Sulfur reduction geobacter capable of fixing CO2 to produce 1-acetone as well as construction method and application of sulfur reduction geobacter

Abstract

The invention discloses a sulfur reduction geobacter strain capable of fixing CO2 to produce 1-acetone as well as a construction method and application of the sulfur reduction geobacter strain, and belongs to the technical field of biological genetic engineering. According to the invention, plasmid DNA (deoxyribonucleic acid) carrying an exogenous related gene for producing 1-acetone is introduced into the strain through a genetic engineering method, and a metabolic enzyme related to synthesis of 1-acetone is successfully expressed, so that a pure culture sulfur reduction geobacter strain TZ-19 capable of fixing CO2 and synthesizing 1-acetone is obtained; the problem that the process of producing 1-acetone through CO2 fixation by using the sulfur reduction geobacter is changed from nothing to nothing is solved, and a development prospect is provided for large-scale production by using a bioreactor and microbial fermentation in the fields of green chemistry and sustainable energy.

Description

Technical Field

[0001] The invention relates to a Geobacter sulphurreducens strain capable of fixing CO2 to produce 1-acetone and a construction method and application thereof, belonging to the technical field of biological gene engineering. Background Art

[0002] Microbial fermentation is the process of converting raw materials into organic compounds through metabolism by microorganisms under specific conditions, and plays an important role in modern biomanufacturing.

[0003] The microbial fermentation technology of ketone compounds is becoming more and more mature, especially in the fields of biofuels, bio-based chemicals and medicine. In order to improve the yield and efficiency of fermentation products, modern genetic engineering technology is widely used to transform the metabolic pathways of microorganisms through gene editing or synthetic biology, so that they can more efficiently convert carbon sources into target products.

[0004] For the production of ketone compounds, the traditional fermentation yield is low, but by constructing microbial strains with strong tolerance and high metabolic efficiency, it is expected to increase the concentration of the target product. In recent years, the industrialization trend of microbial fermentation technology for producing ketone compounds has gradually strengthened, especially some anaerobic fermentation technologies and anaerobic bacterial co-culture systems have made significant progress in solving the problem of by-product inhibition. The optimization of bioreactors also provides technical support for large-scale production, making microbial fermentation have broad prospects in the fields of green chemistry and sustainable energy.

[0005] Most of the research and application of microbial fermentation to produce ketones and other compounds remain on traditional model microorganisms such as Escherichia coli and yeast, and the fermentation production is carried out under laboratory conditions, which is costly and difficult to apply to large-scale industrial production. In recent years, the anaerobic microorganism Geobacillus sulfurreducens, as an electrogenic microorganism with a genetic operating system, has attracted extensive research due to its excellent electrical conductivity, ability to absorb electrons from electron donors in the environment and synthesize organic matter in its body. Since most of the research on Geobacillus sulfurreducens is still at the stage of wild-type strains and mixed cultures of multiple strains, it is impossible to artificially control the metabolism and production of the strains, let alone apply them to industrial production. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a strain of Geobacter sulphurreducens capable of fixing CO2 to produce 1-acetone and a construction method and application thereof.

[0007] In order to solve the above technical problems, the technical solution principle adopted by the present invention is: Traditional microbial fermentation to produce alcohol, ketones and other organic substances mostly uses microorganisms such as yeast. This type of microorganism needs to use organic carbon in the environment as a carbon source for heterotrophic growth, and has strict requirements on the culture environment. As an environmental autotrophic microorganism, Geobacter sulfur-reducing has a strong ability to adapt to the environment and absorb electron donors in the environment, which can effectively reduce the impact of the environment on microbial fermentation and the cost of culture and fermentation. After adaptive evolution of environmental pressure, the pure cultured Geobacter sulfur-reducing CO2-fixing strain was introduced into a plasmid pWU54 that can express the gene kivD encoding α-ketoisovalerate decarboxylase and the alcohol dehydrogenase gene adh2 by genetic engineering. The evolved strain successfully fixed CO2 and produced 1-acetone.

[0008] Specifically, the present invention provides an application of a gene kivD encoding α-ketoisovalerate decarboxylase and a gene adh2 for alcohol dehydrogenase to genetically modify a microorganism, wherein the nucleic acid sequence of the gene kivD encoding α-ketoisovalerate decarboxylase is shown as SEQ ID NO.1, and the nucleic acid sequence of the gene adh2 for alcohol dehydrogenase is shown as SEQ ID NO.2.

[0009] The present invention provides a plasmid pWU56, which contains the nucleic acid sequences shown in SEQ ID NO.1 and SEQ ID NO.2. The construction method of the plasmid pWU56 comprises the following steps: KpnI2 and BamHI restriction sites were added upstream and downstream of kivD by PCR, and BamHI and HindIII restriction sites were added upstream and downstream of adh2 gene. The two genes were fused by ligase reaction to form kivD-adh2 fusion sequence; the fusion sequence was then ligated to pTrc99a plasmid by enzyme ligation reaction to express the new plasmid pWU56.

[0010] The present invention provides a recombinant strain comprising the above plasmid. Preferably, the strain is Geobacter sulfurreducens, which can grow adaptively in a formate-ferric citrate medium (FCF) with formate as the only electron donor and carbon source and ferric citrate as the electron acceptor. The Geobacter sulfurreducens can fix CO2 and synthesize 1-acetone.

[0011] The present invention also provides a strain of Geobacter sulfurreducens that fixes CO2 to produce 1-acetone and a construction method thereof, the process steps comprising: 1) Laboratory adaptive evolution of the strain, 2) Construction of 1-acetone synthesis gene expression vector, 3) Electroporation of the vector into the adaptively evolved strain to form strain TZ-19, 4) Testing the acetone synthesis ability of strain TZ-19.

[0012] Beneficial effects of the present invention: In order to obtain strains suitable for industrial production in microbial reactors, the present invention transforms the metabolic pathways of microorganisms through gene editing or synthetic biology to more efficiently convert carbon sources into target products. By conducting laboratory adaptive evolution on the pure cultured wild-type strain PCA of Geobacter sulfurreducens, an evolved strain that can adaptively grow in formate-ferric citrate medium (FCF) with formate as the only electron donor and carbon source and ferric citrate as the electron acceptor was obtained.

[0013] The strain was sequenced and its metabolism was mutated. The carbon fixation ability of the strain was tested in a culture medium with H2 as the electron donor and CO2 as the sole carbon source. Finally, a plasmid DNA carrying exogenous 1-acetone-related genes was introduced into the strain through genetic engineering and the enzymes related to the synthesis of 1-acetone were successfully expressed. A pure cultured Geobacter sulfurreducens strain TZ-19 capable of fixing CO2 and synthesizing 1-acetone was obtained. The problem of using Geobacter sulfurreducens to fix CO2 and produce 1-acetone from scratch was solved, providing development prospects for large-scale production using bioreactors and microbial fermentation in the fields of green chemistry and sustainable energy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0015] Figure 1 It is a diagram of the adaptive evolution process of microorganisms through repeated generations.

[0016] Figure 2 This is an information chart of 15 single nucleotide mutation sites.

[0017] Figure 3 It is a plasmid sequence map.

[0018] Figure 4 This is a TEM electron microscope image of strain TZ-19.

[0019] Figure 5 This is a diagram of the metabolic process of the TZ-19 strain in synthesizing 1-acetone.

[0020] Figure 6 is the acetone synthesis ability of strain TZ-19.

[0021] Figure 6 A is a graph showing the results of detecting the concentration and titer of 1-acetone in FCF medium; Figure 6 B is a graph showing the results of detecting the concentration and titer of 1-acetone in FCHC medium. DETAILED DESCRIPTION

[0022] Example 1 This embodiment provides a method for constructing Geobacter sulfurreducens that can fix CO2 to produce 1-acetone. The strain constructed in this embodiment is named strain TZ-19, and the process steps include: 1) Laboratory adaptive evolution of the strain, 2) Construction of 1-acetone synthesis gene expression vector, 3) Electroporation of the vector into the adaptively evolved strain to form strain TZ-19, 4) Testing the acetone synthesis ability of strain TZ-19.

[0023] 1. Laboratory Adaptive Evolution of Strains Strains: Wild-type Geobacter sulfurreducens PCA; Formate ferric citrate (FcF) medium formulation; Basic elements: NaHCO3 (sodium bicarbonate): 2.5 g / L, NH4Cl (ammonium chloride): 0.25 g / L, NaH2PO4·H2O (sodium dihydrogen phosphate monohydrate): 0.6 g / L, KCl (potassium chloride): 0.1 g / L, trace element solution: 10 mL / L, vitamin solution: 10 mL / L; Trace element solution: Nitrilotriacetic acid (NTA): 1.5 g / L, MgSO4·7H2O: 3 g / L, MnSO4·H2O: 0.5 g / L, NaCl: 1 g / L, FeSO4·7H2O: 0.1 g / L, CoCl2·6H2O: 0.1 g / L, CaCl2·2H2O: 0.1 g / L, ZnCl2: 0.1 g / L, CuSO4·5H2O: 0.01 g / L, AlK(SO4)2·12H2O: 0.01 g / L, H3BO3: 0.01 g / L, Na2MoO4·2H2O: 0.01 g / L, NiCl2·6H2O: 0.02 g / L, Na2SeO3: 0.02 g / L, Na2WO4·2H2O: 0.02 g / L.

[0024] Formate ferric citrate: sodium formate 10mM, ferric citrate 50mM; Adjust pH to between 6.8 and 7.2.

[0025] FcHC medium: FcF medium with H2 and CO2 added.

[0026] Through repeated passages in a culture medium with formate as the sole carbon source, the process Figure 1, which forced it to mutate and change its metabolism under environmental pressure, turned on the expression of related enzyme genes, enabled it to use formate as an electron donor, and also improved the ability of sulfur-reducing bacteria to absorb electrons. After repeated passages, the strain was sequenced for its whole genome. The ability of the evolved strain to fix CO2 was further tested in FCFHC medium.

[0027] 2.1-Construction of acetone synthesis gene expression plasmid vector Target gene acquisition The Lactococcus lactis gene kivD encoding α-ketoisovalerate decarboxylase and the Saccharomyces cerevisiae alcohol dehydrogenase gene adh2 were downloaded from the KEGG database.

[0028] The KivD gene sequence is shown in SEQ ID NO.1: Adh2 gene is shown in SEQ ID NO.2 KpnI2 and BamHI restriction sites were added to the upstream and downstream of kivD by PCR, and BamHI and HindIII restriction sites were added to the upstream and downstream of adh2 gene, and the two genes were fused by ligase reaction to form a kivD-adh2 fusion sequence. The fusion sequence was then ligated to the pTrc99a plasmid by enzyme digestion and ligation reaction to form a new plasmid pWU56.

[0029] PCR reaction primers and reaction conditions are as follows: kivDupKpnI2:CTggtaccAAATAAAGGAGGGAACATGTATACAGTAGGAGATTAC; kivDdnBamHI:GAggatccTTATGATTTATTTTGTTCAGC; adh2upBamHI:GAggatccAAATAAAGGAGGGAACATGTCTATTCCAGAAACTCAA; adh2dnXbaI:GAtctagaTTATTTAGAAGTGTCAACAACG; transsexual: Temperature: 94-95℃; Time: 15-30 seconds; annealing: Temperature: 50-65°C (set according to the Tm value of the primer); Time: 15-30 seconds; the purpose is to allow the primer to bind to the complementary region of the template DNA.

[0030] extend: Temperature: 72°C (optimal temperature for Taq DNA polymerase); Time: 1 min / kb (based on the target fragment length); The polymerase synthesizes a new DNA strand along the template.

[0031] 3. The vector was electroporated into the strain to form strain TZ-19 after adaptive evolution Electroporation pretreatment: Cell culture: Geobacter cells were cultured in acetate-Fe(III) citrate-rich medium (FcF) until the logarithmic growth phase (OD 600 About 0.4-0.6).

[0032] Cool cells: Cool the cultured cells on ice for 10-15 minutes.

[0033] Collect cells: Centrifuge the cells at 6500 rpm for 10 min at 4°C and remove the supernatant.

[0034] Wash cells: Wash the cells three times with sterile ice-cold electroporation buffer, centrifuging each time (4°C, 6500 rpm, 10 min) and discarding the supernatant to reduce the salt ion concentration in the cells.

[0035] Resuspend cells: Finally, resuspend the cells in an appropriate amount of ice-cold electroporation buffer to the desired concentration, usually a high concentration cell suspension.

[0036] Preparation of electroporation mixture: Prepare plasmid DNA: Use highly pure plasmid DNA (usually a few hundred nanograms to 1 µg).

[0037] Mix: Mix 20-50 µL of cell suspension with 1-5 µL of plasmid DNA (avoid bubbles) and transfer to an ice-cold electroporation cuvette (usually a cuvette with 0.2 cm electrode gap).

[0038] Post-electroporation processing: Immediately add FcF medium: After the electroporation is completed, immediately add 0.5-1 mL of ice-cold SOC medium (or anaerobic medium) to the electroporation cup and mix gently.

[0039] Recovery: Transfer the mixture to an anaerobic culture flask and recover at 30°C for 4-6 hours to allow cells to resume growth and expression of exogenous DNA.

[0040] Screening: The revived cells are inoculated into a solid culture medium containing selective antibiotics or markers, cultured for several days, and successfully transformed colonies are screened.

[0041] 4. Test the acetone synthesis ability of strain TZ-19.

[0042] The TZ-19 strain was inoculated into FCF medium and FCHC medium respectively, and the OD 600 When the concentration reached 0.2-0.3, its ability to produce 1-acetone was tested by high performance liquid chromatography.

[0043] The following are the test conditions for the production of 1-acetone by HPLC: Column type: Reversed phase C18 column (a common choice, suitable for separation of non-polar compounds) For example: Phenomenex Luna C18 (250 mm × 4.6 mm, 5 µm) or Agilent ZorbaxEclipse Plus C18 Column temperature: 30-40°C (to maintain the stability of the column and avoid peak tailing) Mobile phase Water / Methanol: Water / methanol (60:40, volume ratio) can be used as the mobile phase.

[0044] Mobile phase pH: Usually controlled under acidic conditions, pH about 2-4, adjusted using sulfuric acid or phosphoric acid.

[0045] Flow rate: Typically 0.5-1.0 mL / min. Higher flow rates reduce analysis time but may reduce separation; lower flow rates help improve resolution.

[0046] Detector: Commonly used HPLC detectors include ultraviolet-visible light detectors (UV-VIS) 1-UV absorption of acetone: The wavelength was set to 215 nm.

[0047] 5 strains TZ-19 test results 1. Laboratory Adaptive Evolution of Strains The results of whole genome sequencing of the evolved strain showed that compared with the wild-type Geobacter sulfurreducens PCA, the adaptively evolved strain had 15 single nucleotide mutation sites. Figure 2 The information shown.

[0048] 2.1-Construction of acetone synthesis gene expression plasmid vector Figure 3 Plasmid sequence map 3. The vector was electroporated into the adaptively evolved strain to form strain TZ-19. Figure 4 This is a TEM electron microscope image of strain TZ-19. Figure 5 This is the metabolic process of the TZ-19 strain synthesizing 1-acetone.

[0049] 4. Test the acetone synthesis ability of strain TZ-19. See Figure 6 , Figure 6 is the acetone synthesis ability of strain TZ-19, Figure 6 A and Figure 6 B is the result of detecting the concentration and titer of 1-acetone in FCF medium and FCHC medium, respectively. The results show that strain TZ-19 can use formate as an electron donor to synthesize 1-acetone in FCF, and can also fix CO2 to synthesize 1-acetone in FCHC medium.

[0050] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A Geobacter sulfurreducens, characterized in that The Geobacter sulfurreducens can adaptably grow in a formate-ferric citrate medium with formate as the only electron donor and carbon source and ferric citrate as the electron acceptor, and contains the nucleic acid sequences shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The Geobacter sulfurreducens according to claim 1, characterized in that The Geobacter sulfurreducens is capable of fixing CO2 and synthesizing 1-acetone.

3. The method for constructing Geobacter sulfurreducens according to claim 1, characterized in that: The steps include: 1) Laboratory adaptive evolution of strains; 2) Construction of 1-acetone synthesis gene expression vector; 3) The vector was electroporated into the adaptively evolved strain to form strain TZ-19; 4) Test the acetone synthesis ability of strain TZ-19.

4. The method for constructing Geobacter sulfurreducens according to claim 3, characterized in that: The specific steps are as follows: 1) Laboratory adaptive evolution of strains Strains: Wild-type Geobacter sulfurreducens PCA; Prepare ferric formate citrate medium with the following formula: Basic elements: sodium bicarbonate: 2.5 g / L, ammonium chloride: 0.25 g / L, sodium dihydrogen phosphate monohydrate: 0.6 g / L, potassium chloride: 0.1 g / L, trace element solution: 10 mL / L, vitamin solution: 10 mL / L; Trace element solution: Nitrilotriacetic acid: 1.5 g / L, MgSO4·7H2O: 3 g / L, MnSO4·H2O: 0.5 g / L, NaCl: 1 g / L, FeSO4·7H2O: 0.1 g / L, CoCl2·6H2O: 0.1 g / L, CaCl2·2H2O: 0.1 g / L, ZnCl2: 0.1 g / L, CuSO4·5H2O: 0.01 g / L, AlK(SO4)2·12H2O: 0.01 g / L, H3BO3: 0.01 g / L, Na2MoO4·2H2O: 0.01 g / L, NiCl2·6H2O: 0.02 g / L, Na2SeO3: 0.02 g / L, Na2WO4·2H2O: 0.02 g / L; Formate ferric citrate: 10 mM sodium formate, 50 mM ferric citrate, adjust pH to between 6.8 and 7.2; FcHC medium: H2 and CO2 are added to the FcF medium. 2) Construction of 1-acetone synthesis gene expression plasmid vector Obtaining target gene: The Lactococcus lactis gene kivD encoding α-ketoisovalerate decarboxylase and the Saccharomyces cerevisiae alcohol dehydrogenase gene adh2 were downloaded from the KEGG database, and the sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; KpnI2 and BamHI restriction sites were added to the upstream and downstream of kivD gene by PCR, and BamHI and HindIII restriction sites were added to the upstream and downstream of adh2 gene. The two genes were fused by ligase reaction to form a kivD-adh2 fusion sequence; the fusion sequence was then ligated to the pTrc99a plasmid by enzyme digestion and ligation reaction to form a new plasmid pWU56; PCR reaction primers and reaction conditions are as follows: kivDupKpnI2:CTggtaccAAATAAAGGAGGGAACATGTATACAGTAGGAGATTAC kivDdnBamHI:GAggatccTTATGATTTATTTTGTTCAGC adh2upBamHI:GAggatccAAATAAAGGAGGGAACATGTCTATTCCAGAAACTCAA adh2dnXbaI:GAtctagaTTATTTAGAAGTGTCAACAACG Denaturation: Temperature: 94-95°C; Time: 15-30 seconds Annealing: temperature: 50-65°C; time: 15-30 seconds; the purpose is to allow the primer to bind to the complementary region of the template DNA; Extension: temperature: 72°C; time: 1 min / kb; 3) The vector was electroporated into the strain to form strain TZ-19 after adaptive evolution Electroporation pretreatment: Cell culture: in an acetic acid-Fe 3+ Geobacter cells were cultured in citrate medium to the logarithmic growth phase; Cooling cells: Cool the cultured cells on ice for 10-15 minutes; Collect cells: centrifuge the cells at 4°C, 6500 rpm for 10 min and remove the supernatant; Wash cells: Wash cells 3 times with sterile ice-cold electroporation buffer, centrifuge each time and discard the supernatant to reduce the concentration of salt ions in the cells; Resuspend cells: Finally, resuspend the cells in an appropriate amount of ice-cold electroporation buffer to the desired concentration; Preparation of electroporation mixture: Prepare plasmid DNA: Use highly pure plasmid DNA; Mix: Mix 20-50 µL of cell suspension with 1-5 µL of plasmid DNA and transfer to an ice-cold electroporation cuvette; Post-electroporation processing: Immediately add FcF medium: After the electroporation is completed, immediately add 0.5-1 mL of ice-cold SOC medium to the electroporation cup and mix gently; Recovery: Transfer the mixture to an anaerobic culture bottle and recover at 30°C for 4-6 hours to allow cells to resume growth and expression of exogenous DNA; Screening: The revived cells are inoculated into a solid culture medium containing selective antibiotics or markers, cultured for several days, and successfully transformed colonies are screened; 4) Testing the acetone synthesis ability of strain TZ-19 The TZ-19 strain was inoculated into FCF medium and FCHC medium respectively, and the OD 600 When it reached 0.2-0.3, its ability to produce 1-acetone was tested by high performance liquid chromatography; The following are the test conditions for the production of 1-acetone by HPLC: Column type: reverse phase C18 column; Column temperature: 30-40°C; Mobile phase: water / methanol: Use water / methanol with a volume ratio of 60:40 as the mobile phase; Mobile phase pH: controlled under acidic conditions, pH 2-4, adjusted with sulfuric acid or phosphoric acid; Flow rate: 0.5-1.0 mL / min; Detector: UV-visible detector; 1-UV absorption of acetone: The wavelength was set to 215 nm.

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