JP4614053B2 - Light-driven substance production method using a conjugate of oxidoreductase and photosynthetic reaction center - Google Patents

Description

The present invention relates to a light-driven material production method using photosynthesis.

In recent years, modern societies that depend on fossil fuels have faced various environmental problems, and there is a growing trend toward an environmentally compatible society. However, in order to shift to an environmentally friendly society, a clean, efficient and non-waste natural material production method that does not emit greenhouse gases and environmental pollutants is indispensable. Not.

As an effective method for producing substances with low environmental impact and low cost, microorganisms or plants (photosynthetic organisms) with photosynthetic ability are cultured, and various substances are produced enzymatically in photosynthetic organisms using sunlight as an energy source. The method of doing can be mentioned. Since photosynthetic organisms can grow on electron donors such as water and electron acceptors such as carbon dioxide and sunlight, the production of materials by photosynthetic organisms does not require expensive equipment and does not consume resources. Have In addition to the target substance, the product is not polluted with only biological substances such as oxygen and carbohydrates, and no greenhouse gases are generated. In particular, the photosynthetic reaction center that converts sunlight into chemical energy in photosynthetic organisms has the potential to produce substances with high photoconversion efficiency because it causes a photocharge separation reaction with a quantum yield close to 100%. Moreover, since a low reduction potential of about −500 mV and a high oxidation potential of about +800 mV are generated, it is possible to carry out substance production by a wide range of redox reactions.

However, when substance production is actually attempted using photosynthetic organisms, the production efficiency of the target substance is often low, and a problem that it varies greatly depending on the culture environment has been reported. For example, attempts to produce hydrogen using the hydrogen-producing ability of cyanobacteria or algae have the problems of low photoconversion efficiency of hydrogen generation to sunlight and the problem of hydrogen generation only in special culture environments. Has no practical technology.

Currently, as a technique for improving production efficiency in substance production using living organisms, genetic recombination in which a gene encoding an enzyme that catalyzes a target enzyme reaction is introduced into the organism is widely performed. The introduced gene is excessively expressed in the organism, and as a result, the target enzyme reaction is promoted. Many successful examples have been reported as represented by genetically modified crops. However, despite attempts to increase hydrogen generation efficiency by overexpressing hydrogenase, an enzyme directly involved in hydrogen generation, such as hydrogen production using cyanobacteria, In many cases, sufficient results cannot be achieved. This is because natural enzymes and intracellular metabolic mechanisms do not necessarily have properties suitable for production of the target substance.

In the photosynthesis process, photoinduced charge separation reaction takes place first in photosystem II, which is a photosynthetic reaction center protein. The resulting oxidizing power draws electrons from water molecules, and the reducing power is another photosynthesis reaction center protein. Electron transfer to I. In the photosystem I, the electrons received from the photosystem II are further photoexcited, and the resulting reducing power is normally transmitted to ferredoxin, and then distributed to various enzymes in the cell for use in carbonic acid fixation and the like.

It is known that hydrogen generation in cyanobacteria or algae occurs when a part of the extra reducing power generated by photosystem I is transferred to hydrogenase or nitrogenase, which is a proton reductase, via an electron carrier such as ferredoxin. It has been. However, because the transfer of reducing power to hydrogenase or nitrogenase is of low priority in the cell, only a small amount of solar energy absorbed by cyanobacteria or algae is used for hydrogen synthesis. Since the priorities of such metabolic pathways and electron transfer pathways in organisms depend on specific interactions between proteins, it is not always sufficient to artificially modify the expression level of enzymes. The production efficiency of the target substance cannot be improved.

Therefore, in order to increase production efficiency, molecular design at the protein molecule level is necessary. In other words, it is possible to design powerful metabolic pathways or electron transfer pathways by artificially creating specific interactions between proteins or by creating molecular devices by combining enzymes involved in target substance production. desired.

Until now, a general and powerful method for improving production efficiency in substance production using photosynthetic organisms has not been proposed. However, the present invention creates an artificial electron transfer pathway by designing a molecular device in which a photosynthetic reaction center and an oxidoreductase are linked at the molecular level, and efficiently reduces and / or oxidizes the photosynthesis reaction center. The objective is to construct an efficient light-driven system for the target redox reaction.

According to the present invention of claim 1 described in “Claims”, the oxidoreductase is chemically bonded and / or near the electron acceptor binding site and / or the electron donor binding site of the photosynthesis reaction center. Alternatively, the reduction force and / or the oxidation force generated by the photosynthesis reaction are directly oxidized by hydrogen bonding and / or electrostatic interaction and / or hydrophobic interaction and / or van der Waals interaction. Since it can be efficiently transmitted to the reductase, a method for producing an enzyme reaction that requires an oxidation-reduction process by light driving with high light conversion efficiency is provided (FIG. 1). As shown in FIG. 1, when an enzyme that performs a reduction reaction is linked to the vicinity of the electron acceptor binding site of the photosynthetic reaction center (FIG. 1a), and an enzyme that performs the oxidation reaction is near the electron donor binding site of the photosynthetic reaction center. When an enzyme that performs a reduction reaction and an enzyme that performs an oxidation reaction are coupled to both the vicinity of the electron acceptor binding site and the vicinity of the electron donor binding site of the photosynthetic reaction center (FIG. 1b). Is mentioned. In addition, the oxidoreductase and the photosynthetic reaction center described in the specification indicate a protein or protein complex characterized by each function, and artificially mutate natural and natural amino acid sequences. Including the introduced mutant type and the induced type in which a small molecule or polypeptide is bound to the natural type or mutant type.

Photosynthesis reaction centers include, for example, reaction centers present in cyanobacteria and algae, photochemical systems I and II existing in plants, red photosynthetic bacteria such as red sulfur bacteria and red non-sulfur bacteria, green sulfur bacteria and filamentous properties. Examples include reaction centers present in green photosynthetic bacteria such as fluffy green bacteria.

The oxidoreductase is preferably an oxidoreductase that directly exchanges electrons with an electron carrier protein such as cytochromes or ferredoxin, but a hydride derived from a coenzyme such as NAD (P) H, flavins, or quinones. In some cases, an oxidoreductase accompanying the exchange of ions can also be used. For example, hydrogenase, cytochrome P450, NADP ⁺ reductase, methane monooxygenase, cytochrome C peroxidase, nitrogenase, nitrate reductase, nitrite reductase, nitrous oxide reductase, nitric oxide reductase, sulfate reductase, sulfite reductase , Pyruvate dehydrogenase, pyruvate ferredoxin oxidoreductase, aldehyde ferredoxin oxidoreductase, formaldehyde ferredoxin oxidoreductase, keto acid ferredoxin oxidoreductase, ketovalerate ferredoxin oxidoreductase, glyceraldehyde 3-phosphate ferredoxin oxidoreductase Enzyme, ketovaline ferredoxin oxidoreductase, oxoglutarate synthase, glutamate synthase, indolepyruvate ferredoxin oxidoreductase, biliverdin reductase, Thiamine dehydrogenase, toluene dioxygenase, biphenyl dioxygenase, alcohol oxidase, glucose oxidase, lactate oxidase, lysyl oxidase, cholesterol oxidase, pyranose oxidase, benzoate dioxygenase, naphthalene dioxygenase, xylene monooxygenase, phenylpropionate dioxygenase, biphenyl dioxygenase And benzene dioxygenase.

As a method of linking an oxidoreductase and a photosynthetic reaction center, for example, as shown in FIG. 2, a fused oxidoreductase (13) in which any subunit (12) of the photosynthetic reaction center and the oxidoreductase (2) are fused. ) May spontaneously associate with the photosynthetic reaction center (14) lacking the subunit (12). This method utilizes the fact that subunits of the photosynthetic reaction center self-assemble with specific and strong affinity. The fusion oxidoreductase (13) is obtained by combining a gene (9) encoding the oxidoreductase (2) and a gene (10) encoding the subunit (12) with an appropriate promoter. It can be synthesized as a single polypeptide in which the subunit (12) and the oxidoreductase (2) are linked by introducing the plasmid into the cell by transformation using a plasmid or homologous recombination method. Methods for designing the fusion gene (11) include a method of linking and / or inserting the other gene at the end and / or in the middle of one of the genes, or dividing two or more genes at one or more locations. And a method of combining them in a mosaic shape (see FIG. 2). The fusion oxidoreductase (13) can be self-assembled with the photosynthetic reaction center by mixing with the photosynthetic reaction center (14) outside the cell. In addition, when the fusion oxidoreductase (13) is expressed in a photosynthetic organism in which the gene (10) encoding the subunit (12) has been deleted by a marker gene replacement method using a suicide vector, intracellular photosynthesis It is also possible to form a linking body with the reaction center (14). In addition, when the fusion oxidoreductase (13) is expressed in a natural photosynthetic organism, the fusion oxidoreductase (13) and the natural subunit (12) compete with each other in the photosynthetic reaction center ( In order to bind to 14), a part of the photosynthetic reaction center in the cell can be used as a conjugate with the fusion oxidoreductase (13). Examples of subunits of the photosynthesis reaction center include PsaA, PsaB, PsaC, PsaD, PsaE of photosystem I, D1, D2, CP43, CP47 of photosystem II, H subunit and cytochrome C domain in the reaction center of red photosynthesis bacteria. , Etc.

As another method for linking the oxidoreductase and the photosynthetic reaction center, for example, as shown in FIG. 3, a polypeptide (17) having an affinity for the photosynthetic reaction center (1) and the oxidoreductase (2) are used. The fused fusion oxidoreductase (18) is mixed with the photosynthetic reaction center (1) isolated outside the cell, or expressed in a photosynthetic organism having the photosynthetic reaction center (1), so that it can be expressed inside or outside the cell. A method of self-assembling a conjugate of the fusion oxidoreductase (18) and the photosynthetic reaction center (1) can be mentioned. The method for expressing the fusion oxidoreductase (18) and the method for fusing genes are the same as those described in [0013] above. Examples of the polypeptide (17) include ferredoxin, flavodoxin, plastocyanin, antibodies, polypeptides screened from polypeptide libraries, and the like. In addition, the definition of the photosynthetic reaction center in the present specification includes the inductive type in which a small molecule or a polypeptide is bound to the photosynthetic reaction center, but it has an affinity for antibodies bound to the photosynthetic reaction center. A polypeptide such as an antibody having a phenotype can also be mentioned as the polypeptide (17). Further, when using a photosynthetic reaction center modified by biotinylation or sugar chain modification by introducing a recognition sequence of a modifying enzyme such as biotinylated enzyme or glycosyltransferase as a photosynthetic reaction center, polypeptide (17) Examples thereof include avidin, which is a biotin-binding protein, and lectin, which is a sugar chain-binding protein.

As another method of linking an oxidoreductase and a photosynthetic reaction center, for example, an oxidoreductase mutant having improved affinity for the photosynthetic reaction center by substitution and / or insertion and / or deletion of a large number of amino acids is used. A method is mentioned. The oxidoreductase mutant can interact with the photosynthetic reaction center by electrostatic interaction, hydrophobic interaction, or van der Waals interaction without being linked to the photosynthetic reaction center by a covalent bond. The oxidoreductase mutant can be self-assembled with the photosynthesis reaction center inside or outside the cell by mixing with the photosynthesis reaction center isolated outside the cell or by expressing it in the photosynthesis organism. . As an example of amino acid sequence substitution, for example, a number of amino acid residues near the electron transfer site of hydrogenase are replaced with negatively charged aspartic acid or glutamic acid, and the positively charged photosystem I electron acceptor binding site is replaced. For example, the affinity can be improved.

As another method for linking the oxidoreductase and the photosynthetic reaction center, for example, as shown in FIG. 4, a fused photosynthetic reaction center in which a polypeptide (21) having affinity for the oxidoreductase (2) is fused ( 23) may be combined with oxidoreductase (2). The fused photosynthetic reaction center (23) is a fusion gene (20) in which a gene (10) encoding any subunit (12) of the photosynthetic reaction center and a gene (19) encoding a polypeptide (21) are fused. Is introduced into a photosynthetic organism by transformation using a plasmid or homologous recombination, whereby a fusion subunit (22) in which the subunit (12) is fused with the polypeptide (21) is expressed, and the rest of the photosynthetic reaction center. It can be obtained by self-assembly with the composite structure (14). When the photosynthetic organism into which the fusion gene (20) has been introduced is deficient in the gene (10) encoding the subunit (12), all the photosynthetic reaction centers in the cell become fusion photosynthesis reaction centers (23). When the gene (10) is not deleted, a part of the photosynthetic reaction center becomes the fusion photosynthetic reaction center (23). The fused photosynthesis reaction center (23) and the oxidoreductase (2) can be obtained by mixing the fusion photosynthesis reaction center (23) and the oxidoreductase (2) isolated outside the cell, Self-accumulation is achieved by co-expressing oxidoreductase (2) in the cell expressing the center (23). The method for fusing genes is the same as the method described in [0013] above. Examples of the polypeptide (21) include polypeptides screened from antibodies and polypeptide libraries. In addition, the definition of oxidoreductase in the present specification includes the inductive form in which a small molecule or polypeptide is bound to oxidoreductase, but it has an affinity for antibodies bound to oxidoreductase. Polypeptides such as an antibody having a can also be mentioned as the polypeptide (21). Furthermore, when an oxidoreductase modified by biotinylation or sugar chain modification by introducing a recognition sequence of a modification enzyme such as biotinylation enzyme or glycosyltransferase is used as the oxidoreductase, polypeptide (21) Examples thereof include avidin, which is a biotin-binding protein, and lectin, which is a sugar chain-binding protein.

As another method for linking the oxidoreductase and the photosynthetic reaction center, for example, by artificially introducing a number of amino acid substitutions and / or insertions and / or deletions, the affinity for the target oxidoreductase is improved. And a method using the photosynthetic reaction center mutant. The photosynthetic reaction center mutant can interact with the oxidoreductase by electrostatic interaction, hydrophobic interaction, or van der Waals interaction without being linked to the photosynthetic reaction center by a covalent bond. The photosynthetic reaction center mutant is a conjugate with the oxidoreductase inside or outside the cell by mixing with the oxidoreductase isolated outside the cell or by co-expressing with the oxidoreductase in a photosynthetic organism. Are self-assembled. As an example of substitution of the amino acid sequence in the photosynthetic reaction center, for example, a number of amino acid residues in the electron acceptor binding site of photosystem I are converted to lysine or arginine charged to positive charge, and electron transfer of negatively charged hydrogenase Examples thereof include improving the affinity for the site.

As another method for linking the oxidoreductase and the photosynthetic reaction center, for example, as shown in FIG. 5, a polypeptide (24) having affinity for both the oxidoreductase (2) and the photosynthetic reaction center (1). Can be exemplified by a method of binding the oxidoreductase to the photosynthetic reaction center. The polypeptide (24) is mixed with both the oxidoreductase (2) and the photosynthetic reaction center (1) isolated outside the cell, or the oxidoreductase (24) in the photosynthetic organism having the photosynthetic reaction center (1). By co-expression with 2), a linked body of the oxidoreductase (2) and the photosynthetic reaction center (1) can be constructed inside and outside the cell. Polypeptide (24) has an affinity for, for example, an antibody capable of binding to oxidoreductase at one of the two binding sites and the photosynthetic reaction center at the other binding site, or oxidoreductase Examples thereof include a fusion protein obtained by fusing a polypeptide and a polypeptide having affinity for the photosynthetic reaction center. In addition, when the oxidoreductase and the photosynthetic reaction center are biotinylated by incorporating a biotinylated enzyme recognition sequence, avidin capable of binding a plurality of biotins is included in the polypeptide (24).

As another method for linking an oxidoreductase and a photosynthetic reaction center, for example, by fusing a recognition sequence of a protein cross-linking enzyme to both the oxidoreductase and the photosynthetic reaction center, the oxidoreductase and the photosynthetic reaction center can be combined. An example is a method that utilizes recognition by the protein cross-linking enzyme and specific cross-linking at the recognition sequence portion. The cross-linked product of the oxidoreductase and the photosynthetic reaction center is obtained by mixing the oxidoreductase isolated outside the cell and the photosynthesis reaction center and the protein cross-linking enzyme, By co-expressing the photosynthetic reaction center and the protein cross-linking enzyme, they can self-assemble inside and outside the cell. An example of a protein cross-linking enzyme is transglutaminase, and an example of a recognition sequence thereof is ribonuclease A-derived S-peptide.

Outside the cell, an oxidoreductase substrate and an electron donor or electron acceptor were added to a conjugate of the oxidoreductase and the photosynthetic reaction center constructed by the method described in [0013] to [0019]. By performing light irradiation under conditions, a reaction product of oxidoreductase can be produced with high photoconversion efficiency. In addition, a photosynthetic organism that has been genetically engineered so that a conjugate of an oxidoreductase and a photosynthetic reaction center is self-assembled in a cell by the method described in the above [0013] to [0019] It becomes possible to produce a reaction product of oxidoreductase with high photoconversion efficiency only by culturing.

The invention according to claim 2 described in “Claims” is the presence of an electron donor in a conjugate of a hydrogenase and a photosynthetic reaction center prepared inside and outside a cell by the method described in [0013] to [0019]. By irradiating with light, direct and efficient electron transfer from the photosynthetic reaction center to hydrogenase occurs, and therefore, a method for producing photohydrogen with high photoconversion efficiency is provided. As a photosynthetic reaction center linked to hydrogenase, it exists in cyanobacteria and algae that generate a reduction potential sufficient to supply electrons to hydrogenase, in photosystem I existing in plants, and in green photosynthetic bacteria such as green sulfur bacteria and filamentous fluffy green bacteria. A reaction center is preferred. In addition, a conjugate of a hydrogenase and a photosynthetic reaction center is constructed in a photosynthetic organism, and photolysis of water is possible when the photosystem II, which is an oxygen-generating photosynthesis reaction center, is used as an electron donor source (FIG. 6). reference). In the photosystem II, a potential sufficient to oxidize water molecules is generated by photoinduced electron transfer caused by sunlight irradiation, and electrons can be supplied from the water molecules. Therefore, when the photosystem II is combined with the hydrogenase-photosystem I conjugate, the water molecule is oxidized and oxygen and protons are generated by the photoinduced charge separation reaction in the first stage of the photosystem II. The extracted electrons are transferred from photosystem II to photosystem I by an electron carrier such as plastoquinone, and then transferred to hydrogenase by a second-stage photoinduced charge separation reaction in photosystem I (see FIG. 6). ). The hydrogenase that receives the electrons reduces the protons generated by the decomposition of water molecules to produce hydrogen. Therefore, hydrogen and oxygen can be generated by photolysis of water molecules as a whole reaction.

In the present invention, by connecting an oxidoreductase to the photosynthetic reaction center, the reducing power and / or oxidizing power generated by light irradiation to the photosynthetic reaction center is preferentially and effectively transmitted to the target oxidoreductase. enable. Furthermore, a conjugate of an oxidoreductase and a photosynthetic reaction center can be constructed in a cell by genetic manipulation of a photosynthetic organism. By cultivating photosynthetic organisms having a conjugate of oxidoreductase and photosynthetic reaction center under sunlight, the efficiency of the desired redox reaction product can be reduced at low cost without using fossil fuels and without environmental pollution. Production can be performed. Until now, the only technology to artificially improve the substance-producing ability of natural photosynthetic organisms has been to introduce the gene of the target enzyme into the cell. However, the present invention modifies the electron transport pathway of photosynthesis. To make the method possible, a more universal and powerful technology was provided. Therefore, the present invention is a very useful method in the industry.

Hereinafter, a hydrogen production method using a conjugate of a hydrogenase and a photosynthetic reaction center will be described in detail as an example of the best mode of substance production using a conjugate of the oxidoreductase and the photosynthetic reaction center of the present invention. However, the technical scope of the present invention is not limited by the following description.

In recent years, hydrogen has attracted attention as a clean energy source that has a high energy content, does not generate greenhouse gases, and does not pollute the environment. However, even now, an efficient method for producing hydrogen that does not depend on fossil fuels has been established. Not. One of the energy production methods suitable for an environment-friendly society is a method for producing hydrogen using natural energy with an inexhaustible amount of resources such as sunlight.

As an ideal hydrogen production system using sunlight, a photo-hydrogen production method combining a photosynthesis reaction center and hydrogenase can be mentioned. Because the photosynthesis reaction center is the most efficient molecular device that can generate photocharge separation reaction most efficiently by sunlight, and convert light energy into chemical energy by chemical synthesis using the resulting reduction power and oxidation power. On the other hand, hydrogenase can catalyze the reduction power and the reaction from protons to hydrogen molecules at room temperature with a very high turnover number. However, the transfer of reducing power from the photosynthetic reaction center to hydrogenase is a reaction that does not occur directly in nature and is very inefficient. Therefore, by linking hydrogenase to photosystem I, which is one of the photosynthetic reaction centers, the collision frequency between photosystem I and hydrogenase can be dramatically improved, and the electrons between photosystem I and hydrogenase can be improved. It is thought that transmission can be made efficient. Therefore, by using such a hydrogenase and photosystem I conjugate, the solar energy absorbed in the photosystem I can be preferentially converted to hydrogen energy, and the photoconversion efficiency and hydrogen generation rate can be reduced. Can be improved.

The linkage between photosystem I and hydrogenase is designed so that the electron acceptor binding site of photosystem I and the electron transfer site of hydrogenase are closer to each other, based on the molecular design based on the crystal structure of photosystem I and hydrogenase. Is preferred.

Photosystem I has a clear crystal structure, is a membrane protein composed of more than ten subunits, and has several chromophores such as chlorophyll in subunits called PsaA and PsaB. It is known that photoinduced electron transfer occurs in order to generate oxidizing power and reducing power (see FIG. 7). The oxidizing power takes electrons from an electron donor such as plastocyanin, and the reducing power is transmitted from an electron acceptor binding site formed by subunits called PsaC, PsaD and PsaE to an electron acceptor such as ferredoxin. In particular, the N-terminus of PsaE is close to the electron acceptor binding site. On the other hand, hydrogenase has been extensively studied, especially since iron nickel-hydrogenase having both iron and nickel has a clear crystal structure. Iron-nickel-hydrogenase consists of two subunits, a large subunit and a small subunit, which accepts electrons from the electron transfer site located near the C-terminus of the small subunit, and then the iron of the small subunit. It is clear that electrons are sent to the iron nickel center of the sulfur cluster and large subunit, where protons are reduced and hydrogen molecules are released.

From the molecular structure of photosystem I and hydrogenase, the N-terminus of PsaE, which is a subunit of photosystem I, is linked to the C-terminus of the small subunit of hydrogenase, so that the electron acceptor binding site of photosystem I and the hydrogenase It is expected that the electron transfer sites will be linked closer and efficient electron transfer will occur between photosystem I and hydrogenase (see FIG. 7).

The C-terminal of the small subunit of hydrogenase and the N-terminal of PsaE, which is a subunit of photosystem I, are desirably linked at the gene level. That is, a mature form in which PsaE is fused to the C-terminal of the small subunit only by expressing in vivo the gene in which the gene sequence encoding PsaE is fused to the 3 ′ end of the gene encoding the small subunit of hydrogenase Hydrogenase can be obtained. This PsaE fusion hydrogenase can bind to the photosystem I lacking PsaE while retaining the hydrogenase activity, and is further linked so that the electron acceptor binding site of the photosystem I and the electron transfer site of the hydrogenase are in close proximity. (See FIG. 7).

Therefore, the hydrogenase-photosystem I conjugate prepared by combining the PsaE fusion hydrogenase and PsaE-deficient photosystem I in the extracellular system can be obtained in the presence of an electron donor to the photosystem I such as ascorbic acid. By performing light irradiation, it is possible to perform light-driven hydrogen production with high light conversion efficiency (see FIG. 7). In addition, a gene sequence encoding PsaE is inserted into the 3 ′ end of a gene encoding a small subunit of iron nickel-hydrogenase in a microorganism having photosynthetic ability such as cyanobacteria by homologous recombination, and the original PsaE gene is further inserted. When disrupted by using a suicide vector linked to a marker gene, a hydrogenase-photosystem I conjugate is constructed in the cell simply by culturing the microorganism under light irradiation, and efficient hydrogen production is performed. Can do.

Hereinafter, the present invention will be described in more detail with reference to examples of photo-hydrogen generation systems in which the PsaE fusion hydrogenase and the photosystem I deficient in PsaE are combined outside the cell. However, the technical scope of the present invention is not limited by this embodiment.

[Method for Producing PsaE Fusion Hydrogenase] PsaE fusion hydrogenase was produced using hydrogen bacterium Ralstonia eutropha. Details of genetic manipulation of Ralstonia eutropha and purification of hydrogenase are described below. Ralstonia eutropha is an aerobic microorganism that uses hydrogen as an energy source, and has been studied very vigorously, and thus has accumulated a lot of knowledge about its hydrogen metabolism and genetic manipulation. There are three types of Ralstonia eutropha: membrane-bound hydrogenase, soluble hydrogenase and sensor hydrogenase. Membrane-bound hydrogenase plays a role of receiving electrons from hydrogen molecules and transferring them to transmembrane cytochrome b. The soluble hydrogenase is a hydrogenase that catalyzes the reduction reaction of NAD ⁺ by hydrogen molecules, and the sensor hydrogenase is a hydrogenase having a function of activating hydrogen metabolism by binding hydrogen molecules. It is considered that a membrane-bound hydrogenase that exchanges electrons between proteins is suitable for the purpose of creating a conjugate with photosystem I, and the Ralstonia eutropha mutant HF387 (Prof. An attempt was made to create a PsaE fusion hydrogenase using the membrane-bound hydrogenase gene of Friedrich (assigned from Techunisch University Site Germany).

Membrane-bound hydrogenase is highly homologous to iron nickel-hydrogenase derived from Desulfobrio vulgaris whose crystal structure has been clarified so far, but the C-terminal of its small subunit gene. Has a binding site with a transmembrane cytochrome b consisting of about fifty amino acids which is not present in iron nickel-hydrogenase derived from Desulfovibrio vulgaris (see FIG. 8). Therefore, the PsaE fusion hydrogenase (see FIG. 8) in which the cytochrome b binding site of the small subunit of the membrane-bound hydrogenase is removed, and a linker of three residues (Ser-Gly-Gly) and PsaE are linked is replaced with photohydrogen. It was thought to form the hydrogenase-photosystem I conjugate most suitable for development.

Removal of the cytochrome b binding site of the membrane-bound hydrogenase small subunit gene present on the intracellular giant plasmid pHG1 and ligation of the linker and the PsaE gene were performed by homologous recombination using a suicide vector.

The gene coding for the PsaE fusion small subunit is obtained by first synthesizing the PsaE gene of Synechococcus elongatus (produced from synthetic DNA) into the gene fragment of the natural small subunit subcloned into the general-purpose vector for Escherichia coli pUc18. It was prepared by ligation using restriction enzyme and DNA ligase. Thereafter, a fragment of the gene encoding the small subunit fused with PsaE was recombined into the suicide vector pLO3 (transferred from Friedrich (Technische University), Germany) and introduced into the Ralstonia eutropha mutant HF387. Suicide vector pLO3 has two marker genes, a telocycline resistance gene and a sucrose-dependent lethal gene, and a clone in which the entire region of the suicide vector has been integrated on pHG1 is selected from telocycline resistance. By the second homologous recombination, it was possible to select a clone lacking the vector portion by leaving only the PsaE fusion small subunit gene on pHG1 from sucrose-dependent lethality. It was confirmed by DNA sequencing that the gene encoding the PsaE fusion small subunit was replaced with the gene encoding the small subunit of native membrane-bound hydrogenase.

A Ralstonia eutropha recombinant having a gene encoding a PsaE fusion small subunit is inoculated into a 10-liter jar fermenter containing 8 liters of FGN medium having the following composition, and the resulting mixture is used at 30 ° C. For about 3 days under aeration conditions of about 2 liters / min. Thereafter, the culture solution was centrifuged (6000 × g, 30 minutes) to recover the cells, and 500 ml of Tris buffer (50 mM Tris-HCl (pH 8.3), 5% sucrose, 0.2 mM manganese chloride, 1 mM 4- (2-aminoethyl) -benzenesulfonyl fluoride, 3 μg / ml phosphoramidon, 3 μg / ml pepstatin A, 100 μM bestatin hydrochloride, 10 μM E64, 3 μg / ml leupeptin). The cell suspension was lysed on ice with an ultrasonic cell disrupter (1 min × 40 times), and the cell extract was centrifuged (200000 × g, 60 minutes) to remove insoluble components. The cell extract is sufficiently added to an anion exchange column (φ50 mm × 110 mm, Q-sepharose, manufactured by Amersham Biosciences) equilibrated with Tris buffer (50 mM Tris-HCl (pH 8.2), 5% sucrose). Washed. After that, the pH 7.3 phosphate buffer (50 mM Na-Phosphate, 1M NaCl, 5% sucrose) was mixed as the eluent to the Tris buffer (50 mM Tris-HCl, 5% sucrose) pH 8.2. The hydrogenase adsorbed on the anion exchange column was eluted by linearly increasing the ratio from 0% to 50%. The hydrogenase activity of each fraction of the eluate was measured in a test tube containing 4 ml of Tris buffer (50 mM Tris-HCl (pH 7.4), 3 mM methylviologen, 4 mg / ml dithionite) for activity measurement, which was degassed under reduced pressure and purged with argon. Then, 50 μl sampled was added, and the amount of hydrogen generated after 30 minutes at 30 ° C. was estimated by measuring with a gas chromatograph. A fraction containing hydrogenase was collected, concentrated by ultrafiltration, and then a gel filtration column (φ16 mm × 600 mm, Superdex 200, Amersham Bio, equilibrated with Tris buffer (50 mM Tris-HCl (pH 7.4), 5% sucrose). Added to Science). Hydrogenase was eluted by flowing Tris buffer (50 mM Tris-HCl (pH 7.4), 5% sucrose) at a flow rate of 0.8 ml / min. The fraction containing hydrogenase was identified in the same manner as the ion exchange column. Fractions with high hydrogenase activity were collected and concentrated, glycerose was added to a final concentration of 10%, dispensed in 1 ml portions, and stored at -80%.

[Composition of Ralstonia eutropha FGN medium (per liter)] ・ 9 g of disodium monohydrogen phosphate dodecahydrate, 1.5 g of dipotassium dihydrogen phosphate, 2 g of ammonium chloride, 2 g of ammonium chloride, seven magnesium sulfate Hydrate aqueous solution (20% (w / v)) 1 ml, calcium chloride dihydrate aqueous solution (1% (w / v)) 1 ml, iron chloride hexahydrate (III) (0.5% (w / v)) v)) 1 ml, nickel chloride hexahydrate (II) (1 mM) 1 ml, fructose aqueous solution (20% (w / v)) 10 ml, glycerol aqueous solution (20% (w / v)) 10 ml.

[Isolation of PsaE-deficient photosystem I from Synechocystis sp. PCC6803 PsaE gene-deficient strain] To about 700 ml flask, 500 ml of BG11 medium having the following composition and 0.5 ml of 20 mg / ml kanamycin solution were added, and Synecocystis sp. (Synechocystis sp.) PCC6803 PsaE gene deficient strain (assigned from prof. Nelson (Tel Aviv University Israel)) was inoculated, and the resulting mixture was irradiated with light at 30 ° C. and 30 μE / m ⁻² / s ^−1. And maintained for 4-5 days with aeration. Thereafter, the cells were collected by centrifuging the culture (6000 × g, 30 minutes), and 3 ml of Tris buffer (50 mM Tris-HCl (pH 8.0), 10 mM sodium chloride, 10 mM magnesium chloride, 10 mM calcium chloride, 0.4 M sucrose, 1 mM 4- (2-aminoethyl) -benzenesulfonyl fluoride, 5 mM benzamide). The cell suspension was disrupted by a French press (20000 psi, 2 times), and the thylakoid membrane component was recovered by centrifuging the cell extract (18000 × g, 30 minutes). Further, thylakoid membrane components were washed by repeating suspension and centrifugation three times. This washed thylakoid membrane component was dissolved in 3 ml of a solubilizing buffer (25 mM Tris-HCl (pH 8.0), 5 mM sodium chloride, 5 mM magnesium chloride, 5 mM calcium chloride, 0.2 M sucrose, 1.6% dodecyl-β). After suspension in (D-maltoside), the mixture was maintained at 4 ° C. for 1 hour and centrifuged (18000 × g, 30 minutes) to remove thylakoid membrane insoluble components. The thylakoid membrane solubilizing component is a 10% to 30% sucrose density gradient solution (10 mM MOPS-NaOH (pH 7.0), 0.01% dodecyl-β-D-maltoside, about a liquid height) prepared in a super centrifuge tube. The photosystem I and other proteins were separated by layering them 5 cm) and centrifuging (200000 × g, 4 hours) using an angle rotor. By collecting the green band containing photosystem I, almost uniform photosystem I could be obtained and stored at -30%.

[Composition of BG11 medium (per liter)] 5 ml of 1M HEPES-KOH (pH 8.0) 0.03 g of dipotassium hydrogen phosphate 0.05 g of sodium carbonate 10 ml of a trace element solution (shown below). [Trace element solution (per liter)] ・ Boric acid 0.286 g ・ Manganese chloride tetrahydrate 0.18 g ・ Zinc sulfate heptahydrate 0.022 g ・ Sodium molybdate dihydrate 0.039 g ・ Copper sulfate 0.0079 g of pentahydrate, 0.00494 g of cobalt nitrate hexahydrate, 149.58 g of anhydrous sodium nitrate, 7.49 g of magnesium sulfate heptahydrate, 3.6 g of calcium chloride dihydrate, 0.6 g of citric acid -1.12 ml of 2.5M ethylenediamine tetrasodium tetraacetate aqueous solution (pH 8.0).

[Photohydrogen generation experiment using complex of PsaE fusion hydrogenase and photosystem I] The conditions of the photohydrogen generation experiment conducted using the PsaE fusion hydrogenase and PsaE deficient photosystem I prepared as described above are shown below. 4 ml of a solution for measuring hydrogen generation (12.5 mM Tris-HCl (pH 7.4), 1.2 units PsaE fusion hydrogenase (1 unit: 1 μmol when kept at 30 ° C. for 1 hour in the presence of 28 mM dithionite and 3 mM methylviologen) Hydrogenase amount that generates hydrogen), PsaE-deficient photosystem I corresponding to 150 μg in chlorophyll content, 5 mM magnesium chloride, 3 mM 2,2,6,6-tetramethyl-4-piperidone, 120 mM sodium ascorbate, 25 mM dithiothreitol , 90 μM dichlorophenyldimethylurea, 300 μg / ml glucose oxidase, 50 mM glucose, 100 μg / ml catalase, 1% ethanol, 0.005% dodecyl-β-D-maltoside) Was added to and left on ice for 30 minutes, after repeated five times under reduced pressure using argon flow and vacuum pump, it was irradiated with light of about 30000lux held at 30 ° C.. The gas phase was sampled with a gas tight syringe every 20 to 30 minutes, and hydrogen was separated with a gas chromatograph (Shimadzu GC-14B). The amount of hydrogen generation was estimated by comparing the peak area derived from hydrogen in the gas chromatograph with a correction curve prepared in advance. A hydrogenase deficient in the PsaE portion of the PsaE fusion hydrogenase (a mutant lacking the cytochrome b binding site of the small subunit of Ralstonia eutropha, see FIG. 8) and from Desulfovibrio vulgaris Hydrogenase, a natural photosystem I, was prepared and the reference experiment was performed in the same manner.

As shown in Table 1, the highest hydrogen generation was confirmed in the case of the combination of PsaE fusion hydrogenase and PsaE-deficient photosystem I, and cytochrome b binding site-deficient hydrogenase derived from Ralstonia eutropha not fused with PsaE. The hydrogen generation rate was about 5 to several tens of times that of the system using Desulfobrio vulgaris-derived hydrogenase, natural photosystem I. In addition, when PsaE fusion hydrogenase and PsaE-deficient photosystem I were combined, the rate of hydrogen generation decreased when free PsaE protein was added. These results indicate that the PsaE fusion hydrogenase was incorporated into the photosystem I via PsaE as shown in FIG. 7, and the electron transfer between the hydrogenase and the photosystem I was made efficient.

Table 2 shows a comparison between the photosynthesis reaction center reported so far and an extracellular hydrogen generation system using hydrogenase. The hydrogen generation rate is normalized by the amount of hydrogenase activity and the amount of chlorophyll added. The best comparison with our experimental system is the observation of hydrogen generation by direct electron transfer between free hydrogenase and photosystem I performed by McTavish (J. Biochem. 123, 644-649 (1998)). Obtained from the experimental system. McTavish uses hydrogenase from Clostridium pasteurianum and spinach-derived photosystem I to observe hydrogen generation in the absence of electron mediators such as ferredoxin. The generation rate is about four hundred times the value of McTavish. This also supports the dramatic improvement in electron transfer due to the connection between hydrogenase and photosystem I. Further, as a hydrogen generation system when ferredoxin is used as an electron mediator, Benemann (Proc. Nat. Acad. Sci. USA, 70, 2317-2320 (1973)), Rao (Biochimie, 60, 291-296 (1978) ), Fitzgerald (Biochem. J., 192, 665-672 (1980)) and the like. In these experiments, hydrogen evolution was observed in the presence of free hydrogenase and ferredoxin using chloroplasts with photosystems I and II extracted from plants as photosynthetic reaction centers. Their hydrogen generation rates are almost the same as those we observed this time, and the electron transfer between the linked hydrogenase and photosystem I is equivalent to the electron transfer between ferredoxin, the original electron transfer partner, and photosystem I. It was suggested that it is efficient.

From the above results and discussion, it can be concluded that by fusing PsaE, hydrogenase can link photosystem I, and efficient and direct electron transfer between hydrogenase and photosystem I has been realized. Such a conjugate of drogenase and photosystem I can be constructed in vivo, and a strong electron transfer pathway can be created between hydrogenase and photosystem I.

The figure which shows the light-driven substance production by the coupling body of the photosynthetic reaction center and oxidoreductase of this invention The figure which shows the oxidoreductase fused with the subunit of the photosynthetic reaction center and its gene structure, and the mode of linkage between the oxidoreductase and the photosynthetic reaction center The figure which shows the oxidoreductase which fused the polypeptide which has affinity with respect to a photosynthetic reaction center, its gene structure, and the connection mode of this oxidoreductase and a photosynthetic reaction center A photosynthetic reaction center subunit fused with a polypeptide having affinity for oxidoreductase and its gene structure, a photosynthetic reaction center incorporating the subunit, and a coupling mode of the photosynthesis reaction center and the oxidoreductase Illustration Diagram showing the coupling mode between the photosynthetic reaction center and oxidoreductase using a polypeptide having affinity for both the photosynthetic reaction center and oxidoreductase The figure which shows the photohydrolysis which combined photosystem II with the coupling body of photosystem I and hydrogenase The figure which shows PsaE deficiency photosystem I and PsaE fusion hydrogenase, and those connection modes Diagram showing membrane binding site deficiency and PsaE fusion of hydrogenase from Ralstonia eutropha

Explanation of symbols

1, photosynthesis reaction center 2, oxidoreductase 3, electron donor 4, electron acceptor 5, electron flow (photo-induced charge separation reaction)
6. Light irradiation 7, substrate 8, reactant 9, gene 10 encoding oxidoreductase, gene 11 encoding subunit of photosynthesis reaction center, gene encoding oxidoreductase and subunit of photosynthesis reaction center Gene fusion gene 12, subunit protein 13 of the photosynthetic reaction center, fusion oxidoreductase 14 fused with the subunit protein of the photosynthetic reaction center, photosynthetic reaction center 15 lacking one of the subunits of the photosynthetic reaction center, photosynthetic reaction A gene 16 encoding a polypeptide having affinity for the center, a fusion gene 17 of a gene encoding a redox enzyme and a gene encoding a polypeptide having affinity for the photosynthetic reaction center, and the photosynthetic reaction center Affinity to polypeptide 18, photosynthesis reaction center A fusion oxidoreductase 19 fused with a peptide, a gene 20 encoding a polypeptide having affinity for the oxidoreductase, a gene encoding a subunit of the photosynthetic reaction center and a polypeptide having affinity for the oxidoreductase A fusion gene 21 of a gene encoding a peptide, a polypeptide 22 having an affinity for an oxidoreductase, a fusion subunit 23 in which a polypeptide having an affinity for an oxidoreductase is fused with a subunit of a photosynthetic reaction center , Fusion photosynthesis reaction center 24 fused with polypeptide having affinity for oxidoreductase, polypeptide having affinity for both photosynthesis reaction center and oxidoreductase

Claims (2)

A method for producing hydrogen, comprising irradiating a conjugate of photosystem I, which is a photosynthetic reaction center protein, and nickel / iron hydrogenase with light. 2. The method for producing hydrogen according to claim 1, wherein the hydrogenase is a nickel-iron hydrogenase derived from Ralstonia eutropha.

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