Recombinant vector for enhanced oil recovery, engineering strain for enhanced oil recovery and construction method and application thereof

Abstract

This invention relates to the field of microbial genetic engineering technology, specifically to a recombinant vector for enhanced oil recovery (EOR), engineered bacterial strains for EOR, their construction methods and applications, engineered bacterial strain agents and their applications, and methods for crude oil recovery. The recombinant vector for EOR of this invention includes a starting vector and an expression gene integrated into the starting vector; the expression gene includes a surfactant synthesis gene and a heat-resistant gene; the surfactant synthesis gene is the srfA gene; the heat-resistant gene is selected from the hsp70 gene, hsp60 gene, or dnaK gene. In this invention, a dual-gene synergistic modification strategy is employed, simultaneously introducing a heat-resistant gene and a surfactant synthesis gene and optimizing their expression, thereby achieving a dual enhancement of the engineered strain's environmental adaptability and metabolic function.

Description

Technical Field

[0001] This invention relates to the field of microbial genetic modification technology, specifically to a recombinant vector for enhanced oil recovery, an engineered strain for enhanced oil recovery and its construction method and application, an engineered strain agent and its application, and a method for crude oil recovery. Background Technology

[0002] Microbial enhanced oil recovery (MEOR), as an emerging method for enhancing oil extraction, leverages the diverse metabolic functions of microorganisms to achieve biochemical regulation of crude oil production. However, existing technologies face systemic technical bottlenecks. From a microbial community ecology perspective, traditional MEOR microorganisms face fundamental challenges such as low diversity of indigenous microbial communities, a severe shortage of functional species, a simple community structure, and limited metabolic capacity. These limitations directly restrict the biotransformation efficiency and mechanism of action of MEOR.

[0003] Microorganisms face extremely harsh survival conditions in oil reservoir environments, including high temperatures (45-80℃), high pressures (10-50 MPa), high salinity (3-10% NaCl), highly corrosive environments, and complex geological micro-ecological environments. Existing microorganisms struggle to simultaneously meet multiple stringent requirements, such as maintaining stable metabolic activity, sustaining efficient surfactant synthesis, adapting to extreme environments, and long-term stable survival. Traditional microorganisms often rapidly inactivate under these extreme conditions, experiencing a sharp decline in metabolic capacity, which severely restricts the practical application value of oil recovery technologies.

[0004] Genetically modified microorganisms were intended to offer new solutions to these problems, but they still face numerous technical bottlenecks. Gene introduction efficiency is extremely low, with conventional transformation methods generally below 0.1%. The lack of suitable gene tools for non-model strains further hinders successful modification. Even more challenging is gene expression regulation; introduced functional genes are often highly unstable in microorganisms, exhibiting insufficient promoter transcription strength, poor regulatory system responsiveness, and excessive metabolic burden, directly impacting microbial growth. Gene stability is equally critical; exogenous genes are prone to mutation, recombination, or loss in the complex environment of oil reservoirs, severely affecting the long-term stability of microorganisms.

[0005] More complexly, interactions between different genes can lead to potential disruptions in metabolic pathways and imbalances in energy distribution, fundamentally affecting the overall performance of microorganisms. This systemic imbalance in the coordination of multiple genes and functions makes traditional single-gene modification methods insufficient to fundamentally solve the problem of microbial adaptation in extreme reservoir environments. The challenges facing microbial enhanced oil recovery (MEOR) technology have transcended simple gene modification, requiring a systematic rethinking of microbial functional optimization strategies at the molecular level. How to achieve synergistic optimization of microbial multifunctionality, high efficiency, and strong environmental adaptability at the molecular level has become a key scientific problem restricting technological breakthroughs. Summary of the Invention

[0006] To overcome the problem of poor adaptability of existing engineered strains to extreme reservoir environments such as high temperature, high pressure, and high salinity, this invention provides a recombinant vector for enhanced oil recovery, an engineered strain for enhanced oil recovery, its construction method, and its application. The engineered strain constructed using the recombinant vector of this invention can function stably and continuously in different types of reservoirs, producing sufficient amounts of surfactants and other effective substances, ultimately achieving the goal of improving oil recovery. At the same time, the engineered strain of this invention can function stably and continuously under extreme reservoir conditions such as high temperature and high salinity.

[0007] To achieve the above objectives, a first aspect of the present invention provides a recombinant vector for enhanced oil recovery, the vector comprising a starting vector and an expression gene integrated into the starting vector; The expressed genes include: surfactant synthesis genes and heat resistance genes; The surfactant synthesis gene is the srfA gene; The heat-resistant gene is selected from the hsp70 gene, hsp60 gene, or dnaK gene.

[0008] A second aspect of the present invention provides an engineered strain for enhanced oil recovery, wherein the engineered strain is capable of expressing surfactant synthesis genes and heat resistance genes; The surfactant synthesis gene and the heat resistance gene are derived from the recombinant vector described in this invention.

[0009] A third aspect of the present invention provides a method for constructing the engineered strain of the present invention, the method comprising: introducing the recombinant vector of the present invention into an initial strain for transformation to obtain an engineered strain.

[0010] A fourth aspect of the present invention provides a bacterial agent of the engineered strain described in the present invention.

[0011] The fifth aspect of this invention provides the application of the engineered strain or the bacterial agent described herein in crude oil recovery from oil reservoirs.

[0012] The sixth aspect of the present invention provides a method for crude oil recovery, the method comprising: inoculating the engineered strain or the bacterial agent of the present invention into an oil reservoir.

[0013] Through the above technical solutions, the recombinant vector of this invention can simultaneously introduce heat-resistant genes (such as hsp70, hsp60, dnaK) and surfactant synthesis genes and optimize their expression, achieving a dual improvement in the environmental adaptability and metabolic function of engineered strains. This enables engineered strains to acquire stronger environmental adaptability and metabolic activity, significantly improving the engineering application effect of microbial enhanced oil recovery while ensuring safety, and providing more efficient technical support for oilfield enhanced oil recovery. On the one hand, this invention improves the transformation efficiency of target genes through genetic engineering technology, achieving a transformation efficiency of 5-10%, which is 3-5 times higher than traditional methods. On the other hand, this invention optimizes the gene expression regulation system, enhancing gene stability, and ensuring stable expression of exogenous genes even after more than 40 generations of continuous passage. Simultaneously, this invention achieves synergistic expression of functional genes.

[0014] This invention employs a dual-gene synergistic modification strategy. By simultaneously introducing heat-resistant genes (such as hsp70, hsp60, and dnaK) and surfactant synthesis genes and optimizing their expression, a dual enhancement of the environmental adaptability and metabolic function of the engineered strain is achieved. This strategy enables the engineered strain to maintain over 80% growth activity at 60°C, achieve a survival rate of 65-75% at 10% NaCl concentration, expand its pH adaptation range to 5.0-10.0, and maintain good metabolic activity under anaerobic conditions. Simultaneously, it maintains stable surfactant production, significantly overcoming the limitations of traditional single-gene modification in extreme environment applications. The inventors unexpectedly discovered that the dual-gene synergistic modification strategy of this invention can improve the salt tolerance of the engineered strain. This is speculated to be due to two factors: firstly, the cross-protective effect of heat shock proteins introduced by the heat-resistant genes: heat shock proteins not only provide heat resistance to help bacteria cope with high-temperature stress but also help the strain cope with environmental stress caused by changes in osmotic pressure; secondly, the salt tolerance of the engineered strain is enhanced by the dual-gene modification described in this invention, which strengthens the overall metabolic capacity of the strain.

[0015] In this invention, the synergistic effect of the two genes not only balances the metabolic burden but also forms a virtuous cycle of environmental tolerance and functional expression. This enables the modified strain to function stably and continuously under extreme reservoir conditions such as high temperature and high salinity, significantly enhancing its application value in complex reservoir environments and providing new ideas for the development of microbial enhanced oil recovery technology.

[0016] Biological Preservation The Bacillus subtilis of the present invention ( Bacillus subtilis.Strain OPUS-001 was deposited on March 22, 2013, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCC No. 7361. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the spectrum of the pNZT1 vector; Figure 2 This is a schematic diagram of the spectrum of the vector pBBR1MCS-2; Figure 3 This is a schematic diagram of the pUCG18 vector. Figure 4 This is a scanning electron microscope image of the engineered strain of Example 2 of the present invention after 40 generations of passage. Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] A first aspect of the present invention provides a recombinant vector for enhanced oil recovery, the vector comprising a starting vector and an expression gene integrated into the starting vector; The expressed genes include: surfactant synthesis genes and heat resistance genes; The surfactant synthesis gene is the srfA gene; The heat-resistant gene is selected from the hsp70 gene, hsp60 gene, or dnaK gene.

[0020] In this invention, the expressed gene is a complete fusion sequence designed by a surfactant synthesis gene and a temperature-resistant gene in the correct order of expression elements.

[0021] In this invention, the nucleotide sequence of the hsp70 gene is shown in SEQ ID NO: 1.

[0022] In this invention, the nucleotide sequence of the hsp60 gene is shown in SEQ ID NO: 2.

[0023] In this invention, the nucleotide sequence of the dnaK gene is shown in SEQ ID NO: 3.

[0024] In this invention, the nucleotide sequence of the srfA gene is shown in SEQ ID NO: 4.

[0025] In this invention, there is no particular limitation on the type of the starting carrier. According to a preferred embodiment of the invention, the starting carrier is selected from pNZT1, pBBR1MCS-2 or pUCG18.

[0026] In this invention, as long as the surfactant synthesis gene and the heat resistance gene are linked to the starting vector, there is no particular limitation on the order of the surfactant synthesis gene and the heat resistance gene. For example, the expression gene may sequentially include the surfactant synthesis gene and the heat resistance gene, or the expression gene may sequentially include the heat resistance gene and the surfactant synthesis gene, both of which can achieve the purpose of this invention. According to a preferred embodiment of this invention, the expression gene sequentially includes the surfactant synthesis gene and the heat resistance gene, which can improve the expression efficiency of the surfactant synthesis gene and thus increase the yield of surfactant.

[0027] According to a preferred embodiment of the present invention, when the expressed genes are the srfA gene and the hsp70 gene, the expressed genes are located between the EcoRI and HindIII restriction sites of the pNZT1 vector.

[0028] According to a preferred embodiment of the present invention, when the expressed genes are the srfA gene and the hsp60 gene, the expressed genes are located between the NdeI and BamHI restriction sites of the pUCG18 vector.

[0029] According to a preferred embodiment of the present invention, when the expressed genes are the srfA gene and the dnaK gene, the expressed genes are located between the EcoRI and HindIII restriction sites of the pNZT1 vector, or between the BamHI and XhoI restriction sites of the pBBR1MCS-2 vector.

[0030] To further improve the expression efficiency and expression level of the recombinant vector provided by the present invention, the recombinant vector may further contain at least one gene element selected from promoter, operon, terminator, etc. According to a preferred embodiment of the present invention, the promoter is a promoter located at the 5' end of the coding sequence of the surfactant synthesis gene.

[0031] According to another preferred embodiment of the present invention, the promoter is derived from a strong promoter of the recombinant vector host; more preferably, the promoter is the strong promoter Pbac of Bacillus.

[0032] According to a preferred embodiment of the present invention, the promoter sequence of the srfA gene is shown in SEQ ID NO: 5.

[0033] According to a preferred embodiment of the present invention, the strong promoter sequence of Bacillus is Pbac, as shown in SEQ ID NO: 6.

[0034] In this invention, the nucleotide sequence of the recombinant vector is shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0035] In this invention, any commonly used recombinant vector construction method in the art can be applied. For example, the starting vector and the expression gene fragment can be digested with enzymes and ligated to obtain a recombinant plasmid.

[0036] A second aspect of the present invention provides an engineered strain for enhanced oil recovery, wherein the engineered strain is capable of expressing surfactant synthesis genes and heat resistance genes; The surfactant synthesis gene and heat resistance gene are derived from the recombinant vector described in this invention. The engineered strains described in this invention are particularly suitable for microbial enhanced oil recovery in high-temperature, high-salinity reservoirs, providing more efficient technical support for improving oil recovery rates in oilfields.

[0037] In this invention, conventional oil-producing strains in the art, such as Bacillus, can achieve the purpose of this invention by introducing the recombinant vector described in this invention, and are not limited to those purchased or isolated by the individual.

[0038] According to a preferred embodiment of the present invention, the initial strain is a dominant oil production strain isolated from oilfield produced fluid.

[0039] In addition to dominant bacterial strains adapted to the reservoir environment, oilfield produced fluids also contain various types of bacterial strains, including common indigenous bacteria and exogenous bacteria introduced during the production process. In this invention, the dominant oil-producing bacterial strains are obtained through multi-step screening. According to a preferred embodiment of this invention, preliminary screening is conducted under selective culture conditions (3-7% NaCl, 35-45℃, 48-72h), followed by bacterial identification through morphological observation, physiological and biochemical analysis, and 16S rDNA sequencing. Finally, the dominant bacterial strains are obtained by verifying their oil-producing-related functions.

[0040] According to a preferred embodiment of the present invention, the initial strain is Bacillus.

[0041] The engineered strains constructed by this invention using genetic engineering technology can improve the transformation efficiency of the target gene, reaching 5-10%, which is 3-5 times higher than traditional methods. On the other hand, this invention optimizes the gene expression regulation system, enhances gene stability, and allows exogenous genes to maintain stable expression even after more than 40 generations of continuous passage. At the same time, this invention achieves the synergistic expression of functional genes.

[0042] A third aspect of the present invention provides a method for constructing the engineered strain of the present invention, the method comprising: introducing the recombinant vector of the present invention into an initial strain for transformation to obtain an engineered strain.

[0043] According to a particularly preferred embodiment of the present invention, the method for constructing the engineered strain of the present invention includes: (1) obtaining the dominant strain microorganism: the oilfield produced fluid is inoculated into a liquid culture medium and cultured for 48-72 hours, and the single colony is obtained by streak plate separation and purification. The isolated strain is identified to determine that it is an oil production strain. (2) Obtaining target genes: Genes for biosurfactant synthesis were screened using the homologous sequence—Bacillus srfA gene—as templates, and thermostable genes were screened using the thermophilic bacteria hsp70 gene, hsp60 gene, and dnaK gene as templates. Sequences with similarity ≥70%, coverage ≥80%, and synthesis purity ≥95% were obtained. Codon optimization was performed, and restriction endonuclease sites were added to both ends of the sequences. (3) Construction of recombinant plasmid: The vector and the target gene fragment are digested with enzymes and ligated to obtain the recombinant plasmid; (4) Gene introduction: Prepare competent cells of the strain by chemical or electroporation method, and co-transform the competent cells of the strain with recombinant plasmid by heat shock or electroporation method to obtain strain cells containing recombinant plasmid; (5) Screening and verification of engineered strains: After the transformed bacterial culture is revived, screening is carried out. First, the transformation of the recombinant plasmid is verified to be successful, and then the function of the engineered strain is verified.

[0044] A fourth aspect of the present invention provides a bacterial agent of the engineered strain described in the present invention.

[0045] The fifth aspect of this invention provides the application of the engineered strain or the bacterial agent described herein in oil reservoir crude oil recovery. The engineered strain obtained by the construction method described herein is particularly suitable for microbial enhanced oil recovery in high-temperature, high-salinity oil reservoirs, providing more efficient technical support for improving oil recovery rates in oil fields.

[0046] According to a preferred embodiment of the present invention, the engineered strain or the bacterial agent is used in crude oil recovery from reservoirs at 45-65℃.

[0047] According to a preferred embodiment of the present invention, the engineered strain or the bacterial agent is used in the recovery of crude oil from reservoirs with a salinity of 60-200 g / L.

[0048] The sixth aspect of the present invention provides a method for crude oil recovery, the method comprising: inoculating the engineered strain or the bacterial agent of the present invention into an oil reservoir.

[0049] In the context of this invention, including the following examples, the method for testing the growth activity of the strain is as follows: after culturing for 24 hours under different temperature conditions, the growth activity is expressed as the optical density (OD600) value measured at 600 nm. Growth activity (%) = (OD600 of treatment group / OD600 of control group) × 100%.

[0050] In the context of this invention, including the following embodiments, the method for testing the survival rate of bacterial strains is as follows: The bacterial suspension is serially diluted and spread onto plates, then incubated for 24 hours before counting. Survival rate (%) = (Number of colonies in the treatment group / Number of colonies in the control group) × 100% In the context of this invention specification, the following embodiments are included, where the surface tension is measured using a KRÜSS K100 fully automated surface tension meter and the du Noüy ring method at 25°C. Each sample is measured three times and the average value is taken.

[0051] In the context of this invention specification, the following embodiments are included, including a method for quantitative determination of surfactants: using a methanol-chloroform (1:2) extraction system, the surfactant is extracted, concentrated, and then redissolved in methanol. The absorbance is measured at a wavelength of 215 nm using a UV spectrophotometer, and the concentration is calculated based on a standard curve to obtain the surfactant concentration.

[0052] In the context of this invention, the following embodiments illustrate the use of the displacement efficiency method to test oil recovery: crude oil and a bacterial fermentation broth are mixed at a ratio of 1:9, shaken at 37°C for 48 hours, and then centrifuged to separate the mixture. The residual oil content in the aqueous phase is then determined. Recovery rate (%) = (original oil content - residual oil content) / original oil content × 100%.

[0053] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0054] In the following embodiments, the methods for obtaining dominant strains of microorganisms, obtaining target genes, constructing recombinant plasmids, constructing engineered strains, and screening and validating engineered strains are as follows.

[0055] Obtaining dominant strains of microorganisms: Produced fluid from the oilfield was inoculated at a volume of 2% into LB or TSB liquid medium containing 3-7% NaCl (LB medium: 10.0 g / L tryptone, 5.0 g / L yeast extract, 30-70 g / L NaCl, with optional 10 g / L glycine; TSB medium: 17.0 g / L tryptone, 3.0 g / L soybean peptone, 2.5 g / L glucose, 2.5 g / L K2HPO4, 30-70 g / L NaCl). The medium was incubated at 35-45℃ for 48-72 hours. Single colonies were obtained by streak plating and purification. The isolated strains were then identified by morphological observation, physiological and biochemical experiments, and 16S rDNA sequencing.

[0056] In the following examples, produced fluid samples from the Ci 12-155 well in Liaohe Oilfield were inoculated into LB medium containing 5% NaCl and cultured at 37°C for 48 hours before plate isolation to obtain Bacillus subtilis OPUS-001. Bacillus subtilis. This strain can produce biosurfactants at 37°C, but its growth is significantly inhibited at temperatures above 55°C and at a NaCl concentration of 8%, which cannot meet the needs of reservoir development.

[0057] Obtaining the target gene: After obtaining the dominant strains, homologous sequence alignment of functional genes was performed using the NCBI BLAST tool (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) based on the differences in the dominant strains. Different surfactant synthesis genes and thermostable genes were selected as templates for screening target genes. The alignment conditions were set with an E-value threshold of 1e. -5 -1e -10 The sequence similarity must be ≥70% and the coverage ≥80%. The target gene sequence obtained from the screening will be submitted to a gene synthesis company to design a complete fusion sequence with a purity of ≥95%. Codon optimization will be performed, and appropriate restriction endonuclease sites will be added to both ends of the sequence.

[0058] Construction of recombinant plasmids: (1) Prepare a 45 or 50 μL double enzyme digestion reaction system: 1-5 μg plasmid DNA, 1-5 μg target gene fragment, 10-20 U of each restriction endonuclease, 5 μL 10× buffer, and make up the rest with nuclease-free water.

[0059] (2) Place the reaction system in a 37°C water bath for 2-4 hours for enzyme digestion; separate the target band of the enzyme digestion product by 1% agarose gel electrophoresis, purify the DNA fragment using a gel recovery kit, and determine the DNA concentration and purity, requiring OD260 / 280 to be between 1.8 and 2.0.

[0060] (3) Add 50-100 ng of vector DNA to an 18 or 20 μL reaction system, add the digested target gene fragment at a molar ratio of 3-5:1, 5-10 U of T4 DNA ligase, 2 μL of 10× ligation buffer, and make up the remainder with nuclease-free water. Incubate the reaction system at 16℃ for 8-12 hours to complete the ligation.

[0061] Construction of engineered strains: (1) Preparation of initial competent cells of the strain: The strain was inoculated into LB or TSB medium with 3-7% NaCl and cultured at 30-45℃ (select the appropriate temperature according to the characteristics of the strain) with shaking at 180-220 rpm until the OD600 reached 0.4-0.6; 1.1) Chemical preparation: Treat the aforementioned solution with pre-cooled 0.05-0.2 M CaCl2 solution at 0-4℃ for 20-30 minutes; 1.2) Electrochemical preparation: Wash the above solution 3-4 times with pre-cooled 10% glycerol solution, centrifuge at 3000-4000 rpm for 5 minutes each time, and finally resuspend the bacteria in 10% glycerol solution, with the resuspended volume being 1 / 100-1 / 200 of the above solution.

[0062] (2) Co-transform the competent cells of the strain with the recombinant plasmid. Different co-transformation methods are selected according to the different initial strains to obtain engineered strains: (I) For Gram-negative bacteria, co-transformation is performed by heat shock: 100-500 ng DNA is mixed with 100-200 μL of competent cells, incubated on ice for 20-30 minutes, then heat-shocked at 42°C for 60-90 seconds, followed immediately by incubation on ice for 2-5 minutes.

[0063] (II) For Gram-positive bacteria, co-conversion was performed by electroconversion: the electroconversion parameters were set as follows: voltage 1.8-2.5 kV, capacitance 25-50 μF, resistance 200-400 Ω, pulse time 4-6 ms, and sample volume 40-100 μL.

[0064] In the following embodiments, OPUS-001 ( Bacillus subtilis. The cells were inoculated into LB medium containing 5% NaCl and cultured at 37°C until the OD600 reached 0.5. After washing three times with pre-cooled 10% glycerol solution, the cells were resuspended in 1 / 150 of the original volume of glycerol solution to prepare competent cells. The competent cells were then co-transformed with the recombinant plasmid using electroporation. The electroporation conditions were set as follows: voltage 2.0 kV, capacitance 35 μF, resistance 300 Ω, and pulse duration 5 ms.

[0065] Screening and validation of engineered strains: (1) The transformed bacterial culture was shaken and revived in 1 mL of antibiotic-free LB medium at 35-45℃ for 45-60 minutes, and then spread on LB plates containing the corresponding antibiotics and cultured at 35-45℃ for 16-24 hours for preliminary screening.

[0066] (2) PCR verification of the obtained positive transformants was performed by adding single-clone colony or plasmid DNA as template, 0.2-0.5 μM primers, 0.2 mM dNTPs and 1-2.5 U Taq enzyme to a 25 μL reaction system. The PCR program was as follows: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 55-65℃ annealing for 30 seconds, 72℃ extension for 1 minute / kb, for a total of 30-35 cycles; and a final extension at 72℃ for 10 minutes.

[0067] The primers used to verify each gene are shown in Table 1: Table 1

[0068] (3) Functional verification of the engineered strain: surfactant production was determined (by oil spreading experiment or surface tension meter), temperature resistance was tested (growth curves were measured at temperature gradients of 30, 40, 50, 60, and 70 °C), and salt tolerance was evaluated (survival rate was measured at salinity gradients of 3%, 5%, 7%, and 10%). The above verification experiments ensured that the obtained engineered strain had the expected function and environmental adaptability.

[0069] Example 1 Using the NCBI BLAST tool, thermophilic Bacillus... Geobacillus stearothermophilus Homologous sequence alignment was performed using the heat shock protein gene hsp70 as a template (E-value set to 1e). -8 With a sequence similarity of 75% and a coverage of 85%, the target thermostable gene sequence shown in SEQ ID NO: 1 was obtained. Simultaneously, a highly efficient promoter and coding sequence (E-value of 1e) of the surfactantin synthase gene srfA in Bacillus strains were screened. -6 (The sequence similarity was 76%, and the coverage was 84%). (The efficient promoter sequence of srfA is shown in SEQ ID NO: 5, and the coding sequence of srfA is shown in SEQ ID NO: 4). These sequences were submitted to Thermo Fisher for gene synthesis (sequence purity was 98%), and codon optimization was performed by adding EcoRI and HindIII restriction sites to both ends of the sequences.

[0070] In a 50 μL reaction system, EcoRI and HindIII were used to treat... Figure 1The pNZT1 vector and the synthesized target gene were double-digested with enzymes (37℃, 3 hours). The digestion products were separated and purified by 1% agarose gel electrophoresis (vector DNA concentration: 85 ng / μL, target gene fragment concentration: 45 ng / μL), and ligated in a 20 μL ligation system at a 4:1 molar ratio (16℃, 10 hours) to obtain the recombinant vector with the nucleotide sequence shown in SEQ ID NO: 7. The ligation product was transformed into *E. coli* DH5α competent cells for amplification. Positive clones were obtained through ampicillin (100 μg / mL) resistance selection, and plasmids were extracted and sequenced to verify the correct insertion sequence.

[0071] Obtain OPUS-001 ( Bacillus subtilis. Competent cells were co-transformed with recombinant plasmids. The transformed bacterial culture was revived in antibiotic-free LB medium at 37°C for 1 hour, and then plated on LB plates containing kanamycin (30 μg / mL) for selection.

[0072] The obtained transformants were verified by PCR, and the results showed that the target gene had been successfully integrated.

[0073] Functional validation results showed that the engineered strain maintained over 80% of its growth activity at 60℃, a 2.5-fold increase compared to the initial strain; and its survival rate reached 65% at a 10% NaCl concentration, significantly better than the initial strain. Oil spreading experiments showed that the engineered strain produced 4.2 g / L of surfactant at 55℃, 2.3 times that of the initial strain. After 48 hours of fermentation, the surface tension of the culture medium was measured at 28.5 mN / m (compared to 38.7 mN / m for the initial strain).

[0074] The obtained engineered strain was subjected to oil displacement experiments under simulated reservoir conditions (55℃, 8% NaCl). The results showed that the recovery rate was increased by 32.5%, and the engineered strain maintained stable phenotypic characteristics after 50 generations of continuous culture without resistance pressure, proving that the genetic modification effect was good and had genetic stability.

[0075] Example 2 Using the NCBI BLAST tool, thermophilic genes were screened using the heat shock protein gene dnaK from *Thermus thermophilus* (E-value 1e-7, sequence similarity 72%, coverage 82%) to obtain the sequence shown in SEQ ID NO: 3. Simultaneously, using the srfA operon from *Bacillus subtilis* (a high-surfactant producer) as a template (sequence similarity 85%), efficient promoter and coding sequences for biosurfactant synthesis genes were screened (the efficient promoter sequence for srfA is shown in SEQ ID NO: 5, and the coding sequence for srfA is shown in SEQ ID NO: 4). These sequences were synthesized at ThermoFisher (sequence purity 97%), codon optimized, and BamHI and XhoI restriction sites were added to both ends of the sequences, respectively.

[0076] BamHI and XhoI were used in a 45 μL reaction system for... Figure 2 The pBBR1MCS-2 vector and target gene were double-digested (37℃, 2.5 h). The DNA fragments obtained by gel extraction (vector DNA concentration 75 ng / μL, target gene fragment concentration 45 ng / μL) were ligated in an 18 μL ligation system at a 3.5:1 molar ratio (16℃, 8 h) to obtain the recombinant vector with the nucleotide sequence shown in SEQ ID NO: 8. The ligation product was first transformed into *E. coli* DH5α for amplification and verification. Positive clones were obtained through kanamycin (50 μg / mL) resistance selection, and plasmids were extracted and sequenced to verify the correct insertion sequence.

[0077] Obtain OPUS-001 ( Bacillus subtilis. Competent cells were co-transformed with recombinant plasmids. The transformed bacterial culture was incubated in antibiotic-free LB medium at 35°C for 50 minutes, and then plated on plates containing kanamycin (50 μg / mL) for screening.

[0078] The obtained transformants were verified by PCR, and the results showed that the target gene had been successfully integrated.

[0079] Functional validation results showed that the engineered strain maintained a growth activity of over 75% at 50℃ and a survival rate of 70% under 6% NaCl conditions. After 48 hours of fermentation, the surface tension of the culture medium decreased to 29.8 mN / m, and the surfactant yield reached 3.8 g / L.

[0080] The obtained engineered strain was subjected to oil displacement experiments under simulated reservoir conditions (50℃, 6% NaCl). The results showed that the recovery rate of the engineered strain was 38.7% higher than that of the initial strain. The engineered strain was passaged for 40 generations under no resistance stress conditions. PCR detection and functional verification results showed that the exogenous gene remained stable and its expression activity did not decrease significantly. Scanning electron microscopy observations showed... Figure 4 The results showed that the engineered strain maintained intact cell morphology under high temperature conditions, and the cell surface was covered with obvious biosurfactant micelle structures, confirming the effectiveness of the genetic modification.

[0081] Example 3 Using the NCBI BLAST tool, thermophilic Bacillus... Bacillus thermophilus The surfactant synthesis gene srfA serves as a template (E-value is 1e). -6 The surfactant synthesis gene was screened with 76% sequence similarity and 84% coverage to obtain the sequence shown in SEQ ID NO: 4; thermophilic bacteria Thermus thermophilus The heat shock protein gene hsp60 was used as a template (E-value set to 1e). -8 Thermostable genes were screened (with sequence similarity of 77% and coverage of 83%), yielding the sequence shown in SEQ ID NO: 2. The strong promoter sequence Pbac of Bacillus was also obtained, as shown in SEQ ID NO: 6. These sequences were submitted to Thermo Fisher Scientific for gene synthesis (sequence purity of 98%), codon optimization, and the addition of NdeI and BamHI restriction enzyme sites at both ends of the sequences.

[0082] In a 45 μL reaction system, NdeI and BamHI were used to treat... Figure 3 The pUCG18 vector and target gene were double-digested (37℃, 3.5 h). The digestion products were separated and purified by 1% agarose gel electrophoresis (vector DNA concentration 85 ng / μL (OD260 / 280 1.85), target gene fragment concentration 48 ng / μL). Ligation was then performed in a 20 μL ligation system at a molar ratio of 3.8:1 (16℃, 10 h) to obtain the recombinant vector with the nucleotide sequence shown in SEQ ID NO: 9. The ligation product was transformed into *E. coli* Top10 for amplification. Positive clones were obtained through ampicillin (100 μg / mL) resistance selection, and plasmids were extracted and sequenced to verify sequence correctness.

[0083] Obtain OPUS-001 ( Bacillus subtilis. Competent cells were co-transformed with recombinant plasmids. The transformed bacterial culture was then revived in LB medium without antibiotics at 40°C for 55 minutes and then plated on plates containing kanamycin (35 μg / mL) for screening.

[0084] The obtained transformants were verified by PCR, and the results showed that the target gene had been successfully integrated.

[0085] Functional validation results showed that the engineered strain maintained a growth activity of over 85% at 50℃ and a survival rate of 75% at a 9% NaCl concentration. After 48 hours of fermentation, the surface tension of the culture medium decreased to 22.8 mN / m, and the diameter of the transparent ring formed in the oil droplet diffusion experiment increased by 2.3 times. The surfactant yield reached 4.5 g / L.

[0086] The obtained engineered strain was subjected to oil displacement experiments under simulated reservoir conditions (50℃, 8.5% NaCl). The results showed that the recovery rate was increased by 38.2%. After 45 generations of continuous subculturing under no resistance pressure, PCR detection and surface activity assay results showed that the exogenous gene was stably present and its expression activity remained stable, confirming the effectiveness and genetic stability of the gene modification.

[0087] Example 4 Using the NCBI BLAST tool, homologous sequence alignment was performed using the heat shock protein gene dnaK from the thermophilus bacterium Thermus thermophilus as a template (E-value set to 1e). -7 The target thermostable gene sequence, as shown in SEQ ID NO: 3, was obtained with a sequence similarity of 72% and a coverage of 82%. Simultaneously, using the surfactantin synthase gene srfA from Bacillus subtilis as a template (sequence similarity 83%), efficient promoter and coding sequences (E-value of 1e) of surfactant synthesis genes were screened. -6 (The sequence similarity was 76%, and the coverage was 84%). (The efficient promoter sequence of srfA is shown in SEQ ID NO: 5, and the coding sequence of srfA is shown in SEQ ID NO: 4). These sequences were submitted to Thermo Fisher for gene synthesis (sequence purity was 97%), codon optimization was performed, and EcoRI and HindIII restriction sites were added to both ends of the sequences, respectively.

[0088] The pNZT1 vector and target gene were double-digested with EcoRI and HindIII in a 50 μL reaction system (37 °C, 3 h). The digestion products were separated and purified by 1% agarose gel electrophoresis (vector DNA concentration: 90 ng / μL, target gene fragment concentration: 55 ng / μL), and ligated in a 20 μL ligation reaction system at a 4:1 molar ratio (16 °C, 10 h) to obtain the recombinant vector with the nucleotide sequence shown in SEQ ID NO: 10. The ligation product was transformed into *E. coli* DH5α competent cells for amplification. Positive clones were obtained through ampicillin (100 μg / mL) resistance selection, and plasmids were extracted and sequenced to verify the correct insertion sequence.

[0089] Obtain OPUS-001 ( Bacillus subtilis. Competent cells were co-transformed with recombinant plasmids. The transformed bacterial culture was thawed in LB medium at 37°C for 1 hour, and then plated on LB plates containing kanamycin (30 μg / mL) for selection.

[0090] The obtained transformants were verified by PCR, and the results showed that the target gene had been successfully integrated.

[0091] Functional validation results showed that the engineered strain maintained over 82% growth activity at 55℃, significantly higher than the initial strain; and its survival rate reached 72% at a 9% NaCl concentration, significantly better than the initial strain. Oil spreading experiments showed that the engineered strain produced 4.0 g / L of surfactant at 50℃, and the surface tension of the culture medium decreased to 24.2 mN / m after 48 hours of fermentation.

[0092] The obtained engineered strain was subjected to oil displacement experiments under simulated reservoir conditions (55℃, 8% NaCl). The results showed that the recovery rate was increased by 35.8%, and the engineered strain maintained stable phenotypic characteristics after 45 generations of continuous culture without resistance pressure, proving that the genetic modification effect was good and had genetic stability.

[0093] The results of the above embodiments show that the yield and quality of biosurfactants produced by the engineered strain of the present invention are significantly improved. Fermentation yield increased by 2.3-3.5 times, reaching 3.6-4.5 g / L, and the oil-water interfacial tension was reduced to 22-28 mN / m. Even under high-temperature conditions (above 50℃), it maintained more than 75% activity, significantly enhancing the effect of microbial enhanced oil recovery.

[0094] Furthermore, the engineered strain described in this invention exhibits excellent oil recovery performance, increasing crude oil recovery rate by 30-50%, and demonstrates good application results for different types of crude oil. It continues to function effectively even under high temperature and high salinity conditions, exhibiting good strain activity and functional stability, providing efficient technical support for enhancing oil recovery in oilfields.

[0095] This invention overcomes the problems of poor environmental adaptability and functional instability of microorganisms in traditional microbial enhanced oil recovery (MEOR) technologies, and establishes a complete genetic engineering modification technology system. This technology is simple to operate, easy to industrialize, has relatively low production costs, is environmentally friendly, produces no secondary pollution, and the obtained engineered strains have significant practical implications for improving crude oil recovery.

[0096] Comparative Example 1 To further verify the advantages of co-expressing the heat-resistant gene and the surfactant synthesis gene, this comparative example constructed a recombinant plasmid containing only the srfA gene, with other conditions being the same as in Example 1.

[0097] The srfA gene and its promoter were ligated into the pNZT1 vector and transformed into the initial OPUS-001 strain to obtain the recombinant strain OPUS-001 / pNZT1-srfA. Fermentation and oil displacement experiments were performed on this strain under the same conditions as in Example 1, and the results are as follows: At 37°C, the growth activity of the recombinant strain OPUS-001 / pNZT1-srfA was comparable to that of OPUS-001, but its growth was significantly inhibited at 55°C, with a growth activity of only about 30%. In contrast, the engineered strain in Example 1 could still maintain a growth activity of more than 78% at 55°C, indicating that the introduction of the heat-resistant gene hsp70 significantly improved the heat resistance of the engineered strain.

[0098] After 48 hours of fermentation, the surfactant yield of OPUS-001 / pNZT1-srfA was 2.8 g / L at 37°C and 1.2 g / L at 55°C, which were 1.8 times and 1.2 times that of OPUS-001, respectively. This was 26% and 48% lower than that of the engineered strain in Example 1, indicating that the synergistic expression of srfA and hsp70 significantly improved the surfactant synthesis ability, especially under high temperature conditions.

[0099] The surface tension measurements of the fermentation broth showed that at 37℃ and 55℃, the surface tensions of OPUS-001 / pNZT1-srfA were 32.4 mN / m and 37.5 mN / m, respectively, which were 3.9 and 9.0 units higher than those of the engineered strain in Example 1. This indicates that co-expression of srfA and hsp70 is more beneficial for reducing surface tension, thereby improving crude oil emulsification efficiency.

[0100] Oil displacement experiments conducted under simulated reservoir conditions at ambient temperature (30℃) and 55℃ showed that the recovery rate of OPUS-001 / pNZT1-srfA increased by 18.7% and 5.2%, respectively, while the recovery rate of the engineered strain in Example 1 increased by 28.4% and 21.6% under the same conditions, respectively. This indicates that the introduction of the hsp70 gene significantly enhances the oil displacement effect, especially in high-temperature reservoirs.

[0101] In summary, while srfA expression alone can promote surfactant synthesis and improve oil recovery to some extent, its effect is far less significant than co-expression of srfA and the heat-resistant gene, especially under high-temperature conditions. This indicates that the heat-resistant gene not only enhances the host's heat resistance but also has a significant synergistic effect with srfA, jointly promoting the efficient synthesis of surfactants and thus improving oil displacement efficiency. Therefore, the dual-gene synergistic expression strategy of this invention has significant advantages over single-gene expression.

[0102] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A recombinant vector for enhanced oil recovery, characterized by, The vector includes a starting vector and an expression gene integrated into the starting vector; The expressed genes include: surfactant synthesis genes and heat resistance genes; The surfactant synthesis gene is the srfA gene; The heat-resistant gene is selected from the hsp70 gene, hsp60 gene, or dnaK gene.

2. The recombinant vector according to claim 1, characterized in that, The nucleotide sequence of the hsp70 gene is shown in SEQ ID NO: 1; and / or The nucleotide sequence of the hsp60 gene is shown in SEQ ID NO: 2; and / or The nucleotide sequence of the dnaK gene is shown in SEQ ID NO: 3; and / or The nucleotide sequence of the srfA gene is shown in SEQ ID NO:

4.

3. The recombinant vector according to claim 1 or 2, characterized in that, The expressed genes include a surfactant synthesis gene linked to the starting vector and a heat-resistant gene linked to the surfactant synthesis gene.

4. The recombinant vector according to claim 1 or 2, characterized in that, The launch carrier is selected from pNZT1, pBBR1MCS-2 or pUCG18.

5. The recombinant vector according to claim 1 or 2, characterized in that, The recombinant vector further includes a promoter derived from a strong promoter located at the 5' end of the coding sequence of the surfactant synthesis gene; or The promoter originates from the strong promoter of the recombinant vector host.

6. The recombinant vector according to claim 5, characterized in that, The promoter is the strong Bacillus promoter Pbac.

7. The recombinant vector according to claim 5, characterized in that, The promoter sequence of the srfA gene is shown in SEQ ID NO:

5.

8. The recombinant vector according to claim 6, characterized in that, The Pbac strong promoter sequence is shown in SEQ ID NO:

6.

9. The recombinant vector according to claim 1 or 2, characterized in that, The nucleotide sequence of the recombinant vector is shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:

10.

10. An engineered bacterial strain for enhanced oil recovery, characterized in that, The engineered strain can express surfactant synthesis genes and heat resistance genes; The surfactant synthesis gene and the heat resistance gene are derived from the recombinant vector described in any one of claims 1-9.

11. The engineered strain according to claim 10, characterized in that, The initial strain is a dominant oil-producing strain isolated from oilfield produced fluid; preferably Bacillus.

12. The method of constructing an engineered strain of claim 10 or 11, characterized in that, The method includes: introducing a recombinant vector into an initial strain to transform it into an engineered strain.

13. A microbial agent comprising the engineered strain of claim 10 or 11.

14. The application of the engineered strain of claim 10 or 11 or the bacterial agent of claim 12 in crude oil recovery from oil reservoirs.

15. Use according to claim 14, characterized in that, The application of the engineered strain or the bacterial agent in crude oil recovery from reservoirs at 45-65℃.

16. Use according to claim 14 or 15, characterized in that, Application of the engineered strain or the bacterial agent in the recovery of crude oil from reservoirs with a salinity of 60-200 g / L.

17. A method of crude oil recovery characterized by, The method comprises inoculating the engineered strain of claim 10 or 11 or the microbial agent of claim 13 into an oil reservoir.

Images