Construction of anti-micro-nano bubble escherichia coli and application of anti-micro-nano bubble escherichia coli in carbon sequestration synthesis of succinic acid

Abstract

The invention belongs to the field of bioengineering, and discloses construction of anti-micro-nano bubble escherichia coli and application of the anti-micro-nano bubble escherichia coli in efficient carbon sequestration synthesis of succinic acid. Escherichia coli for producing succinic acid is used as a starting strain, amyloid fibrin (Curli) is amplified and constructed to form an overexpression plasmid of a key gene CsgA, and the starting strain is introduced to obtain an overexpression strain. According to the constructed micro-nano bubble resisting escherichia coli, CO2 micro-nano bubbles serve as the only CO2 donor, no carbonate or bicarbonate needs to be supplemented in the anaerobic fermentation process, cells have excellent resistance to the CO2 micro-nano bubbles, growth and metabolism of the cells are not remarkably inhibited, good succinic acid production performance is kept, and the method is suitable for industrial production. The introduction of CsgA greatly improves the actual utilization efficiency of CO2 gas by thalli, is beneficial to reducing the production cost of succinic acid and improving the environmental benefits of succinic acid, and has wide social and economic values.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and specifically relates to the construction of micro-nano bubble-resistant Escherichia coli and the application of the same in the efficient carbon fixation and synthesis of succinic acid. Background Art

[0002] Succinic acid is an important C4 platform compound. Studies have shown that microorganisms can anaerobically fix 1 mol of CO using the TCA reduction pathway. 2 Theoretically, it can participate in the synthesis of 1 mol succinic acid and can be used as industrial CO 2 It is an effective means of reducing waste gas emissions and converting them into high-value products, which has important environmental and economic value. 2 The solubility in water is extremely low, and CO is directly introduced during the fermentation process. 2 Most of the gas escapes quickly in the form of bubbles, resulting in the anaerobic biosynthesis of succinic acid to CO 2 Gas utilization efficiency is extremely low. Therefore, anaerobic fermentation to fix carbon and synthesize succinic acid generally uses carbonate or bicarbonate as the main CO 2 donor, and the CO 2 The gas only plays the role of maintaining the anaerobic state, which not only increases the fermentation cost, but also a large amount of overflowed CO 2 The gas forms secondary greenhouse gas emissions, seriously weakening the environmental value of biocarbon fixation to synthesize succinic acid.

[0003] Micro-nano bubbles (MNBs) are a type of microscopic bubbles with a diameter of 100nm to 50μm. Micro-nano bubbles have physical and chemical properties that conventional bubbles do not have, mainly manifested as: the bubble has a large specific surface area, a high zeta potential on the surface, and the surface repulsion that prevents small bubbles from merging into large bubbles and overflowing; the volume of bubbles dissolved in water gradually decreases, and the surface pressure gradually increases, which accelerates the dissolution of gas; micro-nano bubbles rise very slowly in liquids and can stay in liquids for a long time. Therefore, micro-nano bubbles are beneficial to promoting CO 2 dissolves, significantly reducing CO 2escape (Journal of Cleaner Production, 2024, 457: 142230). However, since the charge density accumulated in the double layer increases sharply when the micro-nano bubbles shrink, once the bubbles burst, a large number of ions gathered at the double layer interface will immediately release their chemical energy, forming a large number of reactive oxygen species and free radicals (Langmuir: the ACS Journal of Surfaces and Colloids, 2021, 37(16): 5005-5011). Zhang et al. reported that the free radicals produced by the rupture of MNBs can damage biological macromolecules including DNA, RNA, proteins and lipids, thereby affecting cell viability (Food Reviews International, 2023, 39(7): 4213-4235). Han Zhenyao found through detection that CO 2 The collapse of nanobubbles produces hydroxyl radicals (Research on the stability of nanobubbles and their mechanism and application of producing hydroxyl radicals [D]. Nanning: Guangxi University, 2023). 2 When the concentration of -MNBs reaches 20%, more hydroxyl radicals will be produced, which has a significant inhibitory effect on the growth of Sinomicrobium oceani WH-15 (Process Biochemistry, 2023, 126: 117-124). It can be seen that enhancing the resistance of bacteria to micro-nano bubbles is very important for improving the utilization of CO by cells. 2 Carbon fixation and synthesis of succinic acid are of great value. Therefore, studying how to improve the tolerance of cells to free radicals produced by MNBs is of great significance for optimizing biological processes and improving biotransformation efficiency. These findings not only help us understand the mechanism of action of micro-nanobubbles in biological systems, but also provide potential strategies for the development of new biotechnologies.

[0004] Biofilm is a biological aggregate formed by bacteria on biological and non-biological surfaces. It is composed of extracellular polysaccharides, proteins, and DNA, which can help bacteria enhance their resistance to external environmental pressure. Biofilm is a common lifestyle of bacteria in most ecosystems, providing favorable protection for the survival of bacteria. Curli pili play an important role in the formation of Escherichia coli biofilms. They are protein aggregates located on the cell surface of the extracellular matrix. CsgA is the main subunit of Curli. After bacterial expression, CsgA soluble monomer protein is secreted outside the bacterial cell. After self-assembly, it forms amyloid fibers with a β-folded structure on the bacterial surface, which is related to the initial adhesion of the contact surface of Escherichia coli. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the succinic acid producing strains synthesized by carbon fixation are difficult to tolerate CO 2 This defect of micro-nano bubbles leads to the anaerobic biosynthesis of succinic acid requiring less CO 2 The present invention aims to solve the problems of extremely low gas utilization efficiency and dependence on carbonate supply, and provide a method for constructing recombinant Escherichia coli resistant to micro-nano bubbles and realize its application in efficient carbon fixation and synthesis of succinic acid. 2 The succinate-producing strains with resistance to micro-nano bubbles are applied to carbon fixation and succinate synthesis in microorganisms, thereby greatly improving the cell's CO 2 The actual utilization efficiency of the gas makes CO 2 The gas can completely replace carbonate or bicarbonate for the synthesis of succinic acid during anaerobic fermentation.

[0006] In order to achieve the purpose of the present invention, the present invention provides a construction of micro-nano bubble resistant Escherichia coli and its application in efficient carbon fixation and synthesis of succinic acid. The specific technical scheme is as follows:

[0007] A method for synthesizing succinic acid by using micro-nano bubble-resistant Escherichia coli; wherein the micro-nano bubble-resistant Escherichia coli expresses the CsgA gene; and the synthesizing succinic acid by using CO 2 Micro-nano bubbles synthesize succinic acid. Among them, the CsgA gene is a key gene for anti-micro-nano bubbles, which is a key gene for the formation of amyloid fibril CsgA subunit and is closely related to the β-folding structure of Curli.

[0008] Preferably, the micro-nano bubbles are the only CO 2 The supply method is used, and no carbonate or bicarbonate is provided during the fermentation process, and the carbon fixation efficiency of Escherichia coli is improved by combining the pressurized fermentation method. The micro-nano bubble-resistant Escherichia coli is an overexpression recombinant strain that has the ability to produce succinic acid and contains the key gene CsgA of amyloid fibrin.

[0009] Wherein, the diameter of the micro-nano bubbles is 100nm-50μm.

[0010] Wherein, the CsgA gene is derived from Escherichia coli. Preferably, the Gene ID of the CsgA gene is 949055.

[0011] Wherein, the starting bacteria of the micro-nano bubble resistant Escherichia coli are succinic acid-producing Escherichia coli.

[0012] Wherein, the starting bacteria is Escherichia coli BER 208, with a deposit number of CCTCCNO:M 2012351, and the depository is China Center for Type Culture Collection, referred to as CCTCC, and the deposit address is: Wuhan University, Wuhan, China.

[0013] The micro-nano bubble-resistant Escherichia coli is constructed according to the following method: amplifying the CsgA gene, cloning it into a plasmid vector to construct a recombinant plasmid, and introducing it into the starting bacteria to obtain Escherichia coli BER208-CsgA.

[0014] Wherein, the plasmid vector is any one of pDonor, pJN105, pUCP20 or pHERD20T, preferably pDonor plasmid.

[0015] Further preferably, the recombinant plasmid is constructed in the following manner:

[0016] (1) Using the Escherichia coli genome as a template, the CsgA gene fragment was amplified using a PCR amplification system, and the PCR product fragment was recovered;

[0017] (2) Using plasmid pDonor as a template, amplifying the vector fragment by PCR system, recovering the PCR product fragment, and connecting the vector fragment and gene fragment recovered after electrophoresis of the PCR product by homologous recombination connection system to connect the vector fragment and gene fragment amplified by PCR;

[0018] (3) The ligation product was treated with CaCl 2 The transformation method was used to transform into E.coli DH5α, and positive clones were selected for colony PCR verification. The correct positive clones were cultured and the recombinant plasmids were extracted.

[0019] The carbon fixation and synthesis of succinic acid by the micro-nano bubble-resistant Escherichia coli is carried out as follows: the micro-nano bubble-resistant Escherichia coli is inoculated into a fermentation tank filled with a fermentation medium, and the initial OD 550 The temperature is 0.4-0.6, an inducer is added, and CO in the form of micro-nano bubbles is introduced into the fermenter. 2 After the air is replaced, CO 2 The gas maintains a constant absolute pressure in the fermentation tank, and fermentation is carried out in a closed-gas constant-pressure mode. When the glucose concentration in the fermentation liquid is lower than 1g / L, fermentation is stopped.

[0020] Wherein, the fermentation medium comprises: betaine 0.07-0.17 g / L, (NH 4 ) 2 HPO 42.1-3.1g / L, KCl 0.1-0.2g / L, NH 4 H 2 PO 4 0.82-0.92 g / L, MgSO 4 0.15-0.24 g / L, FeCl 3 1.1-1.8g / L, CoCl 2 0.2-0.35g / L, CuCl 2 0.1-0.2g / L, ZnCl 2 0.3-0.5g / L, Na 2 MoO 4 0.4-0.6g / L, MnCl 2 0.2-0.45g / L, H 3 BO 3 0.07-0.09 g / L, glucose 60-100 g / L; preferably, the fermentation medium has a formula of: betaine 0.12 g / L, (NH 4 ) 2 HPO 4 2.6g / L, KCl 0.15g / L, NH 4 H 2 PO 4 0.87g / L, MgSO 4 7H 2 O0.37g / L, FeCl 3 6H 2 O 2.4g / L, CoCl 2 ·2H 2 O 0.3g / L, CuCl 2 ·2H 2 O 0.15g / L, ZnCl 2 ·4H 2 O0.5g / L, Na 2 MoO 4 ·2H 2 O 0.5g / L, MnCl 2 ·4H 2 O 0.5g / L, H 3 BO 3 0.075g / L, glucose 60-100g / L.

[0021] The fermentation temperature is 35-38°C and the fermentation pH is 6.5-6.8, preferably 37°C.

[0022] The inducer is D-arabinose, and the concentration added to the culture medium is 3-8 g / L, preferably 5 g / L;

[0023] The absolute pressure is constant at 0.14-0.2Mpa, and the CO 2 The air intake rate was 0.005-0.1 vvm to maintain a constant pressure.

[0024] The fermentation pH is controlled by adding alkali solution, and the alkali solution is a KOH aqueous solution or a NaOH aqueous solution, and its concentration is 5-8 mol / L, preferably 5 mol / L.

[0025] Preferably, the micro-nano bubble resistant Escherichia coli seed solution is inoculated into a fermentation tank, wherein the OD of the seed solution is 550 ≥4.0, inoculation amount is 5%~10% v / v.

[0026] Further preferably, the specific method of synthesizing succinic acid by carbon fixation of the micro-nano bubble resistant Escherichia coli is as follows: fresh fermentation medium is added to a fermentation tank and sterilized by high pressure steam. CO in the form of micro-nano bubbles is introduced into the fermentation tank by a micro-nano bubble generator. 2 The gas was used to replace the air, and then the seed solution of the anti-micro-nano bubble Escherichia coli was introduced into the fermentation tank to make the initial bacterial concentration OD 550 When the temperature reaches 0.4-0.6, the inducer is then added; when all the air in the fermentation tank is replaced by CO 2 After the gas replacement is completed, close the exhaust outlet and replenish CO through the micro-nano bubble generator. 2 The absolute pressure in the fermentation tank is maintained constant at 0.14-0.2Mpa, and the pH is controlled at 6.5-6.8 by adding alkali solution, and anaerobic fermentation is carried out to fix carbon and synthesize succinic acid. For other relevant fermentation operation parameters, please refer to patent CN117551706A;

[0027] Beneficial effects:

[0028] The present invention provides a method for constructing micro-nano bubble resistant Escherichia coli and explores its application in efficient carbon fixation and succinic acid synthesis. By constructing a recombinant plasmid with a key gene plasmid of micro-nano bubble resistance, the recombinant plasmid is transferred into succinic acid-producing Escherichia coli to construct an overexpression strain with strong ability to form amyloid fibrils (Curli). In the process of anaerobic carbon fixation and succinic acid synthesis using the micro-nano bubble resistant Escherichia coli strain, compared with the starting strain, the micro-nano bubble resistant strain can tolerate micro-nano bubbles in a pressurized fermentation mode, can maintain good succinic acid production performance, and CO 2 The utilization efficiency is significantly improved, and no carbonate or bicarbonate is added during the fermentation process, which is beneficial to reducing the fermentation cost of bio-based succinic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0030] Figure 1 This is the electrophoresis band diagram of the amplified gene CsgA and pDonor vector fragment. In the figure, M: 5kb marker; Lane 1 is the product of the amplified gene CsgA; Lane 2 is the amplified pDonor vector fragment.

[0031] Figure 2 It is the electrophoresis band of the recombinant bacterial colony PCR; M in the figure: 5kb marker.

[0032] Figure 3 This is the electrophoresis band of colony PCR of the recombinant bacterium BER208-CsgA; M in the figure: 2kb marker.

[0033] Figure 4 It is a biofilm produced by recombinant Escherichia coli (crystal violet staining), wherein A is a blank culture medium control without access to bacteria, B is E. coli BER208, and C is recombinant E. coli BER208-CsgA.

[0034] Figure 5 This is a diagram of a micro-nano bubble generator device, including a switch valve 1, an air path interface 2, an air guide metal tube 3, and a porous membrane tube 4.

[0035] Figure 6 This is the curve of anaerobic synthesis of succinic acid by pressurized fermentation under micro-nano bubble aeration system. Figure 6 A is E. coli BER208, Figure 6 B is the recombinant E. coli BER208-CsgA.

[0036] Figure 7 This is the fermentation curve of anaerobic carbon fixation of BER208 acid by recombinant E. coli BER208-CsgA. Figure 7 A is bubbling aeration normal pressure fermentation, Figure 7 B is micro-nano bubble ventilation and pressurized fermentation. DETAILED DESCRIPTION

[0037] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0038] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0039] In the following examples, the seed culture medium and fermentation culture medium used for the recombinant Escherichia coli are composed as follows:

[0040] Seed culture medium: betaine 0.12g / L, KH 2 PO 4 3.5g / L, K 2 HPO 4 ·3H 2 O 6.54 g / L, (NH 4 ) 2 HPO 4 3.5g / L, MgSO 4 7H 2 O 0.25 g / L, CaCl 2 ·2H 2 O 15mg / L, FeCl 3 6H 2 O 1.6g / L, CoCl 2 ·2H 2 O0.2g / L,CuCl 2 ·2H 2 O 0.1g / L, ZnCl 2 ·4H 2 O 0.2g / L, Na 2 MoO 4 ·2H 2 O 0.2g / L, H 3 BO 3 0.05g / L, glucose 20g / L (sterilized separately), sterilized at 115℃ for 20min.

[0041] Fermentation medium: betaine 0.12 g / L, (NH 4 ) 2 HPO 4 2.6g / L, KCl 0.15g / L, NH 4 H 2 PO 4 0.87g / L, MgSO 4 7H 2 O 0.37g / L, FeCl 3 6H 2 O 2.4g / L, CoCl 2 ·2H 2 O 0.3g / L, CuCl 2 ·2H 2 O 0.15g / L, ZnCl 2 ·4H 2 O 0.5g / L, Na2 MoO 4 ·2H 2 O 0.5g / L, MnCl 2 ·4H 2 O 0.5g / L, H 3 BO 3 0.075g / L, glucose 60-100g / L (sterilized separately), sterilized at 115℃ for 20min.

[0042] In the following embodiments, the micro-nano bubble generator is a membrane-tube structure. For detailed information, please refer to the Chinese patent application number CN117551706A. Figure 5 As shown, it includes a gate valve 1, a gas circuit interface 2, a gas-conducting metal tube 3, and a porous membrane tube 4. Among them, the material of the porous membrane tube 4 is ceramic or metal, and the gate valve 1, the gas circuit interface 2, and the gas-conducting metal tube 3 are all made of stainless steel. The distal end of the gas-conducting metal tube 3 is connected to the porous membrane tube 4, and the micropores on the porous membrane tube have a diameter of 50 to 500 nm, which can pass the CO2 into the water. 2 The gas is transformed into micro-nano bubbles with an average diameter of less than 50 μm; the proximal end of the gas-conducting metal tube 3 is connected to the gas path interface 2, and the gas pipe is connected to the other end of the gas path interface 2, so that CO 2 The gas flows from the CO2 cylinder through the gas pipe and gas line interface 2 into the gas guide metal tube 3, and then passes through the porous membrane tube 4 to become CO 2 The gas circuit interface 2 is provided with a gate valve, and the gas flow into the gas circuit can be controlled by adjusting the gate valve. 2 The rate of gas.

[0043] In the following embodiments, the relevant calculation method is as follows:

[0044] Succinic acid production rate: V SA =m SA / (V×t);

[0045] Succinic acid yield: Y SA =m SA / m glu ;

[0046] CO in atmospheric fermentation under bubbling aeration system 2 Actual utilization rate (adding carbonate): η 1 =(n 3 -n 4 ) / (n 1 -n 2 );

[0047] CO in atmospheric fermentation under bubbling aeration system 2 Actual utilization rate (without adding carbonate): η 1 =n3 / (n 1 -n 2 );

[0048] CO2 production from pressurized fermentation under micro-nano bubble aeration system 2 Actual utilization rate: η 2 =n 3 / (n 1 -n 2 -n 5 ).

[0049] Among them, V SA is the succinic acid production rate (g / (L . h));m SA is the yield of succinic acid (g); t is the fermentation period (h); V is the final volume of the fermentation broth (L); Y SA is the yield of succinic acid (g / g); m glu is the mass of glucose consumed in fermentation (g); η 1 CO is the CO produced by atmospheric fermentation under bubbling aeration system 2 Actual utilization rate; η 2 CO2 production from pressurized fermentation under micro-nano bubble aeration system 2 Actual utilization rate; n 1 CO is the input during the fermentation process 2 (mol); n 2 CO contained in the volume of the tank above the fermentation liquid level 2 (mol); n 3 is the amount of succinic acid produced (mol); n 4 is the amount of succinate synthesized by cells consuming carbonate (mol); n 5 The CO2 that escapes from the tank due to insufficient sealing of the tank during the pressurized fermentation process 2 Amount (mol). Among them, CO in the pressurized system 2 Volumes were converted to standard atmospheres for calculations.

[0050] In the following examples, the crystal violet staining characterization method of the Escherichia coli product membrane is as follows:

[0051] The overnight cultured E. coli suspension was diluted in fresh LB liquid medium at a volume ratio of 1% and dispensed into wells containing 5 g / L D-arabinose, with a blank LB liquid medium without bacterial cells as a negative control. The well plate was sealed and placed in an incubator at 30°C for 72 hours. At the end of the incubation, the floating bacteria were removed and the wells were incubated with PBS solution (NaCl 8 g / L, KCl 0.2 g / L, Na 2 HPO 4 1.44g / L, KH 2 PO4 The plate was washed three times with PBS solution (NaCl 8 g / L, KCl 0.2 g / L, Na 2 HPO 4 1.44g / L, KH 2 PO 4 0.24 g / L, pH = 7.2) to remove excess crystal violet, washed three times, dried and photographed.

[0052] In the following examples, the Escherichia coli BER208 has a deposit number of CCTCCNO: M 2012351, and the full name of the depository is China Center for Type Culture Collection, abbreviated as CCTCC, and the address is: Wuhan University, Wuhan, China.

[0053] Example 1

[0054] In this embodiment, the specific method for constructing recombinant Escherichia coli is as follows:

[0055] 1. Amplification of CsgA gene fragment:

[0056] Escherichia coli BL21 (DE3) (purchased from General Biotechnology (Anhui) Co., Ltd.) was used as a template to amplify the Escherichia coli CsgA gene fragment (Gene ID: 949055).

[0057] The upstream primer csgA-Primer 1 sequence is:

[0058] 5'-GGGCTAGAAATAATTTTGATGAAACTTTTAAAAGTAGCAGCAATTGCA-3'(SEQ ID NO.1)

[0059] Downstream primer csgA-Primer 2 sequence:

[0060] 5'-CAATAACTGCCTTAAAAAAATTAGTACTGATGAGCGGTCGC-3'(SEQ ID NO.2)

[0061] The PCR amplification system is: 1 μL of E. coli BL21 (DE3) solution and ddHO 2 20 μL of O was boiled in water for 5 min, and 1 μL of the primers were taken out, including 2 μL of upstream csgA-Primer 1 and downstream csgA-Primer 2, 2×Phanta Max Master Mix 25 μL, and ddHO.2 O 20μL, total system 50μL.

[0062] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 30 s, 30 cycles, and complete extension at 72°C for 5 min.

[0063] The PCR product was subjected to PAGE experiment, and the correct amplified band was recovered using the column-type gel recovery kit. After gel recovery, the gel image was identified (such as Figure 1 Lane 1) is shown.

[0064] 2. Amplify pDonor vector fragment

[0065] Plasmid pDonor (purchased from GenScript Biotech Co., Ltd.) was used as a template to amplify the vector fragment. The sequence of the upstream primer pDonor-Primer 1 is:

[0066] 5'-TTTTTTTAAGGCAGTTATTGGTGCCC-3'(SEQ ID NO.3)

[0067] Downstream primer pDonor-Primer 2 sequence:

[0068] 5'-CAAAATTATTTCTAGCCCAAAAAAACGGG-3'(SEQ ID NO.4)

[0069] The PCR amplification system was as follows: 1 μL of plasmid pDonor template, 2 μL of upstream primers pDonor-Primer 1 and downstream primers pDonor-Primer 2, 25 μL of 2×Phanta Max Master Mix, ddH 2 O 20μL, total system 50μL. PCR reaction program: 95℃ pre-denaturation for 3min, 95℃ denaturation for 15s; then 60℃ annealing for 15s, 72℃ extension for 30s, 30 cycles; 72℃ full extension for 5min.

[0070] The PCR product was subjected to PAGE experiment, and the correct amplified band was recovered using the column-type gel recovery kit. After gel recovery, the gel image was identified (such as Figure 1 Lane 2 shown).

[0071] 3. Construction of recombinant plasmid

[0072] The ligation reaction system is: linearized vector pDonor 2μL (about 100ng), gene fragment csgA 1μL (about 40ng), 5×CE II Buffer 4μL, Exnase II 2μL, ddH2 O 11 μL, total volume 20 μL. The recombinant plasmid pDonor-csgA was obtained by ligation.

[0073] 4. Construction of recombinant Escherichia coli

[0074] Transformation competent cell experiment: Take 20μL of recombinant plasmid pDonor-csgA and add it to the prepared 100μL E. coli DH5α competent cells, flick it evenly with your fingers, put it in an ice bath for 30min, heat shock it in a 42℃ water bath for 90s, quickly put it on ice for 2min, then add 800μL of fresh LB medium, shake and culture it at 37℃, 200r / min for 1h, centrifuge it at 5000rpm for 3min, and discard 800μL of supernatant. Resuspend the bacteria with the remaining culture medium, spread it on the surface of LB solid culture medium containing resistance (ampicillin, 100μg / mL) with a sterile coating rod, and culture it in a 37℃ incubator for 12h; perform colony PCR and electrophoresis identification ( Figure 2 ), screen out the correct positive clones; inoculate the verified positive clones in LB test tubes containing resistance (ampicillin, 100 μg / mL) and culture them to extract the recombinant plasmid for sequencing, and then transform the extracted recombinant plasmid into E. coli BER208 competent cells according to the same transformation method, screen the positive clones, that is, culture the colony PCR verification and perform PAGE identification ( Figure 3 ); Through identification, the positive clone containing the recombinant plasmid was successfully screened out, and finally the single clone colony was preserved and stored in a -80℃ refrigerator, named recombinant E. coli BER208-CsgA. The crystal violet staining experiment of the biofilm proved that the recombinant E. coli BER208-CsgA could form a stable biofilm, while the starting strain E. coli BER208 could not form a biofilm ( Figure 4 ).

[0075] Example 2

[0076] In this example, the recombinant bacteria E. coli BER208-CsgA prepared in Example 1 was used to synthesize succinic acid by anaerobic carbon fixation (the starting bacteria E. coli BER208 was used as a control), and the specific steps were as follows:

[0077] Experiment 1: The starting strain E. coli BER208 was activated and inoculated into the seed culture medium for expansion culture to obtain E. coli seed liquid (biomass OD 550≥4.0). Add the prepared fermentation medium to a 5L fermenter with a liquid volume of 3L and a glucose concentration of 60g / L in the fermentation medium. Use a micro-nano bubble generator as a micro-dispersed gas supply device, insert it into the tank through the interface of the fermenter, close the inlet valve of the micro-nano bubble generator, and then perform steam sterilization. After sterilization, connect the micro-nano bubble generator to the CO 2 Connect the gas pipeline, open the inlet valve of the micro-nano bubble generator, and supply CO into the fermenter at a ventilation rate of 0.1 vvm. 2 Gas, forming micro-nano bubbles, the exhaust outlet of the fermentation tank remains open, and after it is cooled to the fermentation temperature of 37°C, the cultured recombinant Escherichia coli seed liquid is transferred into the fermentation tank by flame inoculation according to the inoculation amount of 10% v / v, and ventilation is maintained for 20 to 30 minutes. After all the original gas in the fermentation tank is replaced, all the exhaust outlets of the fermentation tank are closed, the air pressure in the tank is increased to 0.18MPa (absolute pressure), and the fermentation is carried out in a closed air constant pressure mode. The intake rate of the micro-nano bubble generator is adjusted to 0.02-0.1vvm to keep the tank pressure constant. The fermentation temperature is 37°C, and the pH value of the fermentation liquid is controlled to 6.8 with 5mol / L KOH during the fermentation process. The stirring speed is 100rpm. The fermentation is terminated after 70h of fermentation, and the residual glucose is less than 1g / L.

[0078] Experiment 2: After activation, the recombinant E. coli BER208-CsgA was inoculated into the seed culture medium for expansion culture to obtain the recombinant E. coli seed solution (biomass OD 550 ≥4.0). Add the prepared fermentation medium into a 5L fermenter with a liquid volume of 3L. The glucose concentration in the fermentation medium is 60g / L. The initial OD 550 The pH value was 0.615, and 5 g / L D-arabinose was added. Other operations were the same as above.

[0079] Table 1 Effect of different strains on succinic acid yield and CO in Example 2 2 The impact of actual utilization

[0080]

[0081] The results are shown in Table 1. When the starting strain was used for fermentation, CO 2 – MNBs are the only CO 2 Source, fermentation for 60h only produced 1.02g / L succinic acid, biomass OD 550 From 0.489 to 0.304, only a small amount of glucose was consumed during the whole process, and the fermentation failed ( Figure 6A). When the recombinant E. coli BER208-CsgA was used for fermentation, the aeration and pressurization fermentation of MNBs was not weakened. The yield of succinic acid reached 44.89 g / L after 60 h of fermentation. The highest biomass OD 550 Reached 6.85, CO 2 The actual utilization rate reached 27.33% ( Figure 6 B). The results showed that the recombinant E. coli could effectively tolerate the free radicals and shear force generated by micro-nano bubbles, and could significantly improve CO while ensuring normal production performance. 2 utilization efficiency.

[0082] Example 3

[0083] In this example, the recombinant bacteria E. coli BER208-CsgA prepared in Example 1 was used to synthesize succinic acid by anaerobic carbon fixation (the starting bacteria E. coli BER208 was used as a control), and the specific steps were as follows:

[0084] Experiment 1: The starting strain E. coli BER208 was activated and inoculated into the seed culture medium for expansion culture to obtain E. coli seed liquid (biomass OD 550 ≥4.0). The prepared fermentation medium was added to a 5L fermenter with a liquid volume of 3L, and the glucose concentration in the fermentation medium was 50g / L. The micro-nano bubble supply and replacement method was the same as in Example 2. After all the original gas in the fermenter was replaced, all exhaust outlets of the fermenter were closed, and the air pressure in the tank was increased to 0.14MPa (absolute pressure). The fermentation was carried out in a closed air constant pressure mode, and the air intake rate of the micro-nano bubble generator was adjusted to 0.02-0.1vvm to keep the tank pressure constant. The fermentation temperature was 37°C, and 5mol / L NaOH was used to control the pH value of the fermentation liquid to 6.8 during the fermentation process. The stirring speed was 120rpm. The fermentation was terminated after 60h of fermentation when the residual glucose was less than 1g / L.

[0085] Experiment 2: After activation, the recombinant E. coli BER208-CsgA was inoculated into the seed culture medium for expansion culture to obtain the recombinant E. coli seed solution (biomass OD 550 ≥4.0). Add the prepared fermentation medium into a 5L fermenter with a liquid volume of 3L. The glucose concentration in the fermentation medium is 60g / L. The initial OD 550 The pH value was 0.457, and 5 g / L D-arabinose was added. Other operations were the same as above.

[0086] Table 2 Succinic acid yield and CO of different strains in Example 3 2 Actual Utilization

[0087]

[0088] The results are shown in Table 2. When the starting strain E. coli BER208 was used for fermentation, CO 2 – MNBs are the only CO 2 Source, fermentation for 60h only produced 0.98g / L succinic acid, biomass OD 550 The glucose content of MNBs decreased from 0.423 to 0.298, and only a small amount of glucose was consumed during the whole process, resulting in the failure of fermentation. However, when the recombinant E. coli BER208-CsgA was used for fermentation, the MNBs aeration and pressurization fermentation did not weaken, and the yield of succinic acid reached 84.58%, with the highest biomass OD 550 Reached 5.44, CO 2 The actual utilization rate reached 26.55%. The results showed that the recombinant E. coli can effectively tolerate the free radicals and shear force generated by micro-nano bubbles, and can significantly improve CO while ensuring normal production performance. 2 utilization efficiency.

[0089] Example 4

[0090] In this embodiment, a pressurized micro-nano bubble method is used to supply the recombinant bacteria E. coli BER208-CsgA with anaerobic carbon fixation to synthesize succinic acid (using a normal pressure bubbling method as a comparison), and the specific steps are as follows:

[0091] Experiment 1: After activation, the recombinant E. coli BER208-CsgA was inoculated into the seed culture medium for expansion culture to obtain the recombinant E. coli seed solution (biomass OD 550 ≥4.0). The prepared fermentation medium was added to a 5L fermenter, with a liquid volume of 3L and a glucose concentration of 50g / L in the fermentation medium. In a 5L fermenter, the cultured recombinant E. coli seed solution was inoculated into the fermenter at an inoculation rate of 10% v / v, and its initial OD 550 The concentration of D-arabinose was 0.432, and 5 g / L D-arabinose was added. CO was supplied to the fermenter by conventional bubbling ventilation. 2 Gas, namely CO 2 The gas pipeline is passed into the liquid fermentation medium through the conventional air inlet pipe of the fermenter, and millimeter-level bubbles are formed under the shear of the stirring paddle. The air inlet flow rate is controlled at 0.15vvm, the fermentation temperature is 37°C, and 2.5mol / L KOH and 2.5mol / L K 2 CO 3 (The volume ratio is 1:1) The pH value of the fermentation liquid is controlled at 6.8, the stirring speed is 100 rpm, and the fermentation is terminated when the residual glucose is lower than 1 g / L after 72 hours of fermentation.

[0092] Experiment 2: After activation, the recombinant E. coli BER208-CsgA was inoculated into the seed culture medium for expansion culture to obtain the recombinant E. coli seed solution (biomass OD 550 ≥4.0). The configured fermentation medium is added to a 5L fermentor, the liquid volume is 3L, the glucose concentration in the fermentation medium is 50g / L, and 5g / L D-arabinose is added. The micro-nano bubble supply and replacement method are the same as in Example 2. The exhaust outlet of the fermentor is kept open. After it is cooled to the fermentation temperature of 37°C, the cultured recombinant Escherichia coli seed liquid is transferred into the fermentor by flame inoculation according to the inoculation amount of 10% v / v, and ventilation is maintained for 20 to 30min. After all the original gas in the fermenter is replaced, all the exhaust outlets of the fermenter are closed, and the air pressure in the tank is increased to 0.18MPa (absolute pressure). The fermentation is carried out in a closed air constant pressure mode, and the intake rate of the micro-nano bubble generator is adjusted to 0.01-0.08vvm to keep the tank pressure constant. The fermentation temperature is 37°C, and the pH value of the fermentation liquid is controlled to 6.8 by 5mol / L KOH during the fermentation process, and the stirring speed is 100rpm. Fermentation is terminated after 75h, and the residual glucose is less than 1g / L.

[0093] Table 3 Effects of different ventilation modes on succinic acid yield and CO in E.coli BER208-CsgA 2 The impact of actual utilization

[0094]

[0095] The results are shown in Table 3 and Figure 7 As shown in the figure, compared with the bubbling aeration fermentation under normal pressure, the succinic acid production performance of E. coli BER208-CsgA was not weakened by micro-nano bubbles, no carbonate or bicarbonate was added during the pressurized fermentation process, the alkali cost for succinic acid fermentation was reduced by 12%, and the cell's CO 2 The actual utilization efficiency of the gas increased by 5.65 times, significantly reducing the CO 2 Secondary emissions of greenhouse gases caused by gas escape.

[0096] Example 5

[0097] In this embodiment, a pressurized micro-nano bubble method is used to supply the recombinant bacteria E. coli BER208-CsgA with anaerobic carbon fixation to synthesize succinic acid (using a normal pressure bubbling method as a comparison), and the specific steps are as follows:

[0098] Experiment 1: After activation, the recombinant E. coli BER208-CsgA was inoculated into the seed culture medium for expansion culture to obtain the recombinant E. coli seed solution (biomass OD 550≥4.0). The prepared fermentation medium was added to a 5L fermenter, with a liquid volume of 3L and a glucose concentration of 80g / L in the fermentation medium. In a 5L fermenter, the cultured recombinant E. coli seed solution was inoculated into the fermenter at an inoculation rate of 10% v / v, and its initial OD 550 The concentration of D-arabinose was 0.518, and 5 g / L D-arabinose was added. CO was supplied to the fermenter by conventional bubbling ventilation. 2 Gas, namely CO 2 The gas pipeline is introduced into the liquid fermentation medium through the conventional air inlet pipe of the fermenter, and millimeter-level bubbles are formed under the shear of the stirring blade. The air inlet flow rate is controlled at 0.2vvm, the fermentation temperature is 37°C, and 2.5mol / L NaOH and 2.5mol / L Na 2 CO 3 (The volume ratio is 1:1) The pH value of the fermentation liquid is controlled at 6.8, the stirring speed is 80 rpm, and the fermentation is terminated when the residual glucose is lower than 1 g / L after 100 h of fermentation.

[0099] Experiment 2: After activation, the recombinant E. coli BER208-CsgA was inoculated into the seed culture medium for expansion culture to obtain the recombinant E. coli seed solution (biomass OD 550 ≥4.0). The configured fermentation medium is added to a 5L fermentor, the liquid volume is 3L, the glucose concentration in the fermentation medium is 80g / L, and 5g / L D-arabinose is added. The micro-nano bubble supply and replacement method are the same as in Example 2. The exhaust outlet of the fermentor is kept open. After it is cooled to the fermentation temperature of 37°C, the cultured recombinant Escherichia coli seed liquid is transferred into the fermentor by flame inoculation according to the inoculation amount of 10% v / v, and ventilation is maintained for 20 to 30min. After all the original gas in the fermenter is replaced, all the exhaust outlets of the fermenter are closed, and the air pressure in the tank is increased to 0.18MPa (absolute pressure). The fermentation is carried out in a closed air constant pressure mode, and the air intake rate of the micro-nano bubble generator is adjusted to 0.01-0.08vvm to keep the tank pressure constant. The fermentation temperature is 37°C, and the pH value of the fermentation liquid is controlled to 6.8 by 5mol / L NaOH during the fermentation process, and the stirring speed is 80rpm. Fermentation is terminated after 120h, and the residual glucose is less than 1g / L.

[0100] Table 4 Effect of different ventilation modes on the yield of succinic acid and CO of recombinant Escherichia coli in Example 5 2 The impact of actual utilization

[0101]

[0102] The results are shown in Table 4. Compared with the bubbling aeration fermentation under normal pressure, the succinic acid production performance of the recombinant E. coli BER208-CsgA was not significantly weakened by the micro-nano bubbles, and the yield of succinic acid could still reach 85.84%. No carbonate or bicarbonate was added during the fermentation process, the alkali cost for succinic acid fermentation was reduced by 10.3%, and the cell CO 2 The actual utilization efficiency of the gas increased by 6.92 times, significantly reducing the CO 2 Secondary emissions of greenhouse gases caused by gas escape.

[0103] The present invention provides a method for constructing micro-nano bubble-resistant Escherichia coli and its application in efficiently fixing carbon and synthesizing succinic acid. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. Application of micro-nano bubble resistant Escherichia coli in carbon fixation and succinic acid synthesis; in, The micro-nano bubble-resistant Escherichia coli expresses the CsgA gene; The carbon fixation synthesis of succinic acid is to use CO2 micro-nano bubbles to synthesize succinic acid.

2. The use according to claim 1, characterized in that: The diameter of the micro-nano bubbles is 100nm-50μm.

3. The use according to claim 1, characterized in that: The CsgA gene is derived from Escherichia coli.

4. The use according to claim 1, characterized in that: The starting bacteria of the micro-nano bubble resistant Escherichia coli are succinic acid-producing Escherichia coli.

5. The use according to claim 4, characterized in that: The starting bacteria is Escherichia coli BER 208, and the deposit number is CCTCC NO:M 2012351.

6. The use according to claim 1, characterized in that: The micro-nano bubble-resistant Escherichia coli was constructed according to the following method: amplifying the CsgA gene, cloning it into a plasmid vector to construct a recombinant plasmid, and introducing it into the starting bacteria to obtain the micro-nano bubble-resistant Escherichia coli, which was named Escherichia coli BER208-CsgA.

7. The use according to claim 6, characterized in that: The plasmid vector is any one of pDonor, pJN105, pUCP20 or pHERD20T.

8. The use according to any one of claims 1 to 7, characterized in that: The carbon fixation and synthesis of succinic acid by the micro-nano bubble-resistant Escherichia coli is carried out as follows: the micro-nano bubble-resistant Escherichia coli is inoculated into a fermentation tank filled with a fermentation medium, and the initial OD 550 The pressure is 0.4-0.6, an inducer is added, and CO2 gas in the form of micro-nano bubbles is introduced into the fermentation tank. After the air is replaced, the absolute pressure in the fermentation tank is maintained constant by using CO2 gas in the form of micro-nano bubbles. The fermentation is carried out in a closed air and constant pressure mode. When the glucose concentration in the fermentation liquid is lower than 1g / L, the fermentation is stopped.

9. The use according to claim 8, characterized in that: The fermentation medium comprises: betaine 0.07-0.17 g / L, (NH4)2HPO4 2.1-3.1 g / L, KCl 0.1-0.2 g / L, NH4H2PO4 0.82-0.92 g / L, MgSO4 0.15-0.24 g / L, FeCl3 1.1-1.8 g / L, CoCl2 0.2-0.35 g / L, CuCl2 0.1-0.2 g / L, ZnCl2 0.3-0.5 g / L, Na2MoO4 0.4-0.6 g / L, MnCl2 0.2-0.45 g / L, H3BO3 0.07-0.09 g / L, glucose 60-100 g / L; The fermentation temperature is 35-38°C and the pH is 6.5-6.8; The inducer is D-arabinose, and the concentration added to the culture medium is 3-8 g / L; The absolute pressure is constant at 0.14-0.2Mpa, and the constant pressure is maintained by controlling the CO2 inlet rate in the form of micro-nano bubbles to 0.005-0.1vvm.

10. The use according to claim 9, characterized in that: The fermentation pH is controlled by adding alkali solution, and the alkali solution is a KOH aqueous solution or a NaOH aqueous solution, and its concentration is 5-8 mol / L.

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