CN115976089B - Method for enhancing electric energy output by engineering strengthening Shewanella vesicle secretion - Google Patents

Abstract

The invention discloses a method for improving electric energy output by engineering strengthening Shewanella vesicle secretion, which comprises the steps of screening genes ompR, wbpP, gmhB, wbpA for encoding cell outer membranes, peptidoglycan integrity, lipopolysaccharide synthesis and porin; designing 6 targeted sgrnas for each gene, and constructing a recombinant plasmid containing the sgrnas and dCas9 through Golden gate assembly; the recombinant plasmid is transferred into Shewanella through chemical transformation and conjugation to obtain 24 engineering strains; inducing an engineering bacteria system to express CRISPR-dCAs9, inhibiting transcription of target genes so as to promote vesicle secretion, observing the number of vesicles by a scanning electron microscope, and screening engineering strains corresponding to the biggest efficient sgRNA of each gene; the microbial fuel cell based on the engineering strain secondary fermentation liquor and the potassium ferricyanide solution is constructed, and the electric energy output effect is obviously improved.

Description

A method for engineering enhanced Shewanella vesicle secretion to increase electrical energy output

Technical Field

The present invention belongs to the field of bioenergy technology, and more specifically, utilizes synthetic biology means and CRISPR-dCas9 technology to enhance the vesicle secretion of Shewanella oneidensis MR-1, thereby enhancing its power generation output effect in microbial fuel cells.

Background Art

Outer membrane vesicles (OMVs) are closed, unevenly sized spherical particles filled with periplasmic contents that are formed by budding from the outer membrane of Gram-negative bacteria. Their average diameter is usually between 10 and 300 nm. Almost all Gram-negative bacteria can release outer membrane vesicles to the surrounding environment during normal growth, and outer membrane vesicles have multiple physiological and environmental functions such as intercellular information exchange, transfer of cell metabolites, and cell defense. There are many theories about the formation and secretion mechanism of OMVs. The most widely recognized model is membrane cross-linking: the cell wall structure of Gram-negative bacteria is composed of the inner membrane layer, peptidoglycan layer, and outer membrane layer from the inside to the outside. The peptidoglycan in the bacterial wall space and the lipoproteins located in the bacterial outer membrane maintain the integrity of the outer membrane structure through covalent cross-linking. When certain factors (such as external stimuli, etc.) act to cause the loss of covalent cross-linking, part of the outer membrane bulges outward to form vesicles. Based on this mechanism, many studies have promoted the production of E. coli vesicles by reducing the integrity of peptidoglycan and cell wall, thereby increasing the accumulation of lipid-soluble products on the cell membrane. In addition, the content of porins and lipopolysaccharides on the outer membrane surface also plays an important role in outer membrane vesicles. Reducing the synthesis of lipopolysaccharides and porins will cause changes in the curvature of the outer membrane, thereby promoting the secretion of vesicles. It has been reported in the literature that Geobacter can secrete outer membrane vesicles rich in cytochromes and promote electron transfer between Geobacter biofilms and electrodes in a long-distance electrical transmission manner.

Energy shortage and environmental pollution are two major problems facing modern society. All countries are working hard to develop new energy and address environmental problems. Among them, microbial fuel cells (MFCs) based on the bidirectional electron transfer mechanism of electroactive microorganisms have shown excellent performance in power generation and have become a research hotspot for scientists from all over the world. Microbial fuel cells are divided into two types: monopolar chambers and bipolar chambers. The most common type is the bipolar chamber microbial fuel cell. The bipolar chamber MFC is mainly composed of cathode and anode chambers, proton exchange membranes, cathode and anode electrodes. Among them, the electroactive microorganisms in the anode chamber can convert the chemical energy in organic matter into electrical energy and transfer it to the anode electrode in the form of electrons. Then the electrons are transferred to the cathode through an external circuit and captured by the electron acceptors in the cathode chamber. Inside the battery, protons pass from the anode to the cathode through the proton exchange membrane to form a closed loop. At present, there are two main intercellular electron transfer mechanisms between power-producing microorganisms and electrodes that are widely recognized, namely direct electron transfer mediated by cytochrome proteins or conductive nanowires and indirect electron transfer mediated by soluble redox-active molecules. As the most widely studied model electrogenic bacterium, Shewanella has relatively clear research on its metabolic pathways and extracellular electron transfer pathways. People have also constructed various MFC devices based on the electron transfer mechanism of Shewanella. However, there are currently no literature reports on enhancing its electrogenic output by strengthening the secretion of its outer membrane vesicles.

Therefore, the present invention uses Shewanella MR-1 as the starting strain, and utilizes synthetic biology methods to inhibit and screen the genes encoding the integrity of the extracellular membrane or peptidoglycan, as well as the synthesis of porins and lipopolysaccharides on its genome through CRISPR-dCas9 technology, thereby enhancing the vesicle formation of Shewanella and improving the power output of its microbial fuel cell.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art, and to design and construct 6 targeting sgRNAs for the genes related to the integrity of the extracellular membrane or peptidoglycan, as well as the synthesis of porins and lipopolysaccharides on the genome of the efficient electricity-producing microorganism ShewanellaoneidensisMR-1: ompR, wbpP, gmhB and wbpA, and to inhibit the expression of related genes through CRISPR-dCas9 technology to enhance the formation of Shewanella vesicles; and to screen out the engineered bacteria constructed by the four genes ompR5, wbpP3, gmhB4 and wbpA4 with relatively good vesicle formation effects from the 24 engineered strains constructed, and the electrochemical test results show that the engineered bacteria WbpP3 rich in vesicles exhibits a higher power output effect in microbial fuel cells.

The technical solution of the present invention is summarized as follows:

The present invention provides a method for enhancing the secretion of Shewanella vesicles by engineering to improve the output of electrical energy; the method comprises the following steps:

(1) Screening genes related to vesicle secretion in the genome of the model electrogenic microorganism Shewanella oneidensis MR-1, and obtaining genes ompR encoding an extracellular membrane protein regulator, gmhB involved in lipopolysaccharide synthesis, and wbpP and wbpA related to cell membrane or peptidoglycan integrity;

(2) For the four target genes ompR, wbpP, gmhB, and wbpA, six targeting sgRNAs and their upstream and downstream primers were designed and constructed for each gene;

(3) Using the Golden gate technique, different sgRNA sequences were integrated into the original basic plasmid pHG11-dCas9 with dCas9 protein, IPTG-inducible P _tac promoter, and kanamycin resistance to obtain 24 recombinant plasmids containing sgRNA and dCas9;

(4) heat-stimulating the 24 recombinant plasmids constructed in step (3) and transferring them into 2,6-diaminopimelate (DAP) auxotrophic Escherichia coli competent WM3064 for resistance screening;

(5) performing conjugation transfer between the Escherichia coli WM3064 containing the recombinant plasmid obtained by the resistance screening in step (4) and wild Shewanella, introducing the recombinant plasmid in the Escherichia coli into the wild Shewanella, and constructing 24 engineered Shewanella strains;

(6) inducing the expression of the CRISPR-dCas9 system of the 24 engineered Shewanella strains constructed in step (5), performing transcriptional inhibition on each target gene, and observing the number of vesicles by scanning electron microscopy, and screening the engineered strains OmpR5, WbpP3, GmhB4, and WbpA4 corresponding to the most efficient sgRNA for each target gene;

(7) Based on the secondary fermentation broth and potassium ferrocyanide solution of the four engineered strains screened in step (6), dual-chamber microbial fuel cells were constructed and electrochemically characterized. By inducing the expression of the CRISPR-dCas9 system, the transcription of the target gene was inhibited, thereby promoting vesicle secretion and improving its extracellular electron transfer capacity.

Among them, the sequence of the gene ompR screened out in step (1) is SEQ ID NO.1; the sequence of the gene wbpP is SEQ ID NO.2; the sequence of the gene gmhB is SEQ ID NO.3; the sequence of the gene wbpA is SEQ ID NO.4; the sequence of the original basic plasmid pHG11-dCas9 involved in step (3) is SEQ ID NO.5; the recombinant plasmid sequence contained in the engineering strain OmpR5 corresponding to the most efficient sgRNA of the four target genes screened out in step (6) is SEQ ID NO.6; the recombinant plasmid sequence contained in the engineering strain WbpP3 is SEQ ID NO.7; the recombinant plasmid sequence contained in the engineering strain GmhB4 is SEQ ID NO.8; the recombinant plasmid sequence contained in the engineering strain WbpA4 is SEQ ID NO.9.

The preparation method of the secondary fermentation broth in step (7) is as follows: the primary seed liquid is inoculated at a 1% inoculation amount into a 250 mL conical flask containing 100 mL of LB liquid culture medium containing 0.1 mM IPTG and 50 mg/L kanamycin, and the culture is expanded overnight at 30°C and 200 rpm in a shaking incubator to obtain a secondary fermentation broth.

Step (7) The fermentation broth obtained by using the recombinant strain with high vesicle production as an anode microbial catalyst is inoculated into the anode chamber of the microbial fuel cell at OD ₆₀₀ = 0.5, and the cathode chamber is inoculated with a traditional K ₃ [Fe(CN) ₆ ] solution as a cathode electron acceptor to achieve high electrical energy output.

The beneficial effects of the present invention are:

The invention discloses a method for enhancing the vesicle secretion of Shewanella by engineering to improve the output of electric energy. The method comprises the following steps: screening genes ompR, wbpP, gmhB and wbpA encoding the cell outer membrane, peptidoglycan integrity, lipopolysaccharide synthesis and porins; designing 6 targeting sgRNAs for each gene, and constructing a recombinant plasmid containing sgRNA and dCas9 through Golden Gate assembly; introducing the recombinant plasmid into Shewanella through chemical transformation and conjugation transfer to obtain 24 engineered strains; inducing the engineered bacteria system to express CRISPR-dCas9, inhibiting the transcription of the target gene and promoting the secretion of vesicles, observing the number of vesicles through a scanning electron microscope, and screening the engineered strain corresponding to the most efficient sgRNA for each gene; constructing a microbial fuel cell based on the secondary fermentation broth of the engineered strain and a potassium ferrocyanide solution, and the output effect of the electric energy is significantly improved.

The results of electrochemical analysis show that: as shown in the voltage-time curve of Figure 7, the abscissa represents time and the ordinate represents voltage. In general, the power generation voltage of all microbial fuel cells will increase first with time, reach a peak value and then decrease. Among them, WbpP3 has the best power generation performance among the four engineered bacteria, and its maximum output voltage can reach 219mV, which is more than 4 times higher than the maximum output voltage of the original wild-type Shewanella MR-1; as shown in the power density curve obtained by linear sweep voltammetry (LSV) in Figure 8, the abscissa represents current density and the ordinate represents power generation power density. In general, the power generation power density of all microbial fuel cells increases first and then decreases with the increase of current density, presenting a parabolic curve. Among them, the engineered strain WbpP3 that promotes vesicle secretion shows the best power generation output performance, with a maximum current density of about 1654mA/m ² and a power generation power density of 356.77mW/m ² , which is about 7 times higher than the power generation power density of the original wild-type Shewanella MR-1.

In summary, as shown in Figure 9, the present invention uses Shewanella MR-1 as the starting strain, uses synthetic biology methods, and uses CRISPR-dCas9 technology to screen and inhibit the genes (ompR, wbpP, gmhB, wbpA) encoding the integrity of the cell outer membrane or peptidoglycan on its genome, thereby enhancing the vesicle secretion and electrical energy output of Shewanella. This provides a new idea for transforming electroactive microorganisms and promoting their industrial applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Detailed map of the original basic plasmid pHG11-dCas9;

Figure 2. Detailed map information of the recombinant plasmid contained in the engineered strain OmpR5;

Figure 3. Specific map information of the recombinant plasmid contained in the engineered strain WbpP3;

Figure 4. Detailed map information of the recombinant plasmid contained in the engineered strain GmhB4;

Figure 5. Detailed map information of the recombinant plasmid contained in the engineered strain WbpA4;

Figure 6. Scanning electron microscopy (SEM) images of secretory vesicles of wild-type Shewanella strain and four engineered strains;

Figure 7. Voltage-time curve of microbial fuel cell;

Figure 8. Power density curve of microbial fuel cell;

Fig. 9 is a diagram showing the implementation principle of the present invention.

DETAILED DESCRIPTION

(1) Screening of genes related to outer membrane vesicles:

Outer membrane vesicles are closed spherical particles formed by budding from the outer membrane of Gram-negative bacteria. Studies have shown that the reduction of cross-links between peptidoglycan and the outer membrane can trigger the formation of outer membrane vesicles, and reducing the expression of genes encoding proteins related to the integrity of the outer membrane or peptidoglycan can change the cell morphology to different shapes (usually from rod-shaped to spherical), thereby increasing the total membrane area of each cell and promoting the production of bacterial outer membrane vesicles. In addition, reducing the content of lipopolysaccharide and porins in the outer membrane will cause changes in the curvature of the outer membrane, thereby promoting vesicle production. Therefore, in the genome of Shewanella MR-1, four protein-coding genes (ompR, rffD, gmhB and wbpA) related to the integrity of the cell outer membrane or peptidoglycan, as well as porin and lipopolysaccharide synthesis were excavated, including the gene ompR encoding the cell outer membrane protein regulator, the gene wbpP encoding UDP-N-acetyl-D-mannosamine dehydrogenase related to the integrity of the cell membrane and peptidoglycan, the gene gmhB encoding DD-heptose-17-bisphosphate phosphatase involved in lipopolysaccharide (LPS) synthesis, and the gene wbpA encoding ADP-L-glycerol-D-mannose-heptose-6-epimerase involved in the lipopolysaccharide synthesis pathway, which were inhibited at the transcriptional level by CRISPR-dCas9 technology. Among them, the sequence of the gene ompR is SEQ ID NO.1; the sequence of the gene wbpP is SEQ ID NO.2; the sequence of the gene gmhB is SEQ ID NO.3; and the sequence of the gene wbpA is SEQ ID NO.4.

(2) Design sgRNA and its upstream and downstream primers:

For the four genes described in step (1), six single guide RNAs (sgRNAs) were designed for each gene. The specific sgRNA design process was as follows: the gene sequence to be edited was input into the website http://crispor.tefor.net/?org=GCF_000146165.2 , the host Shewanella oneidensis MR-1 was selected, and the NGG sequence was automatically searched in the template strand (non-coding strand, T strand) and the non-template strand (coding strand, NT strand), respectively. The 20 bp at the front end of the NGG 5' sequence was part of the sgRNA. Among the obtained sgRNAs, the sgRNAs with high structural stability and the lowest off-target efficiency and targeting the first 1/3 of the coding sequence were selected, wherein the sgRNAs targeting the non-template strand from 5' to 3' end were sgRNA1, sgRNA2, and sgRNA3, and the sgRNAs targeting the template strand from 5' to 3' end were sgRNA4, sgRNA5, and sgRNA6. Then, primers were designed according to the sgRNA sequence, with TTGC added to the 5' end of the upstream primer and AAAC added to the 5' end of the downstream primer (for the next Golden Gate assembly). Primers were synthesized by Genewise, and the specific primer names and sequence information are shown in Table 1.

Table 1. Primer names and sequence information designed based on sgRNA sequences

Table 1.Primer names and sequence information designed according to tosgRNA sequences

(3) Golden gate one-step self-assembly to construct recombinant plasmid:

Different sgRNAs were integrated into the original basic plasmid pHG11-dCas9 by Golden gate technology, as shown in the specific map of the original basic plasmid pHG11-dCas9 in Figure 1. The original basic plasmid contains the repressor protein LacI encoding gene, the IPTG-inducible promoter _Ptac that starts the dCas9 protein encoding gene, the kanamycin resistance gene, the shuttle element mob, and the replicon pBBR1, as well as the promoter _PCI , to obtain the recombinant plasmid pHG11-dCas9-sgRNA-ompR1, 2, 3, 4, 5, 6/wbpP1, 2, 3, 4, 5, 6/gmhB1, 2, 3, 4, 5, 6/wbpA1, 2, 3, 4, 5, 6, a total of 24 (1-6 correspond to different sgRNAs targeting the target gene, respectively).

Goldengate technology uses the characteristics of TypeIIS restriction endonucleases that the cutting site is outside the recognition site, so that enzyme cutting and ligation can be achieved in one pot, and multiple DNA fragments can be connected at the same time. BsaI is a type II restriction endonuclease that can specifically recognize and cut the 5′-GGTCTC(N1)/(N5)-3′ site of the DNA sequence, and can work together with T4 DNA ligase to achieve one-step enzyme cutting and ligation. The pHG11-dCas9 plasmid contains the sgRNA scaffold structure sequence and a 20bp sequence containing the restriction site. The sgRNA sequence can be connected to the target vector through Goldengate. The specific steps are as follows:

①Insert preparation:

Add 2 μL of upstream primer, 2 μL of downstream primer (concentration of 100 μM), and 36 μL of 30 mM HEPES buffer (PH = 7.8) to a 200 μL PCR tube. The upstream and downstream primers here correspond to the upstream and downstream primers designed in step (2).

The above system was reacted in a PCR instrument: 95°C for 5 min, then the temperature was decreased from 95°C to 4°C at a rate of 0.1°C/s. Finally, 120 μL of ultrapure water was added to dilute it to 4 times the original value, pipetted and mixed, and used as Insert, and stored in a -20°C refrigerator.

②Golden gate one-step assembly is performed in a PCR instrument. The reaction system is shown in Table 2.

Table 2. Golden-gate reaction system

Table 2.Golden-gate reaction system.

The PCR reaction program is shown in Table 3:

Table 3. Golden-gate reaction procedure

Table 3. Golden-gate program.

(4) Transform the ligation product into competent E. coli:

The ligation product prepared in step (3) was transformed into competent Trans1-T1 bacteria, cultured overnight, and the plasmid was extracted for sequencing verification and returned. If the sequencing was correct, the returned plasmid with the correct sequencing was transferred to 2,6-diaminopimelate (DAP) nutrient-deficient Escherichia coli competent WM3064 by heat stimulation, and the heat-stimulated bacterial liquid was spread on a 50μg/mL kanamycin resistance plate and placed in a 37°C constant temperature biochemical incubator for overnight culture for resistance screening; if the sequencing was wrong, repeat the experimental operation of the previous step (3) and re-do the Golden gate one-step assembly to prepare the recombinant plasmid.

The specific operation steps for transforming the ligation product into competent bacteria are as follows: Take 100 μL of competent cells Trans1-T1 melted in an ice bath, add 10 μL of the ligation product prepared in step (3) (i.e., target DNA) in a clean bench, and gently tap twice on the clean bench to mix the target DNA and competent cells. Place in an ice bath for 30 minutes, heat shock in a 42°C water bath for 30 seconds, and then quickly transfer the EP tube containing competent cells and target DNA to an ice bath for 2 minutes. Next, add 1 mL of sterile LB culture medium (without antibiotics) to the EP tube in a clean bench, mix with a pipette, and place in a shaker at 37°C, 200 rpm for 1 hour to allow the bacteria to recover. After centrifugation at 5000 rpm for 2 minutes, pour out the supernatant in the clean bench, resuspend the residual liquid with a 200 μL pipette, and then add all the mixed liquid to the LB agar medium containing the corresponding antibiotics, and spread the cells evenly with a spreader. Finally, the plate was placed upside down in a 37°C constant temperature biochemical incubator for overnight culture.

The detailed operation steps of heat-activated transformation are as follows: Take 50 μL of 2,6-diaminopimelate (DAP) nutrient-deficient Escherichia coli WM3064 melted in an ice bath, add 2-3 μL of plasmid in a clean bench, and gently tap twice on the clean bench to mix the plasmid and competent cells. Place in an ice bath for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and then quickly transfer the EP tube containing competent cells and plasmids to an ice bath for 2 minutes. Next, add 1 mL of sterile LB+60 μg DAP culture solution (without antibiotics) to the EP tube in a clean bench, mix with a pipette, and place at 37°C, 200rpm shaker for 2 hours to allow the bacteria to recover. Then, take 100 μL of the transformed competent cells and add them to the LB agar medium containing the corresponding antibiotics, and spread the cells evenly with a spreader. Finally, place the plate upside down in a 37°C constant temperature biochemical incubator and culture overnight.

(5) Introducing the recombinant plasmid in Escherichia coli into wild Shewanella through conjugation transfer:

The Shewanella engineering strain containing the recombinant plasmid was obtained by conjugation transfer between Escherichia coli containing the recombinant plasmid and wild Shewanella oneidensis MR-1.

The detailed steps of conjugation transfer are as follows: pick the 2,6-diaminopimelate (DAP) nutrient-deficient Escherichia coli engineered strain containing the recombinant plasmid and inoculate it into a shaking tube containing 3mL LB+180μgDAP+1% Kana resistance culture medium, and culture it at 37°C and 220rpm overnight; at the same time, pick a single colony of the wild Shewanella strain MR-1 and inoculate it into a shaking tube containing 3mL LB liquid culture medium (wild-type strains do not add antibiotics), and culture it at 30°C and 200rpm overnight. Take 500μL of the Escherichia coli culture containing the recombinant plasmid and the wild Shewanella culture (without plasmid) after overnight culture in the clean bench, add them to the same sterilized 2mL EP tube, centrifuge at 5000rpm for 3min, and pour out the supernatant in the clean bench to enrich the bacteria. Add 1mL of sterile LB+60μgDAP culture solution (without antibiotics) to resuspend the cells, place it in a 30℃ constant temperature biochemical incubator, and culture it for 2 hours. Take 100μL of the above cultured bacterial solution, add it to the LB agar medium containing kanamycin resistance, and spread the cells evenly with a spreader. Finally, place the plate upside down in a 30℃ constant temperature biochemical incubator, and obtain the Shewanella strain containing the recombinant plasmid through resistance screening.

(6)① Inducible expression of CRISPR-dCas9 system:

The engineered strain introduced with the recombinant plasmid was inoculated into LB liquid culture medium containing kanamycin resistance for overnight culture, and then inoculated into 100 mL LB liquid culture medium containing 0.1 mM IPTG and kanamycin resistance at a 1% inoculation rate for overnight culture to induce the expression of dCas9 protein and achieve transcriptional inhibition of the target gene.

②Scanning electron microscope (SEM) pretreatment:

200 μL of the bacterial solution after overnight culture was taken into a 2 mL EP tube, centrifuged at 5000 rpm for 3 min, and then the supernatant was discarded. The bacterial solution was diluted 10 times with 2 mL PBS and rinsed 1-2 times. After drying on a silicon wafer, the tube was sprayed with gold and a scanning electron microscope (Regulus8100, Hitachi, Japan) was used to take SEM images of the secretory vesicles of the engineered strain.

③Screening the most suitable sgRNA:

According to the scanning electron microscopy images obtained in step ② above, observe whether the 24 engineered bacteria actually secrete vesicles and select the sgRNA strains with the best vesicle production effect from the 6 sgRNAs of each gene, namely OmpR5, WbpP3, GmhB4, and WbpA4, and freeze them in glycerol. Among them, the scanning electron microscopy images of the secreted vesicles of these 4 engineered bacteria and wild-type Shewanella are shown in Figure 6. The cell morphology of strains OmpR5, WbpP3, GmhB4, and WbpA4 changed slightly, and all of them secreted exosomes with a diameter of 50-200nm around the cells, and the shapes were mostly spherical. The specific map information of the recombinant plasmids contained in the engineered strains OmpR5, WbpP3, GmhB4, and WbpA4 are shown in Figures 2, 3, 4, and 5, respectively. The recombinant plasmids all contain the repressor protein LacI encoding gene, the IPTG-inducible promoter _Ptac to initiate the dCas9 protein encoding gene, the kanamycin resistance gene, the shuttle element mob, and the replicon pBBR1, as well as the promoter _PCI to initiate the expression of sgRNA.

(7) Assembly and characterization of microbial fuel cells:

① Battery pretreatment: Microbial fuel cells are mainly composed of anode and cathode chambers, cathode and anode electrodes and proton exchange membranes.

② Pretreatment of Nafion 117 proton exchange membrane: According to the size of the proton exchange membrane required for the experiment, the proton exchange membrane was cut into discs with a diameter of 5 cm, and the cut proton exchange membrane was soaked in 1M HCl aqueous solution overnight and sterilized by ultraviolet irradiation in an ultra-clean bench. Before assembling the battery, it was rinsed three times with sterile water to wash away the hydrochloric acid.

③ Pretreatment and assembly of microbial fuel cells: Clamp the anode and cathode chambers with clips outside the clean bench, and use 1cm×1cm carbon cloth as the anode and 2.5cm×3cm carbon cloth as the cathode. After sterilization at 121℃, add cathode and anode liquids in the clean bench.

④ Prepare fresh anolyte: Take 200mL of 5×M9 mother liquor and place it in a 1L blue-cap bottle, add deionized water to about 900mL, and sterilize at high temperature. In the clean bench, add 50mL of sterilized LB culture medium, 20mL of 1M sterilized sodium lactate solution, 1mL of 0.1M filter-sterilized calcium chloride solution, 1mL of 0.1M filter-sterilized magnesium sulfate solution, 2mL4MNaOH, and add the corresponding concentration of inducer and antibiotic according to the plasmid resistance, and then fill the anolyte to 1L with sterilized water.

⑤ Prepare fresh cathode solution: weigh 32.926g _{K 3} [Fe(CN) ₆ ], 17.418g K ₂ HPO ₄ and 13.609g KH ₂ PO ₄ and place them in a large beaker, add distilled water to 2L, stir with a clean glass rod, dissolve by ultrasonic to make cathode solution.

⑥ Strain culture and fermentation: The 4 engineered bacteria and wild-type MR-1 strains frozen at -80°C were cultured on LB solid medium (LB solid medium corresponding to the engineered strains was supplemented with 1% Kana resistance, and antibiotics were not added to the wild-type strains) until single colonies grew out, and single colonies were picked and inoculated into a shaking tube containing 3mL LB liquid medium (1% Kana resistance was added to the engineered strains, and antibiotics were not added to the wild-type strains), and cultured overnight at 30°C and 200rpm. Then, 1% of the inoculum was inoculated into a 250mL conical flask containing 100mL LB liquid medium containing 0.1mM IPTG and 50mg/L kana antibiotics, and the fermentation liquid was expanded and cultured overnight at 30°C and 200rpm.

⑦ Construct a double-chamber microbial fuel cell with potassium ferricyanide solution as the cathode electron acceptor, the secondary fermentation broth of four engineered bacteria OmpR5, WbpP3, GmhB4, and WbpA4 as the anode inoculation source, and the strain introduced with the empty vector pHG11-dCas9 as the wild-type control (WT). According to the final OD ₆₀₀ of 0.5 and the volume of the cathode and anode chambers of 140 mL, the cathode and anode liquids and bacterial liquids were added respectively, and the closed loop was connected with copper wires and 2kΩ resistors. The battery was placed in a 30℃ incubator for static culture, and the above-mentioned started battery was connected to a data acquisition device to record the voltage change curve over time as shown in Figure 7. The voltage continued to rise with the increase of discharge time. When the voltage reached the peak and remained constant, the battery was scanned by linear sweep voltammetry (LSV), and the initial potential of the scan was set to -0.8 to -0.1V, and the scan rate was set to 0.1mV/s, and the power density curve shown in Figure 8 was obtained.

The results of electrochemical analysis show that: as shown in the voltage-time curve of Figure 7, the abscissa represents time and the ordinate represents voltage. In general, the power generation voltage of all microbial fuel cells will increase first with time, reach a peak value and then decrease. Among them, WbpP3 has the best power generation performance among the four engineered bacteria, and its maximum output voltage can reach 219mV, which is more than 4 times higher than the maximum output voltage of the original wild-type Shewanella MR-1; as shown in the power density curve obtained by linear sweep voltammetry (LSV) in Figure 8, the abscissa represents current density and the ordinate represents power generation power density. In general, the power generation power density of all microbial fuel cells increases first and then decreases with the increase of current density, presenting a parabolic curve. Among them, the engineered strain WbpP3 that promotes vesicle secretion shows the best power generation output performance, with a maximum current density of about 1654mA/m ² and a power generation power density of 356.77mW/m ² , which is about 7 times higher than the power generation power density of the original wild-type Shewanella MR-1.

In summary, as shown in FIG9 , the present invention uses Shewanella MR-1 as the starting strain, and inhibits the genes encoding the integrity of the extracellular membrane or peptidoglycan, as well as the synthesis of porins and lipopolysaccharides on its genome (ompR, wbpP, gmhB, and wbpA) by CRISPR-dCas9 technology, specifically including the gene ompR encoding the extracellular membrane protein regulator, the gene wbpP encoding UDP-N-acetyl-D-mannosamine dehydrogenase related to the integrity of the cell membrane and peptidoglycan, and the gene involved in The gene gmhB encoding DD-heptose-17-bisphosphate phosphatase in lipopolysaccharide (LPS) synthesis and the gene wbpA encoding ADP-L-glycerol-D-mannose-heptose-6-epimerase involved in the lipopolysaccharide synthesis pathway are activated, thereby changing the cell morphology, increasing the cell membrane area, reducing the cross-linking effect between peptidoglycan and the cell membrane, and ultimately strengthening the vesicle secretion of Shewanella, thereby increasing the extracellular electron transfer rate between Shewanella and the electrode. This provides a new idea for transforming electroactive microorganisms and promoting their industrial applications.

The technical solutions disclosed and proposed by the present invention can be realized by those skilled in the art by referring to the contents of this article and appropriately changing the conditions, routes and other links. Although the methods and preparation techniques of the present invention have been described through preferred embodiments, relevant technical personnel can obviously modify or re-combine the methods and technical routes described herein without departing from the content, spirit and scope of the present invention to achieve the final preparation technology. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the spirit, scope and content of the present invention. Matters not covered in the present invention belong to the known technology.

Claims (3)

1. A method for enhancing the secretion of Shewanella vesicles and improving the output of electric energy by engineering; the method is characterized in that: the method comprises the following steps:

(1) Screening related genes which are favorable for vesicle secretion on genome of a mode electrogenesis microorganism Shewanella SHEWANELLA ONEIDENSIS MR-1 to obtain genes wbpP related to the structural integrity of cell outer membranes or peptidoglycans; the sequence of the gene wbpP is SEQ ID NO.2;

(2) 6 targeted sgRNAs and upstream and downstream primers thereof are respectively designed and constructed aiming at a target gene wbpP;

(3) The different sgRNA sequences are respectively integrated on original basic plasmid pHG11-dCAS9 with dCAS9 protein, IPTG induced Ptac promoter and kana antibiotic resistance by Golden gate technology, so as to obtain 6 recombinant plasmids containing sgRNA and dCAS 9; the sequence of the original basic plasmid pHG11-dCAS9 related in the step (3) is SEQ ID NO.5;

(4) Respectively carrying out heat activation on the 6 recombinant plasmids constructed in the step (3) to convert the 6 recombinant plasmids into 2, 6-diaminopimelate auxotroph escherichia coli competent WM3064 and screening resistance;

(5) Performing conjugation transfer on the escherichia coli WM3064 containing the recombinant plasmid obtained in the step (4) resistance screening and the wild Shewanella MR-1, and introducing the recombinant plasmid in the escherichia coli into the wild Shewanella MR-1 to construct 6 engineering Shewanella strains;

(6) Inducing the expression of the CRISPR-dCAS9 system of the 6 engineering Shewanella strains constructed in the step (5), carrying out transcription inhibition on each target gene, observing the number of vesicles by a scanning electron microscope, and screening out the engineering strain corresponding to the most efficient sgRNA of the target gene; the recombinant plasmid sequence contained in the engineering strain is SEQ ID NO.7;

(7) And (3) respectively constructing a double-chamber microbial fuel cell based on the secondary fermentation liquid and the potassium ferricyanide solution of the engineering strain obtained by screening in the step (6) and carrying out electrochemical characterization, and inhibiting transcription of a target gene by inducing CRISPR-dCAS9 system expression so as to promote vesicle secretion and improve extracellular electron transfer capacity of the target gene.

2. The method of claim 1; the method is characterized in that: the preparation method of the secondary fermentation broth in the step (7) comprises the following steps: the primary seed liquid was inoculated in an inoculum size of 1% into a 250mL conical flask containing 100mL of LB liquid medium containing 0.1mM IPTG and 50mg/L kana antibiotic, and subjected to shaking overnight expansion culture at 30℃and 200rpm to obtain a secondary fermentation broth.

3. The method of claim 1; the method is characterized in that: and (3) taking fermentation liquor of the recombinant strain of the high-yield vesicle in the step (7) as an anode microbial catalyst, inoculating the fermentation liquor into an anode chamber of a microbial fuel cell according to OD ₆₀₀ = 0.5, and inoculating a traditional K ₃[Fe(CN)₆ solution into a cathode chamber as a cathode electron acceptor, so that high electric energy output is realized.