CN115725490B - Construction method and application of recombinant Shewanella strain for synthesizing and secreting efficient electron transfer carrier phenazine-1-carboxylic acid - Google Patents

Abstract

The invention discloses a construction method and application of a recombinant Shewanella strain for efficiently synthesizing and secreting an electron transfer vector, and relates to the technical field of biological energy. A construction method of a recombinant Shewanella strain for efficiently synthesizing and secreting an electron transfer vector comprises the following steps: the outer membrane porin gene oprf of pseudomonas and the phenazine-1-carboxylic acid (PCA) biosynthesis gene cluster phzABCDEFG are obtained by PCR, the gene sequence is optimized, a proper promoter, RBS sequence and plasmid expression vector are selected, and the recombinant Shewanella strain SP1 for efficiently synthesizing and secreting the electron transfer vector is obtained by heterologous expression in Shewanella. The recombinant Shewanella strain SP1 for efficiently synthesizing and secreting the electron transfer vector constructed by the invention is a double-plasmid heterologous expression strain, and can synthesize the electron transfer vector PCA in cells, the outer membrane porin expression improves the permeability of cell membranes, promotes the excretion of PCA, further enhances the extracellular electron transfer rate and improves the power density of microbial fuel cells.

Description

A method for constructing a recombinant Shewanella strain that synthesizes and secretes a highly efficient electron transfer carrier, phenazine-1-carboxylic acid, and its use

Technical Field

The invention belongs to the technical field of bioenergy, and in particular relates to a construction method and application of a recombinant Shewanella strain capable of efficiently synthesizing and secreting an electron transfer carrier.

Background technique

Energy shortage and environmental pollution are increasingly serious problems facing us today. Scientists are constantly looking for new technical solutions, among which microbial fuel cells (MFC) are one of them. They are used to generate alternative energy and new devices for environmental waste treatment, and their importance is becoming increasingly apparent.

MFC is a device that uses electrogenic microorganisms as anode catalysts to convert chemical energy in organic matter into electrical energy. The electrogenic ability of microorganisms varies greatly, and electrogenic microorganisms determine the function and application of MFC. Shewanella oneidensis is one of the microorganisms that are widely used in MFC for electrogenic activities. Its metabolic pathway and extracellular electron transfer pathway are well studied. Shewanella oneidensis MR-1 (abbreviated as Shewanella MR-1) is the most widely studied strain in the genus Shewanella in terms of genome sequence annotation and genetic characteristics. Shewanella MR-1 has been shown to be able to use flavin compounds such as riboflavin, phenazine compounds such as phenazine-1-carboxylic acid, and anthraquinone-rich substances such as humic acid as electron transfer carriers to mediate its extracellular electron transfer. However, the ability of wild Shewanella to synthesize electron transfer carriers is limited, and the cell permeability is poor. The electron transfer carriers synthesized intracellularly cannot be quickly transferred to the extracellular space, which severely limits its extracellular electron transfer rate.

Summary of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Shewanella strain that can efficiently synthesize and secrete phenazine-1-carboxylic acid, an electron transfer carrier.

The second object of the present invention is to provide a method for constructing a recombinant Shewanella strain that can efficiently synthesize and secrete electron transfer carriers.

The third object of the present invention is to provide a recombinant Shewanella strain that efficiently synthesizes and secretes electron transfer carriers and its role in electricity production.

The fourth object of the present invention is to provide a recombinant Shewanella strain that can efficiently synthesize and secrete electron transfer carriers for use in promoting cell membrane permeability.

The fifth object of the present invention is to provide a recombinant Shewanella strain that can efficiently synthesize and secrete electron transfer carriers for use in the synthesis of electron carrier phenazine-1-carboxylic acid.

The solution is summarized as follows:

A method for constructing a recombinant Shewanella strain capable of efficiently synthesizing and secreting an electron transfer carrier comprises the following steps: obtaining the outer membrane porin gene oprf of Pseudomonas and the gene cluster phzABCDEFG for controlling the secretion of phenazine-1-carboxylic acid (PCA) from the genome of Pseudomonas aeruginosa PAO1 by PCR, and simultaneously heterologously expressing them in Shewanella to obtain a recombinant Shewanella strain SP1 capable of efficiently generating and transferring electrons.

Beneficial effects: Studies have shown that microbial extracellular electron transfer (EET) is a complex process affected by multiple cellular components. Electron transfer carriers can mediate "intracellular-extracellular" electron transfer, which determines the electron transfer rate between cells and electrodes to a certain extent. PCA, as a highly efficient electron carrier, has a higher efficiency of electron transfer mediated by PCA than riboflavin secreted by MR-1 itself. After PCA is synthesized intracellularly, it is secreted to the extracellular space through the porin OprF and binds to the outer membrane cytochrome MtrC and OmcA. The oxidized PCA outer membrane cytochrome cofactor binds to accept an electron and becomes a reduced PCA. The reduced PCA carries electrons and diffuses to the surface of the anode electrode to release electrons and becomes an oxidized PCA. The oxidized PCA diffuses back to the surface of the cell outer membrane to accept electrons again, and the electrons are transferred between the cell and the electrode in this cycle. Therefore, a recombinant Shewanella strain that efficiently synthesizes and secretes electron transfer carriers is constructed, and the core gene cluster for PCA synthesis and the cell porin OprF are heterologously expressed, so that Shewanella can express porins while synthesizing the electron transfer carrier PCA, thereby enhancing cell permeability and accelerating the cell to secrete the electron transfer carrier PCA to the extracellular space, thereby greatly improving the extracellular electron transfer rate based on "electron transfer carrier mediation", and at the same time improving the electrochemical performance of MFC. The recombinant Shewanella strain SP1 that efficiently synthesizes and secretes electron transfer carriers constructed by the present invention is a double-plasmid heterologous expression strain, which can generate PCA intracellularly, and the expression of the outer membrane porin OprF promotes the efflux of PCA, thereby enhancing extracellular electron transfer and improving the power density of microbial fuel cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a pHG13-oprf plasmid map;

Fig. 2 is a pYYDT-phzABCDEFG plasmid map;

FIG3 is an electrical power density curve of the recombinant Shewanella strain SP1 that efficiently synthesizes and secretes electron transfer carriers;

FIG. 4 is a cell permeability measurement of the recombinant Shewanella strain SP1 that efficiently synthesizes and secretes electron transport carriers.

FIG5 is a concentration standard curve of PCA in HPLC.

FIG. 6 is a HPLC spectrum of the fermentation broth of the recombinant Shewanella strain SP1 that efficiently synthesizes and secretes electron transfer carriers.

Detailed ways

The following is a detailed description of the construction method and use of a recombinant Shewanella strain for efficiently synthesizing and secreting an electron transfer vector provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

The original strain wild-type Shewanella oneidensis MR-1 is referred to as wild-type Shewanella MR-1, which was purchased from ATCC (United States, https://www.atcc.org/) in September 2010 as ATCC 700550 strain.

The present invention will be further described below in conjunction with specific embodiments.

Example 1

A method for constructing a recombinant Shewanella strain that efficiently synthesizes and secretes an electron transfer carrier comprises the following steps: amplifying exogenous genes oprf and phzABCDEFG from the genome of Pseudomonas aeruginosa PAO1. The oprf gene selects the lacUV5 promoter and is connected to the pHG13 vector; the phzABCDEFG gene cluster selects the tac promoter and is connected to the pYYDT vector. Then the constructed plasmid is introduced into wild-type Shewanella to obtain the recombinant Shewanella strain SP1, wherein the nucleotide sequences of the gene oprf and the gene cluster phzABCDEFG are shown in SEQ ID NO.7 and SEQ ID NO.9, the promoter lacUV5 and its RBS sequence are shown in SEQ ID NO.11, and the promoter tac and its RBS sequence are shown in SEQ ID NO.12.

The specific construction method includes the following steps:

1. Amplification of genes oprf and

system:

program:

2. Amplification of the gene cluster phzABCDEFG

system:

program:

3. Construction of expression vector:

Using pHG13 plasmid (SEQ ID NO.5) as template, pHG13-F (SEQ ID NO.13) and pHG13-R (SEQID NO.14) as primers, PCR amplification of linear vector was performed. Finally, the linearized vector fragment pHG13 and the gene of outer membrane porin OprF (SEQ ID NO.7) were connected to obtain the vector pHG13-oprf (SEQ ID NO.8). Sequencing test was correct. The final plasmid map is shown in Figure 1.

Using pYYDT plasmid (SEQ ID NO.6) as template, pYYDT-F (SEQ ID NO.15) and pYYDT-R (SEQID NO.16) as primers, PCR amplification of linear vector was performed. Finally, the linearized vector fragment pYYDT and the gene cluster phzABCDEFG (SEQ ID NO.9) of synthetic PCA were connected to obtain the vector pYYDT-phzABCDEFG (SEQ ID NO.10). Sequencing detection was correct. The final plasmid map is shown in Figure 2.

The constructed plasmid pYYDT-phzABCDEFG was first transferred into Escherichia coli WM3064 (commercial strain), and then combined with Shewanella MR-1 for transfer. The constructed plasmid was transferred into Shewanella MR-1, and colony PCR was performed to verify the growth of a single colony to obtain the recombinant Shewanella strain SO3;

The constructed plasmid pHG13-oprf was first transferred into Escherichia coli WM3064 (commercial strain), and then combined with the constructed Shewanella SO3 to obtain the target strain SP1 with double plasmids.

Shewanella MR-1 needs to be cultured in LB medium at 30°C, while the growth of Escherichia coli WM3064 requires the addition of 0.3M/mL DAP (2,6-diaminopimelic acid) to LB medium at 37°C. When culturing the strains, the corresponding resistance should be added to the medium.

E.coli WM3064 chemical transformation steps:

1. Take 50ul WM3064 competent cells from -80℃ refrigerator and place on ice for 10min;

2. Mix the constructed plasmid pHG13-oprf and plasmid pYYDT-phzABCDEFG with WM3064 competent cells in a 1.5 mL centrifuge tube and place on ice for 30 minutes;

3. Then heat shock in a water bath at 42°C for 90 seconds, and ice bath again for 5 minutes;

4. Add 1 mL of LB medium containing 0.3 M/mL DAP to the above system and culture at 37°C, 200 rpm for 1 hour;

5. Take 200uL of culture solution and spread it on LB plates containing 100mg/L chloramphenicol resistance (plasmid pHG13-oprf) and 50mg/L kanamycin resistance (plasmid pYYDT-phzABCDEFG), and culture at 37℃ for 12 hours;

6. Pick a single colony for PCR verification, and extract the plasmid from the strain with the correct band and perform sequencing verification.

The correct WM3064 strain was verified by overnight culture and then transferred to Shewanella MR-1 by conjugation.

The conjugative transfer steps of the single-plasmid Shewanella strain SO3 are as follows:

1. Take out the E. coli WM3064 glycerol bacteria and S. oneidensis MR-1 glycerol bacteria containing plasmid pYYDT-phzABCDEFG from the -80℃ refrigerator, take an appropriate amount and inoculate it into the corresponding resistance culture medium, and culture it overnight in a shaker at the corresponding temperature and speed (E. coli WM3064, 37℃, 220rpm; S. oneidensis MR-1, 30℃, 200rpm);

2. Pipette 500 μL of E. coli WM3064 and S. oneidensis MR-1 containing plasmid pYYDT-phzABCDEFG into EP tubes, mix well, centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend with LB+100 μg/ml 2,6-diaminopimelic acid (DAP) liquid medium, and place in a 30°C constant temperature incubator for 2 h;

3. Pipette 100 μL of the mixed bacterial solution and spread it evenly on a solid medium containing LB (without DAP, which inhibits the growth of E. coli WM3064) containing 50 mg/L kanamycin resistance, and place it in a constant temperature incubator at 30°C for overnight culture;

4. Use colony PCR to screen the correct transformants, perform sequencing verification, and proceed to the next step of the experiment.

Conjugative transfer of dual-plasmid Shewanella strain SP1:

1. Take out the E. coli WM3064 glycerol bacteria containing the plasmid pHG13-oprf and the SO3 glycerol bacteria containing the pYYDT-phzABCDEFG plasmid from the -80℃ refrigerator, take an appropriate amount and inoculate it into the corresponding resistance culture medium, and culture it overnight in a shaker at the corresponding temperature and speed (E. coli WM3064, 37℃, 220rpm; S. oneidensis MR-1, 30℃, 200rpm);

2. Pipette 500 μL of E. coli WM3064 containing plasmid pHG13-oprf and SO3 containing plasmid pYYDT-phzABCDEFG into EP tubes, mix well, centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend with LB+100 μg/ml 2,6-diaminopimelic acid (DAP) liquid medium, and place in a 30°C constant temperature incubator for 2 h;

3. Pipette 100 μL of the mixed bacterial solution and spread it evenly on a solid medium containing LB (without DAP to inhibit the growth of E. coli WM3064) containing 100 mg/L chloramphenicol resistance and 50 mg/L kanamycin resistance, and place it in a constant temperature incubator at 30°C for overnight culture;

4. Use colony PCR to screen the correct transformants, perform sequencing verification, and proceed to the next step of the experiment.

Example 2: Characterization of the power generation performance of the recombinant Shewanella strain SP1, recombinant Shewanella strain SO3 and wild-type Shewanella MR-1 MFC

1. Strain activation

The recombinant Shewanella strain SP1, recombinant Shewanella strain SO3 and wild-type Shewanella MR-1 that synthesize and secrete efficient electron transfer carriers were taken out of the -80°C refrigerator and cultured overnight at 30°C, 200rpm in LB culture medium containing 100 mg/L chloramphenicol resistance and 50 mg/L kanamycin resistance, LB culture medium containing 50 mg/L kanamycin resistance and LB culture medium without resistance.

The bacterial liquid at the end of overnight culture was inoculated into anode liquid containing 0.5 mM IPTG, 100 mg/L chloramphenicol resistance, 50 mg/L kanamycin resistance, and no resistance at a ratio of 1%, and cultured at 30°C, 200 rpm for 10 hours. The _OD600 was measured, the volume was calculated ( _OD600 in MFC = 0.7), the anode liquid was added to _OD600 = 0.7, and added to the MFC anode chamber.

The anode solution contains 6 g/L disodium hydrogen phosphate, 3 g/L potassium dihydrogen phosphate, 0.5 g/L sodium chloride, 1 g/L ammonium chloride, 1 mM magnesium sulfate, 0.1 mM calcium chloride, 20 mM sodium lactate, 5% LB medium, and the balance is water.

2. Characterization of MFC power generation performance

The experimental device uses a double-chamber MFC, a 110mL anode chamber (containing 110mL of bacteria and anode liquid in total) and a 110mL cathode chamber. The size of the anode carbon cloth electrode is 1cm×1cm, and the size of the cathode carbon cloth electrode is 2.5cm×3cm. The two chambers are separated by a proton exchange membrane. The proton exchange membrane was soaked in 1M hydrochloric acid aqueous solution overnight and rinsed three times with sterile distilled water.

The cathode solution contains 50mM potassium ferrocyanide, 50mM potassium dihydrogen phosphate and 50mM potassium dihydrogen phosphate, and the balance is water. The MFC is placed in a 30°C incubator, and the positive and negative electrodes are connected to a 2KΩ external resistor.

3. Electrochemical performance analysis

Linear sweep voltammetry (LSV) was performed from -0.87 V to -0.1 V at a scan rate of 0.1 mV/s using a multi-channel electrochemical workstation CHI1000C.

4. Results

Bioelectrochemical analysis can be used to study the efficiency of extracellular electron transfer in MFC. Figure 3 shows the linear sweep voltammogram (LSV), i.e., polarization curve, at a sweep rate of 0.1 mV/s. It can be seen from the figure that the maximum current density of MFC power generation using the power-producing recombinant Shewanella SP1 is about 6584 mA/m ² , and the maximum power density is 2252.9 mW/m ² , which is greatly improved compared with the original strain wild-type Shewanella MR-1 and the recombinant Shewanella strain SO3.

Example 3: Characterization of cell membrane permeability of the electrogenic recombinant Shewanella strain SP1 and the recombinant Shewanella SO3 strain that does not express porins

1. Strain activation

The recombinant Shewanella strain SP1 and recombinant Shewanella SO3 that synthesize and secrete efficient electron transfer carriers were taken out of the -80°C refrigerator and cultured overnight at 30°C, 200 rpm in LB culture media containing 100 mg/L chloramphenicol resistance and 50 mg/L kanamycin resistance, respectively.

2. NPN fluorescence measurement

After 10 h of culture, the recombinant Shewanella strain SP1 and wild-type Shewanella MR-1 were washed three times with 10 mM PBS buffer (pH = 7.4), and an appropriate amount of NPN (1-N-phenylnaphylamine, 6 μM) was added to 1 mL of the bacterial solution and incubated for 10 min. 200 μL of the bacterial solution was added to a transparent 96-well plate, placed in a microplate reader, and the fluorescence value of 400-500 nm was measured at an excitation wavelength of 355 nm.

3. Results

The non-polar fluorescent probe 1-N-phenylnaphthylamine (NPN) is a mature fluorescent probe for measuring cell membrane permeability. NPN can be used to quantify the efficiency of molecular transport into cells. It has strong fluorescence in the phospholipid environment, but weak fluorescence in the water environment. NPN can enter the phospholipid layer of the cell membrane, thereby producing obvious fluorescence. As shown in Figure 4, after adding NPN to the cell suspension, at an excitation wavelength of 355nm, the engineered Shewanella SP1 has a strong absorption light at 415nm, which is about 2 times that of the recombinant Shewanella SO3 that does not express porins, indicating that the cell membrane permeability of the recombinant Shewanella SP1 has increased.

Example 4: Determination of the Fermentation Yield of the Electron Transfer Vector PCA of the Electrogenic Recombinant Shewanella Strain SP1

1. Strain activation

The recombinant Shewanella strain SP1 that efficiently synthesizes and secretes an electron transporter was taken out of a -80°C freezer and cultured overnight at 30°C and 200 rpm in an LB medium containing 100 mg/L chloramphenicol resistance and 50 mg/L kanamycin resistance.

2. Expand training

The bacterial solution after overnight culture was inoculated into the anode solution containing 0.5 mM IPTG with 100 mg/L chloramphenicol resistance and 50 mg/L kanamycin resistance at a ratio of 1%, and cultured at 30°C, 200 rpm for 10 hours.

The anode solution contains 6 g/L disodium hydrogen phosphate, 3 g/L potassium dihydrogen phosphate, 0.5 g/L sodium chloride, 1 g/L ammonium chloride, 1 mM magnesium sulfate, 0.1 mM calcium chloride, 20 mM sodium lactate, 5% LB medium, and the balance is water.

3. Sample processing

Take 270μL of fermentation broth from expanded culture, add 30μL of 6M hydrochloric acid, shake for 10 seconds, mix well, add 270μL of ethyl acetate, shake for 2 minutes, then centrifuge at 12000rpm for 10 minutes, take 100μL of ethyl acetate phase to a new 1.5ml centrifuge tube, centrifuge and concentrate to dryness. After centrifugation, concentration and evaporation, redissolve the sample in 300μL of methanol, and after it is fully dissolved, centrifuge at 13000rpm for 10 minutes to remove impurities, take 100μL of the upper solution, pass through the membrane, and use it as the HPLC sample.

5. HPLC determination

Instrument model: Shimadzu HPLC; analytical column: Hypersil ^TM BDS C18 5um, 4.6x150 mm; mobile phase: 40% A: 5mM ammonium acetate 60% B: methanol; injection volume: 2u1; column temperature: 30°C; UV detection wavelength: 254nm; flow rate: 0.7ml/min.

6. Results

The retention time of PCA is 2.87, and the PCA standard curve (Figure 5) is y=11770x-1704.9 (y is the peak area, x is the sample PCA concentration μM, R ² =0.9998). Based on the sample preparation process, the fermentation broth concentration (μM) = sample PCA concentration * 300/100. The peak area of the sample measured at the retention time of 2.87 is 325391 (Figure 6), and the PCA concentration of the fermentation broth is calculated to be 82.5 μM.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (12)

1. A method for constructing a recombinant Shewanella oneidensis strain that efficiently synthesizes and secretes an electron transfer carrier, characterized in that it comprises the following steps: connecting the outer membrane porin gene oprf of Pseudomonas and the gene cluster phzABCDEFG that controls phenazine-1-carboxylic acid synthesis to a vector, transforming Shewanella with the vector, and constructing a recombinant Shewanella strain SP1. 2. The construction method according to claim 1 is characterized in that the outer membrane porin gene oprf of Pseudomonas and the gene cluster phzABCDEFG controlling the synthesis of phenazine-1-carboxylic acid are amplified from the genome of Pseudomonas eruginosa PAO1 by PCR. 3. The construction method according to claim 2, characterized in that the amplification primers of the outer membrane porin gene oprf are SEQ ID NO.1-2; The amplification primers of the gene cluster phzABCDEFG are SEQ ID NOs. 3-4. 4. The construction method according to claim 2 is characterized in that the amplification program includes: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 1 kb/30 s, 30 cycles; complete extension at 72°C for 5 min; cooling at 16°C for 5 min. 5. The construction method according to claim 1, characterized in that the vector is selected from the pHG13 vector shown in SEQ ID NO.5 and/or the pYYDT vector shown in SEQ ID NO.6. 6. The construction method according to claim 5, characterized in that it comprises selecting the promoter lacUV5 and RBS sequence shown in SEQ ID NO.11, inserting the outer membrane porin gene oprf shown in SEQ ID NO.7 into the pHG13 vector, and obtaining the expression vector pHG13-oprf shown in SEQ ID NO.8; The promoter tac and RBS sequence shown in SEQ ID NO.12 were selected, and the gene cluster phzABCDEFG shown in SEQ ID NO.9 was inserted into the pYYDT vector to obtain the expression vector pYYDT-phzABCDEFG shown in SEQ ID NO.10. 7. The construction method according to claim 6, characterized in that the expression vector pYYDT-phzABCDEFG is transferred into Escherichia coli, and then combined with the wild-type Shewanella oneidensis MR-1 by conjugation transfer, and the recombinant Shewanella strain SO3 is obtained after positive verification; The expression vector pHG13-oprf was transformed into E. coli WM3064, and then combined with the recombinant Shewanella SO3 to obtain the recombinant Shewanella strain SP1. 8. A recombinant Shewanella strain SO3 that efficiently synthesizes and secretes an electron transfer carrier, constructed using the construction method of claim 7. 9. A recombinant Shewanella strain SP1 that efficiently synthesizes and secretes an electron transfer carrier, constructed using the construction method according to any one of claims 1 to 7. 10. Use of the recombinant Shewanella strain SP1 according to claim 9 in electricity generation. 11. Use of the recombinant Shewanella strain SP1 according to claim 9 in promoting cell membrane permeability. 12. Use of the recombinant Shewanella strain SP1 according to claim 9 in the synthesis of electron carriers.