Method for regulating and controlling cell division cycle to strengthen electron transfer capability of shewanella

Abstract

The invention relates to a method for regulating and controlling the cell division cycle to strengthen the electron transfer capability of shewanella. In order to explore the relationship between the cellular morphology and the electron transfer capability of electroactive microorganisms, a synthetic biology strategy is used for carrying out cellular morphology transformation on mode electrogenic bacteria shewanella, and four engineering strains capable of shortening the cell division cycle of shewanella are constructed. Morphological characterization shows that the cell size of the overexpression division protein engineering strain is reduced, and electrochemical characterization shows that the output voltage, current and power density of the engineering strain are gradually increased along with the reduction of the cell volume, which indicates that the overexpression division related protein can shorten the division cycle and accelerate the cell division process; the cells are reduced; and the electron transfer rate of shewanella is improved. The invention provides a new method for transforming electroactive microorganisms by using morphological engineering, enhancing the electron transfer capability of shewanella and improving the application capability of shewanella.

Description

Technical Field

[0001] The present invention belongs to the technical fields of synthetic biology and bioenergy technology. Aiming at the model electricity-producing microorganism Shewanella oneidensis MR-1, the cell morphology is regulated by regulating the cell division cycle to make the cells smaller, thereby enhancing the extracellular electron transfer ability of Shewanella bacteria.

[0002] Background Introduction

[0003] Microbial electrochemical systems based on the bidirectional electron transfer (EET) of electroactive microorganisms have great application potential in the fields of chemical engineering, energy, and environment. At present, the electron transfer rate is the core bottleneck restricting the industrial application of electroactive microorganisms. The model electricity-producing microorganism Shewanella bacteria mainly mediate direct and indirect electron transfer through cytochromes and riboflavin. Based on its EET mechanism, many synthetic biology and materials engineering means have been developed to improve its electron transfer rate from the perspectives of electron generation, transmembrane transfer, and biofilm formation. However, the technical means to regulate the cell size of Shewanella bacteria have not been developed yet, and the influence mechanism of cell size on the electron transfer rate of Shewanella bacteria has not been resolved.

[0004] The regulation of cell size and morphology is closely related to cell division cycle events and the increase in biomass, and is a basic feature of various types of prokaryotic and eukaryotic cells. Bacterial reproduction occurs by binary fission, and the bacterial division process is generally divided into the C period and the D period. The C period refers to the period from the start of DNA replication to the separation of sister chromatids, and the D period refers to the period from the end of replication to the start of cell division. By changing the length of time that bacteria stay in the C period and the D period, the control of the cell size and morphology of bacteria can be achieved. Current research shows that the morphology and size of bacteria are regulated by two modules: cell division proteins and cytoskeletal proteins. Cell division proteins participate in the bacterial division process, regulate the formation of the Z-ring and further assemble into a cell division complex, namely the divisome, to promote septum formation and thus achieve cell division.

[0005] In recent years, a variety of cell division proteins have been identified and characterized. However, further research is needed to understand how they interact precisely with each other and what fine regulations govern the process of divisome assembly and activation. Currently, the research on cell division proteins in Escherichia coli is the most in-depth. Taking Escherichia coli as an example, cell division proteins include scaffold proteins that provide support for the assembly of the entire divisome complex, peptidoglycan synthases and hydrolases used to remodel the peptidoglycan network, DNA translocases that coordinate cell division and chromosome segregation, proteins that coordinate peptidoglycan synthesis and membrane invagination, and many other proteins with regulatory functions. These proteins together assemble into a strictly regulated peptidoglycan synthesis machine that builds a septum between the separated chromosomes and precisely separates the cell into two daughter cells. Therefore, regulating the cell division cycle by controlling cell division proteins is a potential means of directionally regulating cell morphology. Research has found that activating the expression of ribonucleotide reductase NrdAB and division protein FtsZA by blue light and near-infrared light can shorten the C and D phases of Escherichia coli cell division, and its average cell volume is reduced to 59.7% of the wild type, and the specific surface area is increased to 1.15 times that of the wild type, resulting in a 4.96-fold increase in the end product acetoin titer. Some studies have successfully changed the cell morphology of Glutamicibacter by upregulating or downregulating the expression levels of the divIVA gene and ftsZ gene, obtaining cells with various morphologies such as small oval, spherical, long rod-shaped, and dumbbell-shaped, increasing the cell membrane area by 5.2 times, and increasing the single-cell hyaluronic acid production by 13.5 times.

[0006] The present invention overcomes the deficiencies of the prior art and develops a method for directionally modifying the cell morphology of Shewanella by regulating the cell division cycle in combination with existing research theories, and explores the relationship between the cell morphology of electroactive microorganisms and their electron transfer ability. Summary of the Invention

[0007] Problems to be Solved by the Present Invention:

[0008] The object of the present invention is to overcome the deficiencies of the prior art and develop a method for directionally modifying the cell morphology of the model electrogenic bacterium Shewanella by regulating the cell division cycle. Using synthetic biology strategies, the cell morphology of Shewanella is modified, and four engineered strains with shortened cell division cycles of Shewanella are constructed. Morphological characterization shows that the cell size of the engineered strains overexpressing division proteins decreases. Electrochemical characterization shows that the output voltage, current, and power density of the engineered strains gradually increase as the cell volume decreases, indicating that overexpressing division-related proteins can shorten the division cycle and accelerate the cell division process, achieving cell shrinkage and improving the electron transfer rate of Shewanella. The present invention successfully constructs engineered strains with reduced size by using morphological engineering means, realizing the directional modification of the cell morphology of Shewanella, and laying a foundation for analyzing the relationship between the cell morphology of electroactive microorganisms and their electron transfer ability.

[0009] Technical solution of the present invention:

[0010] To solve the above technical problems, the technical solution of the present invention is as follows:

[0011] The present invention provides a method for regulating the cell division cycle to improve the electron transfer ability of Shewanella, comprising the following steps:

[0012] (1) Screening 4 proteins (FtsQ, FtsN, FtsA and FtsZ) related to Shewanella cell division;

[0013] (2) Constructing engineering strains overexpressing their coding genes (ftsQ, ftsN, ftsA and ftsZ). First, obtain the cytokinetic genes ftsQ, ftsN, ftsA and ftsZ from the genome of S. oneidensis MR-1 by PCR amplification, then ligate them with the vector pYYDT plasmid using seamless cloning technology, and finally transfer them into MR-1 by conjugation of auxotrophic Escherichia coli WM3064 to obtain recombinant Shewanella engineering strains FtsQ, FtsN, FtsA and FtsZ.

[0014] (3) Observing the morphological changes of the engineering strain cells using a scanning electron microscope;

[0015] (4) Measuring the cell size of the engineering strain using ImageJ software and calculating the cell volume and specific surface area;

[0016] (5) Electrochemically characterizing the engineering strain using an electrochemical workstation, including measuring the chronoamperometry curve, cyclic voltammetry curve and linear voltammetry curve of the engineering strain, and then calculating the output voltage, current density and power density of the engineering strain.

[0017] Beneficial effects of the present invention:

[0018] The present invention constructs 4 recombinant Shewanella engineering strains FtsZ, FtsQ, FtsA and FtsN that regulate the cell division cycle by overexpressing cell division-related proteins. Morphological characterization shows that the cell size of the engineering strain overexpressing the division protein becomes smaller, mainly manifested as a decrease in length. The results of electrochemical characterization show that the output voltage, current and power density of the engineering strain gradually increase with the decrease of cell volume, indicating that overexpressing division-related proteins shortens the D period of cell division, which can accelerate cell division, make the cells smaller, and at the same time enhance the electron transfer rate of Shewanella. Among them, the engineering strain FtsZ overexpressing the division protein shows the smallest cell length of 0.97 µm, which is only 49.7% of the cell length of the wild type; at the same time, the cell volume of the engineering strain FtsZ also reaches the minimum of 0.059 µm 3, which is only 44.36% of the volume of wild-type cells. The results of electrochemical characterization show that the power density of the minimal strain FtsZ reaches 213.5 mW / m 2 , which is increased to 2.93 times that of the wild-type strain at 72.8 mW / m 2 . Description of the Drawings

[0019] Figure 1 is the schematic design diagram of the engineered strain overexpressing the fission protein.

[0020] Figure 2 is the map of the recombinant plasmids contained in the engineered strain overexpressing the fission protein. Among them, A is plasmid pYYDT-ftsQ; B is plasmid pYYDT-ftsN; C is plasmid pYYDT-ftsA; D is plasmid pYYDT-ftsZ.

[0021] Figure 3 is the cell morphology of the engineered strain overexpressing the fission protein observed by scanning electron microscopy (SEM).

[0022] Figure 4 are the length, width and volume of the engineered strain overexpressing the fission protein. Among them, A is the mean cell length (MCL) and mean cell width (MCW); B is the mean cell volume (MCV) and specific surface area (SSA).

[0023] Figure 5 are the results of electrochemical characterization of the engineered strain overexpressing the fission protein. Among them, A is the output voltage curve of the engineered strain; B is the cyclic voltammetry curve of the engineered strain; C is the linear voltammetry curve of the engineered strain; D is the electrode biomass of the engineered strain. Detailed Embodiments

[0024] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0025] Example 1: Screening of Genes Related to Cell Division in the Present Invention

[0026] The core process in the D period of the cell division cycle is the formation of the divisome. Promoting the formation of the divisome can shorten the D period of the cell, thereby promoting cell division and making the cell smaller. The formation of the cell divisome is responsible for four division proteins: the Z-ring-forming protein FtsZ, the anchoring protein FtsA, the scaffolding protein FtsQ, and the triggering protein FtsN. In the present invention, first, four division-related genes (ftsQ, ftsN, ftsA, and ftsZ) in the D period of the cell division cycle of Shewanella bacteria were screened. By upregulating the expression levels of the four division-related proteins, the formation of the Z-ring was promoted, the time of the D period was shortened, and cell division was accelerated to make the cells smaller. The specific mechanism of action of the genes screened in the present invention for regulating the divisome genes is shown in Table 1.

[0027] Table 1 Genes used in the present invention

[0028] Gene Coding protein function Source ftsQ Two-way membrane protein, which forms a scaffold complex to recruit downstream divisome proteins S. oneidensis MR-1 ftsN Cell division protein, which plays a role in triggering cell contraction S. oneidensis MR-1 ftsA Actin-like protein, which binds to the cell membrane through an amphipathic helix structure S. oneidensis MR-1 ftsZ Z-ring forming protein, which serves as a scaffold to recruit the assembly of other cell division proteins to form a complete divisome S. oneidensis MR-1

[0029] Example 2: Construction of an engineering strain overexpressing division proteins of the present invention

[0030] All plasmids of the present invention were constructed by seamless cloning method. The plasmids and strains involved are shown in Table 2 and Table 3 respectively, and the PCR primers used are shown in SEQ ID NO.1-10.

[0031] Table 2 Plasmids used in the present invention

[0032] Plasmid Description Source pYYDT pBBR1, kanR, mob, lacI Our Lab pYYDT-ftsQ <![CDATA[pBBR1, kanR, mob, lacI, P tac -nrdA-ftsQ]]> This Study pYYDT-ftsN <![CDATA[pBBR1, kanR, mob, lacI, P tac -ftsN]]> This Study pYYDT-ftsA <![CDATA[pBBR1, kanR, mob, lacI, P tac -ftsA]]> This Study pYYDT-ftsZ <![CDATA[pBBR1, kanR, mob, lacI, P tac -nrdA-ftsZ]]> This Study

[0033] Table 3 Strains used in the present invention

[0034] Strain Description Source Shewanella oneidensis MR-1 Lake Oneida isolate Our Lab WT MR-1 harboring pYYDT Our Lab FtsQ MR-1 harboring pYYDT-ftsQ This study FtsN MR-1 harboring pYYDT-ftsN This study FtsA MR-1 harboring pYYDT-ftsA This study FtsZ MR-1 harboring pYYDT-ftsZ This study

[0035] The steps for seamless cloning construction are as follows: First, design seamless cloning PCR primers. The principle is to introduce homologous recombination sequences at the 5' end through the primers, so that the plasmid vector and the gene fragment have 15-20 bp homologous arms, that is, 15-20 bp homologous sequences should be added to the upstream and downstream primers when designing the primers. Then, PCR amplification is used to obtain the vector and fragment with homologous arms. Finally, NovoRec ® Plus PCR One-Step Directional Cloning Kit is used for ligation. The ligation system is shown in Table 4, and the reaction conditions are 50 °C for 30 min. The target gene is ligated to plasmid pYYDT by homologous recombination. The elements carried on the plasmid are shown in Table 2. Finally, recombinant plasmids pYYDT-ftsQ, pYYDT-ftsN, pYYDT-ftsA, and pYYDT-ftsZ are constructed (the plasmid maps are as Figure 2 shown).

[0036] Table 4 Seamless cloning system

[0037] Component Volume (μL) 5× Reaction buffer 2 Fragment 4 Skeleton 3 Recombinase 1

[0038] Example 3: Construction of the microbial fuel cell of the present invention

[0039] The microbial fuel cell (MFC) of the present invention is constructed with a bipolar chamber, which is divided into an anode chamber and a cathode chamber. The connection part is separated by a proton exchange membrane. The anode electrode uses a 1 cm×1 cm carbon cloth, and the cathode electrode uses a 2.5 cm×3 cm carbon cloth. The anode chamber contains a total of 120 mL of anode liquid and secondary seed liquid, so that the final OD of the secondary seed liquid in the anode chamber is unified to 0.5. The anode liquid is composed of 95% M9 buffer, 5% LB medium, 20 mM sodium lactate, 0.1 mM calcium chloride, 1 mM magnesium sulfate, and 4 mM sodium hydroxide; the cathode liquid is composed of 50 mM potassium ferricyanide, 50 mM dipotassium hydrogen phosphate, and 50 mM potassium hydrogen phosphate.

[0040] Example 4: Morphological characterization of the engineered strain overexpressing the fission protein of the present invention

[0041] To observe the morphological changes of the engineered strain, a scanning electron microscope (SEM) was used to perform morphological characterization on the strain on the MFC electrode. The SEM shooting work was provided by Scientific Compass, and the sample treatment method is as follows: Take the electrode in the anode chamber of the MFC, soak it in 3 mL of 2.5% glutaraldehyde at 4 °C overnight, then wash the electrode 3 times with 0.85% NaCl solution, 10 minutes each time, and then soak the electrode in 30%, 50%, 70%, 80%, and 90% ethanol solutions respectively, 10 minutes for each concentration. Finally, the electrode can be freeze-dried for 12 h and then SEM shooting can be carried out.

[0042] After the SEM shooting, ImageJ software was used to measure the length and width of single cells on the electrode. Ten individual cells were selected, and their average cell length (MCL) and average cell width (MCW) were calculated. Furthermore, based on the cell rod model, the average cell volume (MCV) and specific surface area (SSA) were calculated according to formulas (1)-(3).

[0043]

[0044]

[0045]

[0046] Among them, L is the cell length, D is the cell width, S is the cell surface area, and V is the cell volume.

[0047] The SEM observation results show that the cell volume of the engineered strain regulating the cell division body is smaller than that of the wild-type strain and the distribution density on the electrode is larger (as Figure 3As shown). After cell size measurement, it was found that the cell length of the engineered strains decreased significantly, while the width did not change significantly. The average cell lengths of the engineered strains FtsQ, FtsN, FtsA, and FtsZ decreased to 1.53, 1.42, 1.03, and 0.97 µm, respectively (as Figure 4 shown in A). Among them, the cells of strain FtsZ were the shortest, only 49.7% of the cell length of WT cells (1.95 µm). At the same time, the cell length of the engineered strain FtsAZ overexpressing both FtsA and FtsZ was 0.98 µm and did not further decrease, but showed a length similar to that of FtsZ. Further calculation showed that as the length of the engineered strains decreased, the volume of the engineered strains decreased (as Figure 4 shown in B). The average cell volumes of the engineered strains FtsQ, FtsN, FtsA, and FtsZ were 0.095, 0.091, 0.069, and 0.059 µm 3 ³, respectively. Among them, the cell volume of the engineered strain FtsZ reached the minimum, only 44.36% of the cell volume of wild-type cells (0.133 µm 3 ³), and its specific surface area expanded to 15.52 µm -1 ⁻¹, which was 1.1 times that of the WT specific surface area (14.07 µm -1 ⁻¹). The above results indicate that accelerating the cell division process by overexpressing divisome proteins can significantly reduce the cell length, and regulating the cell division cycle can achieve cell size reduction.

[0048] Example 5: Electrochemical Characterization of the Engineered Strain MFC Overexpressing Division Proteins of the Present Invention

[0049] To characterize the electron transfer performance of the engineered strains, the present invention uses an electrochemical workstation for electrochemical characterization, including scanning the chronoamperometric curves, cyclic voltammetry curves (CV), and linear sweep voltammetry curves (LSV) of the engineered strains, and further calculating the output voltage, current density, and power density of the engineered strains.

[0050] The chronoamperometric curve was measured at a constant potential of +0.2 V with a measurement interval of 60 s. The turnover CV scanning potential ranged from -0.7 V to 0.1 V with a scanning rate of 1 mV / s. The LSV scanning potential ranged from -0.8 V to -0.1 V with a scanning rate of 0.1 mV / s. The power density was calculated according to formula (4):

[0051]

[0052] where V is the absolute value of the scanning potential, I is the scanning current, and S is the electrode surface area.

[0053] Electrochemical characterization results showed that the voltage output of MFCs constructed with engineering strains that overexpressed fission-related proteins to regulate the cell cycle was enhanced. The maximum output voltages of engineering strains FtsQ, FtsN, FtsA, and FtsZ were 119.6 mV, 137.9 mV, 145.2 mV, and 151.8 mV, respectively, which were 1.89 times, 2.18 times, 2.29 times, and 2.40 times that of the wild-type strain (63.3 mV) (as Figure 5 shown in Figure 5 Figure A). Among them, the output voltage of strain FtsZ with the largest increase increased by 140%. The overall enhancement trend was consistent with the volume change of the engineering strains, increasing as the cell volume decreased. Further calculation results of current density and power density showed that the maximum current density and power density also showed the same trend (as 2 shown in 2 Figures B and 2 5C). The maximum power densities of engineering strains FtsQ, FtsN, FtsA, and FtsZ were 152.0 mW / m 2 , 182.9 mW / m 2 , 195.4 mW / m 2 , and 213.5 mW / m 2 , respectively, which were 2.09 times, 2.51 times, 2.68 times, and 2.93 times that of the wild-type strain (72.8 mW / m 2 ). Among them, strain FtsZ with the largest increase increased by 193%. Consistent with the morphological changes, the output voltage, current density, and power density of engineering strain FtsAZ that overexpressed FtsA and FtsZ simultaneously did not increase further. By measuring the biomass on the electrode, it was found that the electrode biomass of the corresponding engineering strains was also significantly increased, and the increasing trend was still consistent with the morphological change trend (as Figure 5 shown in Figure D). The above results indicate that obtaining smaller cells by overexpressing fission-related proteins to shorten the D period of the cell division cycle can enhance the electron transfer ability of Shewanella, thereby strengthening the electricity generation performance of the strains.

[0054] For the technical solutions disclosed and proposed in the present invention, those skilled in the art can achieve them by referring to the content of this article and appropriately changing conditions, routes, and other aspects. Although the methods and preparation techniques of the present invention have been described through preferred embodiments, it is obvious that relevant technicians can make changes or re-combinations to the methods and technical routes described in this article without departing from the content, spirit, and scope of the present invention to achieve the final preparation technique. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as being included in the spirit, scope, and content of the present invention. Matters not covered in the present invention belong to well-known technologies.

Claims

1. A method for regulating the cell cycle to enhance the electron transfer ability of Shewanella; characterized by: It includes the following steps: (1) Screen 4 proteins (FtsQ, FtsN, FtsA, and FtsZ) related to Shewanella cell division; (2) Construct engineering strains overexpressing their coding genes (ftsQ, ftsN, ftsA, and ftsZ); (3) Observe the morphological changes of the engineering strain cells using a scanning electron microscope; (4) Use ImageJ software to measure the cell size of the engineering strain and calculate the cell volume and specific surface area; (5) Use an electrochemical workstation to perform electrochemical characterization of the engineering strain, including measuring the chronoamperometric curve, cyclic voltammogram, and linear voltammogram of the engineering strain, and then calculating the output voltage, current density, and power density of the engineering strain.

2. The method according to claim 1, characterized in that: Obtain the cytokinetic genes ftsQ, ftsN, ftsA, and ftsZ from the genome of Shewanella oneidensis MR-1 by PCR amplification.

3. The method according to claim 2, wherein: The amplification primers for the cytokinetic genes ftsQ, ftsN, ftsA, and ftsZ include SEQ ID NO.1-8.

4. The method according to claim 1, characterized in that: The PCR amplification primers for the engineering strain carrying the plasmid pYYDT include SEQ ID NO.9, 10.

5. The method according to claim 3 or 4, characterized in that: The plasmid pYYDT is ligated to the cytokinetic genes ftsQ, ftsN, ftsA, and ftsZ using seamless cloning technology and reacted at 50 °C for 30 min.

6. The method according to claim 5, characterized in that: Chemically transform the recombinant plasmids pYYDT-ftsQ, pYYDT-ftsN, pYYDT-ftsA, and pYYDT-ftsZ into the auxotrophic Escherichia coli WM3064, and then conjugally transfer them into Shewanella oneidensis MR-1. Positively screen to obtain the recombinant Shewanella engineering strains FtsQ, FtsN, FtsA, and FtsZ.

7. Application of the recombinant Shewanella engineering strains FtsQ, FtsN, FtsA, and FtsZ described in claim 6 in regulating the cell division cycle.

8. Application of the recombinant Shewanella engineering strains FtsQ, FtsN, FtsA, and FtsZ described in claim 6 in enhancing the electron transfer ability of Shewanella.

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