C n-hexane to wash the flask and filter paper used for fermentation. Collect the filtrate (including the filter paper) into a round-bottom flask with a constant weight. Place it on a rotary evaporator and evaporate it to dryness. Then weigh it and record it as M. 2 ; Crude oil degradation rate = [(M 1 +1) - M 2 ] × 100%; The degradation rate of SL-1-H1 to crude oil changes with time. Figure 6 As shown, this once again verifies the hydrocarbon-degrading ability of SL-1-H1.

[0044] Example 4 Biomass determination of the heat-resistant engineering strain SL-1-H1 of Geobacillus stearothermophilus The strain culture was cultured overnight in LB medium to a uniform OD 600 To 1.0, take 20 μL to inoculate 1.8 mL fresh LB medium, and culture at 65℃, 960 rpm and 85℃, 960 rpm respectively. Samples were taken every 24 h and diluted to a certain multiple to plate, and the changes in biomass of the wild type and the SL-1-H1 heat-resistant mutant at different temperatures were compared. Figure 7 As shown, when the initial bacterial amount was the same, the biomass of SL-1-H1 was greater than that of the wild-type strain at the same period at both culture temperatures, indicating that the growth ability of the SL-1-H1 mutant was improved in addition to the highest tolerant temperature.

[0045] The above description shows that by combining the expression of native heat shock proteins DnaK, DnaJ and HtpX, the engineered bacteria Geobacillus stearothermophilus SL-1-H1 that can tolerate 85°C is obtained, thereby improving the heat resistance of the native microorganisms of the oil reservoir, and verifying the thermophilic hydrocarbon-decomposing ability of the engineered bacteria, making it suitable for microbial oil recovery; at the same time, the biomass during the fermentation period at different temperatures is also increased compared with the wild type.

[0046] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without creative work are still within the scope of protection of the present invention.

Claims

1. Use of a heat shock protein gene derived from Geobacillus stearothermophilus in any of the following: (1) Application in improving the heat resistance of microorganisms; (2) Applications to increase microbial biomass; (3) Application of improving the hydrocarbon decomposition capacity of microorganisms; (4) Application of thermotolerant engineered bacteria; The heat shock protein includes any one or more of DnaK, DnaJ and HtpX; the gene sequences of DnaK, DnaJ and HtpX are shown in SEQ ID NO.1-3 respectively.

2. A heat-resistant engineering bacterium, characterized in that: The invention comprises two or more heat shock protein genes or expression vectors thereof; the two or more heat shock protein genes or expression vectors thereof are connected in series and then introduced into a host cell; the heat shock protein genes are derived from Bacillus stearothermophilus Geobacillus stearothermophilus The nucleotide sequence of SL-1, heat shock protein gene is shown in any one of SEQ ID NO.1-3.

3. The heat-resistant engineering bacteria according to claim 2, characterized in that: The expression vector also includes a promoter; the nucleotide sequence of the promoter is shown in SEQ ID NO.

4.

4. The heat-resistant engineering bacteria according to claim 2, characterized in that The host is selected from any one of bacteria, fungi and archaea.

5. Use of the heat-resistant engineering bacteria according to any one of claims 2 to 4 in the degradation of crude oil or microbial oil recovery.

6. A method for degrading crude oil or microbial oil recovery, characterized in that: The thermotolerant engineered bacteria, culture fluid or fermented metabolites of any one of claims 2 to 4 are used to replace the displacement fluid to degrade crude oil or perform microbial oil recovery.

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