Application of electron transport protein element group displayed on cell surface and biological photovoltaic device constructed by electron transport protein element group

Abstract

The invention relates to an application of an electron transport protein element group displayed on the surface of a microbial cell in construction of biological photovoltaic, and a biological photovoltaic device, a cathode tank of which comprises microalgae capable of secreting intermediate energy metabolites and a culture medium thereof, and a set of electron transport protein elements that oxidizes the intermediate energy metabolite and releases and transports electrons. According to the invention, microalgae capable of efficiently secreting an intermediate energy metabolite to the outside of a cell is adopted as a light energy capturing body, so that the energy metabolite is transferred from the inside of the cell to the outside of the cell, the light energy conversion efficiency is improved, the efficient transmembrane transfer of an energy substance is realized, and the protein extracellular display technology is adopted. The electron transfer protein elements are anchored on the surfaces of the cells, so that the loss caused by aggregation among the electron transfer protein elements and intracellular metabolism of heterotrophic microorganisms is avoided, the oxidation efficiency and electron transfer efficiency of intermediate energy metabolites are improved, and the energy output of the biological photovoltaic device is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biophotovoltaics, and particularly relates to the application of an electron transfer protein element group displayed on the cell surface and a biophotovoltaic device constructed therefrom. Background Art

[0002] Biophotovoltaics (BPV) is a new solar power generation technology that uses photosynthetic microorganisms such as cyanobacteria and eukaryotic microalgae as optoelectronic conversion materials. Compared with traditional semiconductor photovoltaic technologies, it has the characteristics of low toxicity, recyclability, and strong environmental compatibility, providing a more green and sustainable technical route for solar energy utilization.

[0003] In a biophotovoltaic device, the conversion of solar energy into electrical energy can be divided into two stages, namely: the charging stage (mainly intracellular photoreaction) and the discharging stage (including photoelectron transmembrane transfer and external circuit photocurrent generation). Among them, the process of excited electrons generated by intracellular photoreaction transferring to the external circuit is called extracellular electron transfer (EET), and the efficiency of this process is extremely low, severely limiting the application of biophotovoltaics.

[0004] Although the exogenous addition of artificial electron mediators can improve the electron transfer efficiency, it has potential toxicity to cells and high costs, which is not conducive to long-term and large-scale use. Introducing the EET pathway into photosynthetic microorganisms through genetic engineering may affect the photosynthetic efficiency of photosynthetic microorganisms. Therefore, on the one hand, photosynthetic microorganisms that can secrete energy metabolites to the outside of the cell are needed, and on the other hand, electrogenic heterotrophic microorganisms need to be introduced to internalize energy substances for power generation. Even so, energy is required during the second transmembrane process of energy substances, and other metabolic activities of heterotrophic microorganisms also require energy, and it is difficult to ensure the transmembrane efficiency of electrons generated during the oxidation of energy substances by heterotrophic microorganisms.

[0005] Therefore, how to improve the EET efficiency is one of the key research contents in biophotovoltaics. Summary of the Invention

[0006] To solve the above problems, the present invention provides the application of an electron transfer protein element group displayed on the surface of microbial cells in constructing biophotovoltaics.

[0007] In a specific embodiment, the electron transfer protein element group is an enzyme related to glucose oxidation metabolism.

[0008] In a specific embodiment, the electron transfer protein element group includes an upstream element group, including PPGK, G6PDH, 6PGDH, and PGL.

[0009] In a specific embodiment, the electron transfer protein element group further includes a downstream element group, including RP I, RU5PE, TAL, TK, and PG I.

[0010] The present invention also provides a bio-photovoltaic device, including an anodic chamber, a cathodic chamber, and a proton-permeable semipermeable membrane separating the anodic chamber from the cathodic chamber. The cathodic chamber contains microalgae capable of secreting intermediate energy metabolites and their culture medium, and an electron transfer protein element group capable of oxidizing the intermediate energy metabolites and releasing and transferring electrons, and the main body of the cathodic chamber is made of a transparent material.

[0011] In a specific embodiment, the electron transfer protein elements in the electron transfer protein element group are displayed on the surface of microbial cells.

[0012] In a specific embodiment, the microbial cells are fixed on the surface of the cathode of the cathodic chamber.

[0013] In a specific embodiment, the intermediate energy metabolite is glucose, and the electron transfer protein element group includes PPGK, G6PDH, 6PGDH, and PGL.

[0014] In a specific embodiment, the electron transfer protein element group further includes RP I, RU5PE, TAL, TK, and PG I.

[0015] In a specific embodiment, the ratio of extracellular display bacteria of PPGK, G6PDH, 6PGDH, and PGL is

[0016] 8:1:3:3;

[0017] The ratio of extracellular display bacteria of RP I, RU5PE, TAL, TK, and PG I is 1:5:6:1:1;

[0018] The ratio of extracellular display bacteria of the upstream element group to the extracellular display bacteria of the downstream element group is 1:6.

[0019] In a specific embodiment, the microbial cells are Escherichia coli.

[0020] In a specific embodiment, the microalgae are attached to a solid matrix.

[0021] The present invention has the following advantages:

[0022] 1) By using microalgae capable of efficiently secreting intermediate energy metabolites extracellularly as a light energy capture body, the energy metabolites are transferred from intracellular to extracellular, improving the light energy conversion efficiency and achieving efficient transmembrane transfer of energy substances.

[0023] 2) The protein extracellular display technology is used to anchor the electron transfer protein elements on the cell surface. On the one hand, it avoids the aggregation of electron transfer protein elements, so that they do not interfere with each other in function; on the other hand, it does not need to internalize energy substances into heterotrophic microbial cells, avoiding the loss caused by the intracellular metabolism of heterotrophic microorganisms (used for the growth and metabolism of the microorganisms themselves), thereby improving the oxidation efficiency and electron transfer efficiency of intermediate energy metabolites, further improving the conversion efficiency on the basis of the same substrate level, and further improving the output of the biophotovoltaic device.

[0024] 3) Whole cells displaying proteins are used as catalysts, eliminating the need for complex protein purification during the experimental operation. Using cell number as a counting method simplifies the exploration of the addition ratio between different microorganisms in the microbial community.

[0025] 4) To ensure the stable and efficient photocurrent output of the biophotovoltaic device, the preparation method of the cathode electrode and the cyanobacteria culture method in the device are optimized respectively. Commercial carbon cloth is used as the cathode electrode substrate, and the mixed extracellular display bacterial solution with optimized addition ratio is dripped onto its surface for natural drying. After drying, the cathode electrode is encapsulated with chitosan. The long-term stable operation of the biophotovoltaic device is inseparable from stable and continuous energy input, so cyanobacteria need to maintain good cell activity as energy capturers. The culture medium needs to be continuously updated and supplemented as the energy source for the growth of algae. Under conventional culture methods, the suspended algae are not conducive to the addition of culture medium, so sodium alginate gel is used to embed and culture cyanobacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the metabolic pathway for complete oxidation of glucose.

[0027] Figure 2 This is the electrophoresis diagram of the gene fragments encoding glucose metabolism-related enzymes and anchoring proteins.

[0028] Figure 3 Statistical graphs of enzyme activities for different strain combinations.

[0029] Figure 4 The enzyme activity data of different addition ratios of upstream and downstream strains

[0030] Figure 5 Output difference curve for the device of adding extracellular display bacterial flora.

[0031] Figure 6 This is the culture status of the sodium alginate gel-solidified algae strain and the extracellular glucose content curve.

[0032] Figure 7 Schematic diagram of a biophotovoltaic device in one embodiment of the present invention.

[0033] Figure 8 This is the current output curve of the bio-photovoltaic device in an embodiment of the present invention. Detailed implementation manners

[0034] The principles and features of the present invention will be described below in conjunction with the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0035] 1. Construction of extracellular display strains

[0036] As Figure 1 shown, the complete oxidation metabolic pathway of glucose includes the following enzymes: polyphosphate glucokinase (PPGK), glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), phosphogluconolactonase (PGL), ribulose isomerase (RPI), ribose-5-phosphate isomerase (RU5PE), transaldolase (TAL), transketolase (TK) and phosphohexose isomerase (PGI) multiple enzymes (Table 1). Glucose is phosphorylated by polyphosphate glucokinase (PPGK) and catalyzed by glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) to generate NADH, and the electron transfer of NADH to the anode. Glucose regenerates 6-phosphate glucose through the non-oxidative pentose phosphate pathway and gluconeogenesis. The Faraday efficiency from glucose to electrons through this pathway is as high as 98.8%, and nearly 24 electrons are generated per glucose molecule.

[0037] Table 1 Extracellular display strains of EET components

[0038]

[0039]

[0040] Using the Escherichia coli extracellular display technology, the enzymes involved in the above pathway are anchored to the surface of Escherichia coli using an anchor protein. The method is as follows:

[0041] 1) Amplification of glucose metabolism-related enzymes and anchor proteins

[0042] Amplify the gene fragments encoding the above glucose metabolism-related enzymes. In addition, in order to anchor the above enzymes on the outer cell membrane of the host, the gene fragments of four anchor proteins M i PA, LoT, OmPC, I NP (SEQ ID NO: 10-13) were also amplified. The results are as Figure 2 shown, and the gene fragment amplification was successful.

[0043] 2) Construction of recombinant expression plasmids

[0044] A gene fragment related to glucose metabolism enzyme was ligated with an anchor protein gene fragment to obtain fusion fragments Lot-PPGK, INP-G6PDH, MipA-6PGDH, OmpC-PGL, Lot-RPI, ipA-RU5PE, MipA-TAL, Lot-TK, INP-PGI; the fusion fragments were cloned into the expression vector pET28a to obtain recombinant expression plasmids pET28a-Lot-PPGK, pET28a-INP-G6PDH, pBad-MipA-6PGDH, pET28a-OmpC-PGL, pET28a-Lot-RPI, pET28a-MipA-RU5PE, pET28a-MipA-TAL, pET28a-Lot-TK, pET28a-INP-PGI.

[0045] 3) Construction of extracellular display strains

[0046] The above recombinant expression plasmids were respectively transformed into Escherichia coli BL21 to obtain nine extracellular display bacteria. After activating the extracellular display strains, they were respectively inoculated into a liquid medium and cultured until OD 600 = 0.8, and then IPTG was added for induction expression.

[0047] The results showed that each strain expressed the corresponding protein.

[0048] 2. Enzyme activity verification of extracellular display strains

[0049] After the construction of the extracellular display strains, enzyme activity characterization experiments were carried out on each enzyme in the glucose oxidative metabolism pathway. The measurement principle and method are as follows:

[0050] 1) Determination of PPGK enzyme activity:

[0051] Reaction equation:

[0052] Glucose → (PPGK) → g6p + NAD + → (G6PDH) → 6pg + NADH

[0053] As shown in Table 2, an enzyme activity determination system was prepared, reacted in a 25 °C water bath for 10 min, and the reaction supernatant was taken and placed in a 96-well plate, and its A 340nm absorbance value was measured using a microplate reader.

[0054] Table 2 PPGK enzyme activity determination system

[0055]

[0056] 2) Determination of PG I enzyme activity:

[0057] Reaction equation: F6P → (PG I) → G6P

[0058] G6P + NAD + →(G6PDH)→6 - phosphogluconate + NADH

[0059] Prepare the enzyme activity assay system as shown in Table 3, set the ratio between PGI and G6PDH to 1:9, react in a 37°C water bath for 5 min, take the reaction supernatant and place it in a 96 - well plate, and measure its A 340nm absorbance at this position.

[0060] Table 3 PGI Enzyme Activity Assay System

[0061]

[0062] 3) 6PGDH Enzyme Activity Assay:

[0063] Reaction equation: 6pg + NAD + →(6PGDH)→ru5p + NADH

[0064] Prepare the enzyme activity assay system as shown in Table 4, react in a 37°C water bath for 15 min, after the reaction, centrifuge at 10000 rpm for 5 min, after centrifugation, take the reaction supernatant and place it in a 96 - well plate, and measure its A 340nm absorbance at this position.

[0065] Table 4 6PGDH Enzyme Activity Assay System

[0066]

[0067] For extracellular display strains whose single - enzyme activity cannot be characterized (due to reasons such as inability to obtain specific reaction substrates), couple them with upstream / downstream extracellular display strains to characterize the overall enzyme activity of the extracellular display microbial community. The upstream pathway is the main glucose degradation pathway, and the downstream pathway is the main replenishment pathway of the upstream pathway. Only add the enzyme activity genes involved in the upstream pathway to characterize the enzyme activity, and then add the relevant extracellular display microbial community of the downstream pathway to further characterize the enzyme activity of the system. Measure the A 340 absorbance of the two experimental systems, compare the numerical differences, and analyze whether the downstream pathway plays an enhancing role in the upstream pathway based on this.

[0068] In the enzyme activity characterization experiment of the extracellular display microbial community, use NAD+ as a cofactor, which is reduced to NADH in the enzyme activity reaction system, and characterize the enzyme activity of the extracellular display microbial community by measuring the change in its special absorption peak at 340 nm.

[0069] The enzyme activity of the whole-cell catalyst of the multi-enzyme combination was characterized by the above experimental method, and the enzyme activity was measured. Here, the enzyme activity unit was customized. Here, 1 nmol of NADH catalyzed by the whole-cell catalyst per minute was defined as one enzyme activity unit U. As Figure 3 shown in a, after combining the two extracellular display strains of Lot-PPGK and INP-G6PDH, the activity of the whole-cell catalyst was 6.44 U / OD, which was the first two steps of the glucose degradation pathway. Subsequently, the three extracellular display strains of INP-G6PDH, OmpC-PGL, and MipA-6PGDH were cascaded and combined, and the activity of the whole-cell catalyst was 8.53 U / OD, which was the last two steps of the glucose degradation pathway. These experimental results indicated that cascading these four extracellular display strains could be combined to complete the four-step reaction of glucose degradation, that is, glucose was degraded to ribulose-5-phosphate. Thus, the enzyme activity characterization of the upstream four-enzyme system of the extracellular glucose complete oxidation metabolic pathway was completed.

[0070] For the enzyme activity characterization of the complete glucose oxidation pathway (nine-enzyme system) with an additional anaplerotic pathway, the enzyme activity was characterized by cascading. Only adding the bacterial liquid related to the upstream four-bacteria system, the whole-cell catalytic activity was 6.2 U / OD. On the basis of the upstream, adding the bacterial liquid related to the downstream pathway, the activity of the whole-cell catalyst was 9.9 U / OD. After adding the downstream pathway, the activity of the whole-cell catalyst increased by 1.6 times ( Figure 3 b). Compared with the control group, both the upstream / downstream pathways had significant enzyme activities. It was shown that in the extracellular system, this extracellular display bacterial community could be used as a whole-cell catalyst and had the enzyme activity potential to promote the complete oxidation of glucose.

[0071] 3. Exploration of the proportion of each strain in the extracellular display bacterial community

[0072] The proportion between different microorganisms in the system would affect the degree of substrate oxidation and utilization of the system. Considering that there were many types of enzymes involved in the metabolic pathway of this project, the system was divided into upstream and downstream parts according to the above specific enzyme activity measurement method, and the optimal addition ratio was explored in turn. By comparing the specific enzyme activity data, after exploring the optimal addition ratio of the upstream, the exploration of the downstream addition ratio was carried out. The results were as Figure 4 shown. The optimal addition ratio of the upstream was PPGK:G6PDH:6PGDH:PGL = 8:1:3:3, the optimal addition ratio of the downstream was RPI:Ru5PE:TK:TAL:PGI = 1:5:6:1:1, and the addition ratio between the upstream and downstream was 1:6.

[0073] In subsequent experiments, Escherichia coli was added at this ratio to complete the verification of the enzyme fuel cell and the verification experiment of the artificial designed bacterial community bio-photovoltaic.

[0074] 4. Preparation of the cathode

[0075] In a specific embodiment, a conductor is used as the base material. After centrifugally pre-treating the extracellular-displaying microbial community and then dropping it onto the surface of the cathode electrode, a stable biofilm is formed at room temperature, and then a chitosan solution is dropped to fix the biofilm (modification method).

[0076] In another specific embodiment, a conductor can also be directly used as the cathode electrode, and the above-mentioned extracellular-displaying microbial community is dispersed in the solution of the cathode chamber (mixed bacteria method).

[0077] In the following experiment, carbon cloth was used as the base material of the cathode electrode, and extracellular-displaying Escherichia coli was fixed to the surface of the cathode electrode as EET, shortening the distance between electrons and the electrode, enabling electrons to be directly transferred through the conductor after generation, thereby further improving the electron transfer efficiency and maintaining the high-efficiency output of the device. As Figure 5 shown, the current output level under the mixed bacteria state reaches 120 μA·Cm -2 , and the output of the device under the modification state can be increased by 1.9 times, reaching 228 μA·Cm -2 .

[0078] 2. Construction of the cathode chamber

[0079] The main body of the cathode chamber is made of a light-transmitting material. In addition to the cathode electrode, the cathode chamber is also equipped with microalgae that can secrete glucose extracellularly and a culture medium for culturing the microalgae. In a specific embodiment, the engineered microalgae adhere to the surface of the substrate and are immersed in the culture medium. In another specific embodiment, the engineered microalgae can also be dispersed in the culture medium.

[0080] In the following experiment, the cyanobacterium was embedded and solidified by the method of embedding with sodium alginate gel, so that it adhered to the surface of the substrate.

[0081] The method is as follows: At room temperature, an appropriate amount of algal solution was centrifuged at 6000 rpm for 15 minutes, and then mixed with a 0.5-2.5% w / W sodium alginate solution to prepare a sodium alginate-cyanobacterium mixture. It was solidified with calcium chloride (the sodium ions in sodium alginate can be replaced by calcium ions in calcium chloride to form calcium alginate, which is insoluble in water, thus forming a cross-linked gel system), forming a sodium alginate-cyanobacterium hydrogel, whose structure can support the normal growth and product synthesis of cyanobacteria.

[0082] The strain used in the experiment was the engineered strain YD07 obtained by metabolic engineering transformation of Synechococcus elongatus PCC7002, which has the function of secreting glucose extracellularly.

[0083] The components of the culture medium (1 L) are: 18 g / L NaCl, 0.6 g / L KCl, 1 g / L NaNO3, 5 g / L MgSO4·7H2O, 0.2775 g / L CaCl2, and 1 mL / L trace element stock solution. In a specific embodiment of the present invention, the components of the trace element stock solution are: 2.86 g / L H3BO3, 1.81 g / L MnCl2·4H2O, 0.222 g / L ZnSO4·7H2O, 0.39 g / L Na2MoO4·2H2O, 0.08 g / L CuSO4·5H2O, 0.01 g / L CoCl2·6H2O, 16.8105 g / L EDTA-1Na, and 2.78 g / L FeSO4·7H2O.

[0084] Under the conventional culture method, the above-mentioned cathode chamber can achieve an extracellular sugar production of 405 mg / L ( Figure 6 ), which simplifies the difficulty of replacing the algal strain culture solution on the basis of meeting the experimental requirements of the bio-photovoltaic device. The fresh and abundant culture system will be beneficial to the long-term utilization of the bio-photovoltaic device.

[0085] 3. Assembly of the novel bio-photovoltaic

[0086] The cathode chamber described above is used to assemble the bio-photovoltaic. Based on this, as Figure 7 shown, the bio-photovoltaic device of this embodiment includes an anode chamber, a cathode chamber, and a proton-permeable semi-permeable membrane that separates the anode chamber from the cathode chamber.

[0087] The principle is as follows: In the anode chamber, a conductor (such as metal) electrode is used as the anode electrode. Protons generated in the cathode chamber diffuse through the proton membrane to the surface of the anode electrode, and glucose undergoes an oxidation reaction on the surface of the cathode electrode to generate electrons, thereby forming an electric current in the external circuit.

[0088] The cathode chamber is illuminated to cultivate engineered microalgae. To improve the metabolic efficiency of the engineered microalgae, air containing CO2 is introduced into the cathode chamber to promote its growth and the synthesis of metabolites. A large amount of glucose synthesized by algal cells is excreted into the cathode chamber and is metabolized by heterotrophic microorganisms as a high-energy intermediate to generate protons for the redox reaction in the anode chamber, thereby supporting the continuous current output in the external circuit.

[0089] The above optimization strategies are integrated to build an integrated bio-photovoltaic device. In the cathode chamber, algal strains are embedded in sodium alginate gel to fix light energy through photosynthesis, convert it into glucose, and excrete it into the external system. Glucose is used as a high-energy intermediate metabolite by the extracellular-displayed microbial community, undergoes complete oxidation to generate electrons, thereby realizing the photocurrent output of the integrated bio-photovoltaic device. After the device operates stably for 29 hours, the monitored maximum photocurrent output density can reach 210 μA·cm-2 ( Figure 8 )。

[0090] In the specific embodiments of the present invention, for the purpose of illustration, specific microalgae, the intermediate energy metabolite glucose, and enzymes related to electron transfer are listed to elaborate the principle of the present invention. However, these should not be used to limit the scope of protection of the present invention. After reading this, those skilled in the art can select appropriate microalgae and intermediate energy metabolites, as well as enzymes related to electron transfer adapted to the above-mentioned energy metabolites to achieve the purpose of the present invention. Therefore, these means and their variations should all be covered within the scope of protection of the present invention.

[0091] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Application of the electron transfer protein element group displayed on the surface of microbial cells in constructing bio-photovoltaic cells.

2. The application according to claim 1, wherein The electron transfer protein element group is enzymes related to glucose oxidative metabolism.

3. The application according to claim 2, characterized in that, The electron transfer protein element group includes an upstream element group, including PPGK, G6PDH, 6PGDH, and PGL.

4. The application according to claim 3, wherein The electron transfer protein element group further includes a downstream element group, including RPI, RU5PE, TAL, TK, and PGI.

5. A biological photovoltaic device, comprising an anodic chamber, a cathodic chamber, and a proton-permeable semipermeable membrane separating the anodic chamber from the cathodic chamber, characterized in that, The cathode chamber contains microalgae capable of secreting intermediate energy metabolites and their culture medium, and an electron transfer protein element group capable of oxidizing the intermediate energy metabolites and releasing and transferring electrons, and the main body of the cathode chamber is made of a transparent material.

6. The bio-photovoltaic device according to claim 5, wherein The electron transfer protein elements in the electron transfer protein element group are displayed on the surface of microbial cells.

7. The biological photovoltaic device according to claim 6, characterized in that, The microbial cells are fixed on the surface of the cathode of the cathode chamber.

8. The biological photovoltaic device according to claim 7, characterized in that, The intermediate energy metabolite is glucose, and the electron transfer protein element group includes an upstream element group, including PPGK, G6PDH, 6PGDH, and PGL.

9. The biological photovoltaic device according to claim 8, wherein The electron transfer protein element group further includes a downstream element group, including RPI, RU5PE, TAL, TK, and PGI.

10. The biological photovoltaic device according to claim 9, wherein The proportion of extracellular display bacteria of PPGK, G6PDH, 6PGDH, and PGL is 8:1:3:3; The proportion of extracellular display bacteria of RPI, RU5PE, TAL, TK, and PGI is 1:5:6:1:1; The proportion of extracellular display bacteria of the upstream element group to the extracellular display bacteria of the downstream element group is 1:6.

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