Energy adapter module for a cell

Abstract

The present invention relates to a protein complex that uses electric current to provide biological useful energy to a cell used in biotechnology. The present invention also relates to a method for producing a protein complex as microbial electricity-driven proton pump (MEPP) for a cell and genetically modified host cells used for the production of the energy-delivering protein complex MEPP.

Description

[0001] ‘Energy adapter module for a cell

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a protein complex that uses electric current to provide biological useful energy to a cell used in biotechnology. The present invention also relates to a method for producing a protein complex as microbial electricity-driven proton pump (MEPP) for a cell and genetically modified host cells used for the production of the energy-delivering protein complex MEPP.

[0004] BACKGROUND OF THE INVENTION

[0005] Biotechnology is expected to play an important role in the European Green Deal for establishing a carbon-free economy. Microorganisms could contribute by using regenerative energy to synthesize fuels, fertilizers, chemical building blocks and specialty chemicals and to fix carbon dioxide. Microorganisms offer opportunities to more efficiently use and conserve natural resources while producing many desired organic compounds. These areas can be addressed by using renewable electricity as energy source and utilizing non-limiting sources such as carbon dioxide (CO2), dinitrogen (N2), and water (H2O) for biosyntheses.

[0006] To produce value products, organisms need chemical energy in the form of adenosine triphosphate (ATP). In order to generate ATP, organisms generally can use one of two processes, for example fermentation of energy-rich compounds such as glucose that leads to substrate chain phosphorylation of ADP to ATP or respiration, in which organisms exploit differences in the redox potential of different compounds in their vicinity. Respiration is also described as “electron transfer phosphorylation” to differentiate it from fermentation. In respiration the voltage between two redox active compounds is used to generate an electron flux which is exploited to translocate protons across the membrane, which in turn can be used to regenerate ATP. Humans respire using the redox difference between electron-rich “food” and electron-poor oxygen.

[0007] In a respiratory process, an electron-rich substrate (“electron donor” or “food”) is oxidized (electrons are extracted) and these electrons are channelled through a chain of conducing molecules (“cofactors”) and proteins to a second electron-poor substrate (“electron acceptor”). The electron flux between electron donor and electron acceptor molecules gives rise to an electric current. Specialized proteins through which the electric current flows and their precise orientation couple the electron current to the outward flux of protons through the cell membrane, culminating a proton gradient across the membrane. This electrochemical proton gradient generates a proton motive force (pmf) that is exploited by a universal protein complex “ATPase” to produce ATP from ADP and inorganic phosphate leveraging both the pmf and the influx of protons into the cell.

[0008] In the field of biotechnology, there is a need for a system capable of minimizing energy costs during the formation of the proton gradient, simultaneously enhancing ATP production, while avoiding cellular pathways that have negative feedback loops. Electric energy is an optimal energy source for such a system, however, the challenge of efficiently channelling energy from external grids into bacterial systems remains unresolved. A link between energized electrodes and bacterial organism is required.

[0009] Hence, the object of this invention is to provide a system that enables bacteria to cover their energy demands solely by electricity sources, preferably renewable electricity sources, which would allow them to produce value compounds from inorganic resources such as CO2, N2 and water. Consequently, it is also an objective of the present invention to introduce an “energy adapter module” for bacteria, which is able to translate electric current from the outside into an intracellular source of energy.

[0010] SUMMARY OF THE INVENTION

[0011] The invention is defined by the appended claims.

[0012] The protein complex according to the present invention comprises different functional modules, which are composed of one or several protein subunits that together function as a microbial electricity-driven proton pump, referred to as “Microbial Electricity-driven Proton Pump” (MEPP). Some of the protein subunits have redox-active centres and the redox-centres involved in electron transfer are oriented towards the outside of the cytoplasm permitting to connect the MEPP to extracellular electron donors and extracellular electron acceptors.

[0013] The effect of the present invention is based on the core concept that a membrane-bound protein complex has the ability to emulate a charging adapter for batteries, by charging bacteria with chemical energy in form of ATP. From a mechanistic perspective, this protein complex works like an electricity-driven proton pump. According to the present invention, the MEPP is aligned in the membrane in a manner that allows electric current to flow through the outer protein subunits of the membrane-bound protein complex while pumping protons to the outside of the cell. The pumping of protons results in an electrochemical potential across the cell membrane, generating a proton motive force (pmf) that all bacteria can use as an energy source, e.g., to produce ATP (Fig. 1). Thus, the MEPP according to the invention is a stand-alone protein complex that provides energy to cells when voltage is applied. It can be cloned into a variety of biotechnologically-useful organisms for different applications, for instance for the carbon neutral production of nitrogen fertilizer. Thus, the MEPP according to the invention is able to convert externally supplied electric energy to biologically useable energy without interfering with cellular metabolism and other cellular functions.

[0014] The essential advantage of the MEPP according to the invention is its independence from other energy sources or the reliance on light. With this MEPP as energy delivering system efforts to biotechnologically synthesize value product could fully focus on biosynthetic pathways inside the cell, free from constraints of energy demands. This would translate into the possibility of unidirectional regulation of biosynthetic pathways, eliminating the need for catabolic processes. Consequently, no side products from energy metabolism would accumulate, resulting in pure products. The genes encoding proteins of the MEPP system could be transferred to production strains that can be cultivated in large batches.

[0015] Therefore, the invention relates to the provision of energy within the host cell by the provision of chemical energy in the form of a pmf and ATP.

[0016] In a first aspect, the invention pertains to a redox-active system comprising a protein complex as microbial electricity-driven proton pump (MEPP) for a microbial cell comprising

[0017] A) a central module (CM) comprising

[0018] (i) at least one electron-transfer subunit (FCP) containing at least two iron-sulfur [Fe- S] clusters;

[0019] (ii) at least one membrane-anchor subunit (MAP) linking the (FCP) subunit to the cytoplasmic membrane of the cell; and

[0020] B) at least one electron input module (E_IN) and at least one electron output module (E_OUT) comprising one or more redox-active reaction centres and

[0021] C) at least one extracellular electron donor (ED) and at least one extracellular electron acceptor (EA), wherein the metal clusters of the subunit (i) and the module B) are oriented towards the outside of the cytoplasmic membrane of the cell.

[0022] A further aspect provided herein is an electrochemical reactor comprising the redox-active system based on a protein complex (I) as microbial electricity-driven proton pump (MEPP) according to the first aspect and an anode and cathode. In its primary aspect, the invention pertains to a redox-active system tailored for host cells, comprising the following constituents: A protein complex referred to as MEPP, which is composed of at least three distinct protein modules: an “electron input module” (E_l N), an “electron output module” (E_OUT), and a central module (CM). The protein modules E_IN, E_OUT and CM encompass one or more protein subunits, all oriented towards the outside of the cytoplasmic cell membrane, and bind different redox-active cofactors, including but not limited to Fe-S and Ni-Fe- S clusters, molybdopterin and corrinoid cofactors. The redox-active system according to the invention integrates small redox-active molecules (electron mediators) functioning as either “electron donor” (ED) or “electron acceptor” (EA), facilitating the transfer of electrons between the E_IN and the cathode or between the E_OUT and the anode, respectively.

[0023] A second aspect provided herein is an electrochemical reactor comprising the redox-active system based on a protein complex as microbial electricity-driven proton pump (MEPP) according to the first aspect and an anode and cathode.

[0024] A third aspect provided herein is a method for producing a protein complex as microbial electricity- driven proton pump (MEPP) for a cell expressing a polynucleotide comprising a nucleic acid sequence which is at least 50%, 60%, 70% 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected of the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7 or is an amino acid sequence selected of the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

[0025] The chromosomal and extrachromosomal nucleic acid material of the modified host cells described herein comprise genes that encode proteins involved in the electron and proton transfer.

[0026] Further aspects provided herein is a vector comprising a nucleic acid that encodes a protein having about or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ I D NO. 5, SEQ I D NO. 6 and SEQ I D NO. 7, preferably SEQ I D NO. 6, or said nucleic acid encodes SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, preferably SEQ ID NO. 6 as well as a host cell comprising the vector.

[0027] In a further aspect, the invention provides the use of the MEPP for the production of fuels, fertilizer, chemical building blocks and specialty chemicals.

[0028] Other objects, features, advantages and aspects of the present invention within the spirit and scope of the disclosed invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, which indicate preferred embodiments of the application, are given by way of illustration only.

[0029] DRAWINGS

[0030] Features will become apparent to those of ordinary skill in the art by describing in detail exemplary embodiments with reference to the attached drawings in which:

[0031] Figure 1 illustrates the mechanism of the redox-active system based on a “Microbial Electricity- driven Proton Pump” (MEPP). Electron flow through extracellular subunits of the MEPP protein complex leads to a pumping of protons from the inside to the outside, which consequently generates an electrochemical potential across the cell membrane and a proton motive force (pmf) that all bacteria can use as an energy source, e.g., to produce ATP.

[0032] The redox-active system according to the invention is designed in a way that electrons obtained from electric current from regenerative sources is flowing through the outer parts of the protein complex while it pumps protons to the outside of the cell, very similar to an electric pump, generating a proton motive force (pmf) that all bacteria can use as an energy source, e.g., to produce ATP. The MEPPs are inspired by microbial respiratory protein complexes coupling external electron flow to proton pumping. Respiratory protein complexes are generally composed of several protein subunits some of which are integrated into and others are attached to the inner or outer surface of the cytoplasmic membrane. The catalytic centres of respiratory complexes are usually located in the peripheral subunits and often contain transition metal cofactors such as heme, corrinoids or molybdo-cofactors. Electrons are generally conducted through chains of ironsulphur clusters or heme. Membrane-integrated subunits function as membrane anchor, electron path through the membrane (cytochrome b) or proton path. Catalytic centres in the peripheral subunits can be oriented to the inside of the cell (complex I) or the outside, for example hydrogen uptake hydrogenase or formate dehydrogenase.

[0033] Figure 2 illustrates an embodiment of the redox-active system comprising the MEPP protein complex with all active sites oriented to the outside of the cell, comprising three functional modules including an “electron input module” (E_l N), an “electron output module” (E_OUT), and a central protein module (CM). The protein modules E_IN, E_OUT as well as CM can contain one or more protein subunits, and bind redox-active cofactors. The CM module is preferably composed of three subunits: A “Large Subunit” (CLS), a “Four-Cluster Protein” (FCP) that contains four cubane [Fe-S] clusters, and a “Membrane Anchor Protein” (MAP). In other cases, two or more of these three protein subunits can be merged. Various architectures of respiratory protein complexes have evolved. However, to fulfil the function of coupling an electron flow through the outer parts of the protein complex to proton pumping across the membrane, the MEPP is generally composed of a central module CM, transferring electrons between an “Electron INput module” E_IN and an “Electron OLITput module” E_OUT. The outwards orientation of the modules permits to connect the MEPP to electrodes by using mediator molecules, acting as “Electron Donor” ED or “Electron Acceptor” EA. The function of these mediator molecules is to shuttle electrons between the MEPP and the energized electrodes.

[0034] Figure 3 illustrates an embodiment of the redox-active system comprising the MEPP protein complex based on the organohalide respiratory complex of Dehalococcoides mccartyi. The process begins with the generation of hydrogen, the electron donor (ED), at the cathode. The cathode can be separated from the MEPP by a membrane permeable for protons and hydrogen (not shown in the figure). Hydrogen is then enzymatically oxidized by the large subunit of [Ni-Fe] hydrogenase HupL. Electrons (white dotted line) flow through a “wire” of Fe-S clusters (black / white dots) to reach the cobalt ion of the corrin ring ([Co]) within the electron output module RdhA. At the heart of the MEPP lies a central three-subunit central module (CM), which serves as a structural scaffold, electron conductor, and proton pump. In the preferred embodiment this CM is a “Complex Iron-Sulphur Molybdoenzyme" (CISM). The cumulative effect of this electron transport process establishes a proton motive force (pmf). This pmf not only stands as a potential energy source for ATP synthesis but also fuels endergonic reduction reactions, signifying the pivotal role of the MEPP protein complex in cellular energetics. Abbreviations: [Ni-Fe] - nickeliron centre of HupL; bis-MGD - bis molybdopterin guanine dinucleotide in OmeA; [Co] - corrinoid cofactor in RdhA; other abbreviations as in the text; the two rods represent electrodes.

[0035] Figure 4, part (A) shows the chemical structure and the use of viologens (order from left to right: methylviologen, ethylviologen and benzylviologen) used for monitoring of electron transfer processes. Figure 4, part (B) shows how methyl viologen (MV) is used as an artificial electron mediator and redox dye in biochemistry.

[0036] Figure 5 shows a schematic arrangement of a cyclic voltammetry experiment with three electrodes: the counter electrode (CE), the reference electrode (RE) and the working electrode (WE). Electroactive processes, such as oxidation and reduction of electroactive substances (EA) take place at the WE. Therefore, the potential (E) of the WE is ramped linearly versus time in cyclic phases. If oxidation or reduction of the substance at the WE occur, oxidation or reduction current is flowing at the WE, which is plotted against the applied potential.

[0037] Figure 6 relates to the electron and proton flux though the OHR complex. In part (A) of Figure 6, the deuteration degree (DD1) profile of the dehalogenation product formed during the reductive dehalogenation activity assay, using intact Dehalococcoides mccartyi strain CBDB1 cells is shown. The increasing DD1 observed at the start of the reaction shows that protons, which are incorporated into the dehalogenation product, originate from the inside of the cell. In part (B) of Figure 6, a modelled representation of the OmeA-HupX-OmeB-RdhA submodule is shown. Some highly conserved amino acid residues, which are mostly responsible for proton translocation through the OmeB subunit and their transfer to the active site of RdhA, are shown. Distances between these amino acid residues are given in Angstrom (A).

[0038] Figure 7 shows the reduction of the anode mediator cyanocobalamin by the OHR complex using reduced methyl viologen as electron donor. The y-axis shows the absorption of the remaining reduced methyl viologen. Solid line: Reaction of the OHR complex with cyanocobalamin as proposed electron acceptor. Dashed line: Reaction of the OHR complex with the natural substrate 3,5-dibromo-L-tyrosine, as a positive control. Dotted line: Low background reaction between methyl viologen and cobalamin in the absence of the OHR complex, as a negative control.

[0039] Figure 8 presents data on the cyclic voltammetry and redox characteristics of the cyanocobalamin redox reaction, using either an indium-tin oxide electrode or gold electrode as the working electrode. Midpoint redox potentials of cyanocobalamin versus silver / silver chloride (Ag / AgCI) or standard hydrogen electrode (SHE) reference electrodes are given.

[0040] DEFINITIONS

[0041] As used herein, the following expressions are generally intended to preferably have the meanings as set forth below, except to the extent that the context in which they are used indicates otherwise.

[0042] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. Further, the use of “may” when describing embodiments of the present disclosure refers to “one or more embodiments of the present disclosure." In the following description of embodiments of the present disclosure, the terms of a singular form may include plural forms unless the context clearly indicates otherwise.

[0043] As used herein, the term "substantially," "about," and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent deviations in measured or calculated values that would be recognized by those of ordinary skill in the art. Further, if the term “substantially” is used in combination with a feature that could be expressed using a numeric value, the term “substantially” denotes a range of + / - 5% of the value centred on the value.

[0044] It will be further understood that the terms “include,” “comprise,” “including,” or “comprising” specify a property, a region, a fixed number, a step, a process, an element, a component, and a combination thereof but do not exclude other properties, regions, fixed numbers, steps, processes, elements, components, and combinations thereof.

[0045] As used herein, the term "protein" refers to a single folded polypeptide chain that may or may not contain cofactors or ions in its three-dimensional fold. A protein can contain any of the naturally occurring amino acids as well as artificial amino acids and can be of biologic or synthetic origin. The polypeptide of a protein may be modified, naturally (post-translational modifications) or synthetically, by e.g. glycosylation, acetylation, amidation, carboxylation, hydroxylation and / or phosphorylation, or addition of protein tags. A polypeptide comprises at least two amino acids, but does not have to be of any specific length; this term does not include any size restrictions. Preferably, a protein comprises at least 50 amino acids.

[0046] The term "protein complex" typically refers to an assembly of several proteins / polypeptide chains that transiently interact with each other or persistently bind to each other by different interactions, e.g., non-covalent interactions. As used herein, the term also includes the situation that two or more polypeptides are fused to each other.

[0047] The term “protein subunit” refers to one polypeptide chain / protein that is part of a protein complex. Similarly to the term “protein”, a protein subunit can contain any of the naturally occurring amino acids as well as artificial amino acids and can be of biologic or synthetic origin. The polypeptide chain of a protein subunit may be modified, naturally (post-translational modifications) or synthetically, by, e.g., glycosylation, acetylation, amidation, carboxylation, hydroxylation and / or phosphorylation, or addition of protein tags.

[0048] The term “module” refers to sub-complexes of a protein complex that are generated when a protein complex disintegrates. A module comprises one or more folded proteins that bind to each other and form a functional unit. The terms “electron input module” (E_IN), “electron output module” (E_OUT), and a central protein module CM affiliated with either CISM type I or CISM type II protein complexes (for a detailed description of the composition of CISM protein complexes refer to the subsequent sections) are used herein in accordance with the invention.

[0049] The term "nucleic acid" includes single-stranded and double-stranded nucleic acids and ribonucleic acids as well as deoxyribonucleic acids. It may comprise naturally occurring as well as synthetic nucleotides and can be naturally or synthetically modified, for example by methylation, 5'- and / or 3'-capping.

[0050] The term "vector" is used here in its most general meaning and comprises any intermediary vehicle for a nucleic acid which enables said nucleic acid, for example, to be introduced into prokaryotic and / or eukaryotic cells and, where appropriate, to be integrated into a genome. Vectors of this kind are preferably replicated and / or expressed in the host cells. Vectors comprise plasmids, phagemids, cosmids, bacteriophages or viral genomes. The term "plasmid" as used herein generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.

[0051] According to the invention, the term "host cell" relates to any cell, which can be transformed or transfected with an exogenous nucleic acid. The term "host cells" comprises according to the invention prokaryotic cells, e.g., E. coli, Bacillus megaterium and eukaryotic cells, e.g., yeast. A nucleic acid may be present in the host cell in the form of a single copy or of two or more copies and, in one embodiment, is expressed in the host cell. The nucleic acid may be integrated into the genome of the host cell or present on a circular vector.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] The present invention is based on the development of a membrane-bound protein complex machinery referred to as “Microbial Electricity-driven Proton Pump”, MEPP. In one embodiment the MEPP protein complex may comprise two to seven subunits. The two essential subunits are the FCP and the MAP, responsible for the coupling of electron flow to proton translocation. In one embodiment some of the protein subunits may have redox-active centres which are involved in electron transfer. The redox-active centres of the MEPP may be oriented towards the outside of the cell permitting to connect the MEPP to extracellular electron mediators.

[0054] One promising prototype of such a MEPP was identified in strain CBDB1 of the species Dehalococcoides mccartyi respiring with halogenated contaminants. These bacteria have a membrane-bound protein complex (“organohalide respiration complex”, OHR-complex). Similar protein complexes for example may be found in Dehalogenimonas and Dehalobacter strains.

[0055] A) The central module, CM

[0056] According to the present invention the central module CM comprises (i) at least one electrontransfer subunit FCP containing at least two iron-sulfur [Fe-S] clusters and (ii) at least one membrane-anchor subunit MAP linking the FCP subunit to the cytoplasmic membrane of the cell.

[0057] In one embodiment the central module CM further comprises (iii) at least one subunit with a molybdopterin binding site CLS comprising at least one transition metal cofactor located outside of the cytoplasmic membrane of the cell.

[0058] In another embodiment the membrane-anchor subunit MAP links the central module CM to the cytoplasmic membrane of the cell. According to a preferred embodiment, the central module CM of the protein complex (I) MEPP may comprise three subunits (i), (ii) and (iii).

[0059] The CLS an FCP may be transported to the outside via cellular transporters, preferably by a Twin- Arginine Translocation (TAT) machinery. Therefore, the protein amino acid sequences may have either an N-terminal TAT signal peptide encoded, or may be transported together with another protein subunit containing a TAT signal peptide.

[0060] One example of a central module CM is the “Complex Iron-Sulfur Molybdoenzyme Module”, CISM of the MEPP in strain CBDB1. The CISM protein complexes typically consist of three subunits: i) a “CISM Large Subunit”, some of them contain a molybdopterin binding motif, ii) a Four-Cluster Protein that contains four cubane [Fe-S] clusters, and iii) a membrane anchor protein. The large subunit often exhibits a motif for binding a molybdopterin-type cofactor, although some of the members are described not to bind a molybdopterin cofactor. There are two types of membrane anchor proteins described. CISM type I protein complexes are here defined as CISM assemblies with a cytochrome b-type membrane anchor protein, containing two heme molecules within the transmembrane part of the membrane anchor protein (MAPI). In contrast, CISM type II protein complexes are here defined as CISM complexes with a heme-absent membrane anchor protein forming 8-10 transmembrane helices, thereby acting as membrane anchor and proton path through the membrane (MAP2). i) The electron-transfer subunit, FCP

[0061] In one embodiment the electron-transferring subunit FCP may comprise a tandem ferredoxin fold structure. Preferably the FCP may comprise two or more cubane-like [Fe-S] clusters that collectively form an electron-conducting wire that facilitates the transfer of electrons between the E_IN and E_OUT modules. Concurrent with the flow of electrons within the CM module is the export of protons from the cell.

[0062] In a preferred embodiment a nucleic acid encodes a protein as the FCP subunit with about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 6 or is SEQ ID NO. 6.

[0063] According to a preferred embodiment, the FCP comprises two or more cubane [Fe-S] clusters, and each [Fe-S] cluster may be positioned within a distance of 8 A to 14 A of one another, thereby allowing efficient electron flow between them. Preferably the FCP comprises four cubane ironsulfur [Fe-S] clusters (FCP1 to FCP 4) having a distance between each other of 8 A to 14 A. In the specific example of strain CBDB1 , the FCP is encoded by the hupXgene (locus cbdbA131). The HupX subunit within strain CBDB1 spans 267 amino acids, possesses a molecular weight of approximately 30 kDa, and is equipped to bind four cubane [Fe-S] clusters (Fig. 3). ii) The membrane-anchor protein, MAP

[0064] In one embodiment the CM is a type-ll CISM, in which the membrane-anchor subunit MAP may comprise at least eight transmembrane helices. In a further embodiment the MAP may comprise at least eight transmembrane helices and a quinone binding site. In both embodiments the MAP does not comprise a heme cofactor. The type-ll CISM as CM is preferred because it does not conduct electrons across the membrane but guide protons outside of the cell. This proton export is electrogenic and drives pmf generation.

[0065] A characteristic feature of the MAP is that it is involved in the coupling of the electron current through the peripheral outer MEPP subunits to proton export from the cytoplasm.

[0066] According to a preferred embodiment the MAP may adopt the form of a NrfD family protein, which comprise at least eight transmembrane helices and does not contain a heme cofactor.

[0067] In a preferred embodiment a nucleic acid encodes a protein as the membrane-anchor subunit MAP with about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 7 or is SEQ ID NO. 7.

[0068] In the specific example of strain CBDB1 , the MAP is constituted by the OmeB subunit, encoded by the cbdbA193 locus. The OmeB subunit within strain CBDB1 is an integral membrane protein with a molecular weight of approximately 45 kDa. It spans 403 amino acids. Evidence shows that the OmeB subunit translocates protons across the membrane during organohalide respiration (Fig. 3).

[0069] According to another embodiment, in which the CM is a type-l CISM, the MEPP system may comprise a different type of MAP protein as described above. In this embodiment the MAP may be a cytochrome b binding two heme molecules within the membrane and comprises a quinone binding site at the inside of the cytoplasm membrane. Such a heme-containing MAP obtains electrons from an FCP subunit and transports the electrons through the membrane via the bound heme molecules. In this case, a quinone must be present that is reduced and protonated at the inside of the cell membrane, then diffuses to the outside of the cell membrane where it is reoxidized by a second protein complex with the quinone binding site at the outside of the cell. By oxidizing the quinone, the protons are released to the outside of the membrane, again generating a pmf. Hi) The large subunit, CLS

[0070] According to the invention, the large CLS subunit comprises at least one transition metal cofactor and is located outside of the cytoplasmic membrane of the cell.

[0071] In one embodiment, the transition metal cofactor of the subunit CLS is selected of heme, [Ni-Fe]- centres, corrinoids or molybdopterin-cofactors.

[0072] The CLS is the large subunit of the CM which may be a structural scaffold or may bind a molybdopterin-type cofactor or may have catalytic activity.

[0073] In a preferred embodiment a nucleic acid encodes a protein as the large subunit CLS with about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 5 or is SEQ ID NO. 5.

[0074] In one embodiment, such as the formate dehydrogenase-N (FDH-N) membrane protein complex of Escherichia coli, the CLS exhibits catalytic activity. Then, it can also substitute the function of the E_IN module to oxidize an external electron donor.

[0075] In a preferred embodiment, the CLS comprises a b / s-molybdo-pyranopterin guanine dinucleotide (bis-MGD) and a cubane-like iron-sulphur [Fe-S] cluster.

[0076] In the model organism D. mccartyi strain CBDB1 , the CLS termed as OmeA subunit putatively binds a b / s-MGD and a cubane [Fe-S] cluster and is encoded by the locus cbdbA195 (Fig. 3). OmeA of strain CBDB1 consists of 993 amino acids and has a molecular weight of approximately 105 kDa. Nonetheless, alternate embodiments of the invention may feature the CLS incorporating neither an iron-sulphur [Fe-S] cluster nor a b / s-MGD cofactor.

[0077] B) The electron input module, E_IN and the electron output module, E_OUT

[0078] The E_IN module of the protein complex according to the invention is responsible for electron uptake through the oxidation of an electron donor molecule. The E_IN module may comprise two subunits E_INL and E_INS.

[0079] In a preferred embodiment a nucleic acid encodes a protein as the electron input module E_INL with about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 1 or is SEQ ID NO. 1.

[0080] In a preferred embodiment a nucleic acid encodes a protein as the electron input module E_INS with about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 2 or is SEQ ID NO. 2. In the specific example of strain CBDB1 , the E_IN module encompasses both the large and small subunits, HupL and HupS, respectively, of a [Ni-Fe] hydrogenase enzyme specialized in the oxidation of hydrogen (Fig. 3).

[0081] For example, in strain CBDB1 , the HupL and HupS subunits are encoded within the hup operon by the hupL and hupS genes (loci cbdbA129 and cbdbA130), respectively. The HupL subunit lacks a Twin-Arginine Translocation (TAT) leader sequence and is co-translocated with the HupS subunit, which features a TAT leader sequence. The HupL subunit within strain CBDB1 is comprised of 526 amino acids, having an approximate molecular weight of 58 kDa, and binds both a [Ni-Fe] cluster and a cubane [Fe-S] cluster. The HupS subunit has an approximate molecular weight of 37 kDa, spanning 354 amino acids, and is equipped to bind three cubane [Fe-S] clusters.

[0082] The E_IN module plays a pivotal role in the oxidation of hydrogen, a process initiated at the [Ni-Fe] cluster within this enzyme. Subsequently, these electrons are guided through several cubane [Fe-S] clusters distributed across both the HupL and HupS subunits. The electrons are subsequently transferred to the central electron- and proton-conducting CM module.

[0083] In an embodiment the E_IN modules may interact with diverse electron donors, for example hydrogen, formate or methanol.

[0084] According to the present invention, the E_OUT module comprises at least one subunit comprising a corrinoid as cofactor and adopts the critical function of reducing an electron acceptor molecule. The electrons originate from the action of the E_IN module through the oxidation of an electron donor molecule. The composition of the E_OUT module can vary, featuring either a singular subunit or several protein subunits.

[0085] In one embodiment, the E_OUT module may incorporate periplasmic proteins of the cytochrome c family, for example, MccA (for example described by Eller et al. 2019. “Significance of MccR, MccC, MccD, MccL and 8-methylmenaquinone in sulfite respiration of Wolinella”, doi 10.1016 / j.bbabio.2018.10.002) and NrfA or NrfB (for example described by Clarke et al. 2007. “The crystal structure of the pentahaem c-type cytochrome NrfB and characterization of its solution-state interaction with the pentahaem nitrite reductase NrfA”, Biochem. J., 406, 19-30, doi 10.1042 / BJ20070321) subunits. These periplasmic cytochrome c proteins belong to the phylogenetically diverse family of multiheme cytochromes that are involved, for instance, in the reduction of sulphite (MccA or SirA) or nitrite (NrfA or NrfB). In this embodiment, electrons from E_IN are transferred via FCP to the periplasmic cytochrome c, from where they are shuttled to the anode. In a preferred embodiment, the E_OUT module comprises two subunits E_OUTA and E_OUTB. The subunit E_OUTA may be a subunit that binds two cubane [Fe-S] clusters and a corrinoid cofactor in the active site, preferably a cobalamin cofactor. The subunit E_OUTB may be a membrane subunit as membrane anchor for the active subunit E_OUTA.

[0086] In a preferred embodiment a nucleic acid encodes a protein as the electron output module EJDIITA with about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 3 or is SEQ ID NO. 3.

[0087] In a preferred embodiment a nucleic acid encodes a protein as the electron output module EJDIITB with about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 4 or is SEQ ID NO. 4.

[0088] In the example of strain CBDB1 , the E_OUT module encompasses a reductive dehalogenase (Fig. 3). This enzyme uses various halogenated organic compounds as electron acceptors in vivo. Notably, within strain CBDB1 , there are 32 rhdA genes encoding putative reductive dehalogenases, and alongside them, 32 rdhB genes encoding their membrane anchoring proteins. These genes are organized in operons that are transcriptionally controlled by two- component or MarR-type regulator proteins. The specific choice of RdhA subunit for the E_OUT module depends on the particular electron acceptor molecule being employed. Within strain CBDB1 , the RdhA subunit has an approximate molecular weight of 54 kDa and houses a cobalamin cofactor within its active site, along with two cubane [Fe-S] clusters. The RdhA subunit features a TAT leader sequence, determining its transport across the cytoplasmic membrane via the TAT machinery. In contrast, the RdhB protein is a compact transmembrane protein, characterized by up to three transmembrane helices, comprising approximately 90-100 amino acids and possessing an approximate molecular weight of 10 kDa.

[0089] C) Electron acceptor (EA) and electron donor (ED)

[0090] The redox-active system according to the present invention comprises small redox-active molecules (electron mediators) functioning as either “electron donor” (ED) or “electron acceptor” (EA), facilitating the transfer of electrons between the E_IN and the cathode or between the E_OUT and the anode, respectively.

[0091] According to the present invention EA and ED may be small, water-soluble molecules that can reversibly take up electrons. They can be reduced (electrons are transferred to them) and oxidized (electrons are removed from them). Therefore, they can shuttle electrons between two entities, which can be for example two protein complexes or two electrodes or one electrode and one protein complex. A typical example of an electron mediator are protons. Two protons can be reduced to hydrogen when two electrons are provided according to the reaction scheme: 2 H++ 2 e- H2.

[0092] In one embodiment the electron mediators are redox-active proteins such as ferredoxin or cytochrome, dyes such as viologens, soluble molecules with quinoid structures such as anthraquinone-2,6-disulfonate (AQDS) or metal complexes such as cobalamin.

[0093] In the preferred embodiment of the above described system, protons or hydrogen is the cathode mediator ED that shuttles electrons between the cathode and a hydrogenase activity in the E_IN module. In this case, standard hydrogenase activity can be used as E_IN and hydrogen can easily be removed from the reaction mixture.

[0094] Examples of possible electron acceptors EA include redox dyes such as methylene blue, neutral red, nile red, phenol red and indigo blue; free cofactors such as p-benzoquinone, tetramethyl-p- benzoquinone, p-chloranil, anthraquinone, o-naphthoquinone, 2,5-dibromo-6-isopropyl-3-methyl- p-benzoquinone, methyl-p-benzoquinone, 2,3-dimethoxy-5-methyl-p-benzoquinone, 2,6- dimethoxy-p-benzoquinone and 2-methyl-p-naphthoquinone; proteins with cofactors such as flavins; reversibly reducible halogenated compounds such as Selectofluor II, 2-chloro-1- methylpyridinium iodide and 2,6-dichloro-1-fluor-pyridinium-tetrafluoroborate; metalloproteins such as ferredoxin; or metal complexes such as chloro(pyridine)cobaloxime(lll), methyl(pyridine)cobaloxime(lll), cyanocobalamin (B12), methyl cobalamin, 2,6-bis(N- pyrazolyl)pyridine nickel(ll) bromide and potassium oxalate iron(lll) chloride.

[0095] Preferred electron acceptors EA are selected from the group of potassium oxalate iron(lll) chloride, methyl(pyridine)-cobaloxime(lll), 2,6-bis(N-pyrazolyl)pyridine Ni(ll) bromide, cyanocobalamin, methyl cobalamin, phenol red and indigo blue. More preferred electron acceptors are selected from the group of potassium oxalate iron(lll) chloride, methyl(pyridine)- cobaloxime(lll), 2,6-bis(N-pyrazolyl)pyridine nickel(ll) bromide, cyanocobalamin and methyl cobalamin.

[0096] Suitable electron mediators are characterized by many physicochemical characteristics but an outstandingly important characteristic is the redox mid potential. This redox mid potential describes the mid of the two potentials against a reference electrode where this electron mediator is reduced and oxidized. The redox mid potential is gradually increasing from a more negative value at the cathode (stronger potential) to a less negative redox mid potential of the cathode mediator (CM) though the MEPP to the anode mediator (AM) to the anode. The redox mid potential is quantified by its difference to the potential at a hydrogen standard electrode and is given in millivolt (mV). The preferred redox mid potential of the cathode mediator is at -500 to -300 mV. The preferred redox mid potential of the anode mediator is more positive than +250 mV. Apart from the redox mid potential, electron mediators also have chemical properties due to their orbital configuration that allow interaction with specific protein active centres or electrodes. For example, the cobalt atom in corrinoids (E_OUTA) in its +1 state has a doubly occupied d8orbital that can specifically interact with the sigma hole of the antibonding orbital of halogenated compounds or mediators, for examples described by Cooper et al. 2015, “Anaerobic microbial transformation of halogenated aromatics and fate prediction using electron density modeling “ Environ Sci Technol 49:6018-6028. doi:10.1021 / acs.est.5b00303, allowing specific electron transfer.

[0097] In a specific embodiment of the present invention (“redox substrate mode”) the MEPP interacts with soluble electron mediators on both sides of the complex acting as electron donor ED and electron acceptor EA. This could be, for example, methanol, formate, hydrogen, carbon monoxide or sugars as ED and oxidized inorganic ions, for example, oxygen, nitrate, nitrite, Fe3+, Mn4+, sulfate, carbonate as ES. In this specific embodiment the electron mediators ED and EA may not be regenerated electrically but resupplied after consumption.

[0098] The electron mediators EA and ED react with the E_IN and E_OUT module of the MEPP but not with intermediary sites in the complex to avoid short circuits. In a preferred embodiment, one or both electron mediators EA and ED have a boiling point below 40 °C to be able to remove them easily from products without destroying the MEPP.

[0099] A further aspect of the present invention is a method for producing a protein complex as microbial electricity-driven proton pump (MEPP) for a cell comprising expressing a polynucleotide comprising a nucleic acid sequence which encodes a protein with amino acid sequence identity of is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected of the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7 or is an amino acid sequence selected of the group consisting of SEQ I D NO. 1 , SEQ I D NO. 2, SEQ I D NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

[0100] Electrochemical reactor and electrodes

[0101] Another aspect of the present invention is an electrochemical reactor comprising the redox-active system based on a protein complex as microbial electricity-driven proton pump MEPP according to the present invention and an anode and cathode.

[0102] In one embodiment (“electrode mode”) the microbial production cell with its MEPP is cultivated within an electrochemical device equipped with two electrodes and an appropriate voltage applied. The applied voltage may be between 200 and 2000 mV, depending on the electron mediators ED and EA and the type of MEPP used.

[0103] In a preferred embodiment, the electrode mode comprises two electrodes and two electron mediators ED and EA. The cathode mediator ED carries electrons from the cathode to the E_IN module of the MEPP, while the anode mediator EA carries electrons from the MEPP to the anode. For this, the cathode mediator ED must be reduced by a low-potential cathode and oxidized by the active site of the E_IN module. The anode mediator EA must be reduced by the active centre of the E_OUT module and re-oxidized on the high-potential anode. The potential difference between cathode and anode is applied by an external energy source, preferentially using regenerative energy generated by photovoltaic panels or wind turbines.

[0104] The role of the electron mediators in the electrode mode is to increase the activity of the MEPP without massively increasing electrode surface areas. The electron mediators are preferentially molecules that can be easily separated from synthesis products of the microbial production cell.

[0105] In a preferred embodiment the cathode is separated by a proton-exchange membrane.

[0106] In the preferred embodiment hydrogen is the cathode mediator that shuttles electrons between the cathode and a hydrogenase activity in the E_IN module. In this case, standard hydrogenase activity can be used as E_IN and hydrogen can easily be removed from the reaction mixture. In this preferred embodiment, a proton exchange membrane can be used to separate the cathode from the anode compartment, in which the microbial production cell with the MEPP is located. This proton exchange membrane, e.g., a Nation™ membrane, allows the diffusion of hydrogen from the cathode compartment into the anode compartment and the diffusion of protons from the anode compartment to the cathode compartment but prevents diffusion of microbial cells, proteins, the anode mediator, substrates and products into the cathode compartment. This setup therefore avoids any interactions of the cathode with the MEPP, the anode mediator or other components in the medium, subsequently preventing short circuits.

[0107] Suitable materials for the working electrodes (anode) include but are not restricted to platinum, titanium indium tin oxide and gold as well as a variety of carbon phases. Preferably the working electrode is composed of gold or indium tin oxide.

[0108] The counter electrode (cathode) may be an inert metal such as platinum, titanium or carbon species with a large surface. Preferably the counter electrode is composed of platinum or titanium. As reference electrode a silver / silver chloride electrode (Ag / AgCI) or platinum electrode may be used. Vectors

[0109] Vectors serve as vehicles encoding genes for the individual subunits of a MEPP. These vectors can be used to introduce this DNA into a host cell, a process referred to as transformation. Within the host cell, the genomic material encoded on the vector is either integrated into the host’s genome or the genes encoding for the MEPP subunits are directly expressed from the vector. Integration of the DNA into the genome can occur through either a CRISPR / Cas system or homologous recombination. When genes are expressed and translated from the vector, these vectors must meet specific requirements: (i) They must carry a so-called origin of replication (or / ) to autonomously replicate within the host organism. The o is specific and is dependent on the recipient strain for the plasmid; (ii) The vectors must be selectable and, for example, carry an antibiotic resistance gene; (iii) For transcription initiation and termination, the vectors must have promoter and terminator sequences (promoters can be either inducible or constitutive); (iv) The vectors must possess a multiple cloning site with multiple restriction sites for linearization, allowing the insertion of genes encoding the MEPP subunits into the vectors.

[0110] MEPP is intended to be heterologously produced in a production strain in its functional form. To achieve this, genes encoding the individual MEPP subunits must be inserted / cloned into suitable vectors. These vectors are then transformed into host cells and the encoded proteins are either heterologously expressed directly from the vector or alternatively the vector is integrated into the host’s genome for expression.

[0111] For heterologous production from a vector, vectors with inducible or a constitutive promoter can be used. For heterologous production of MEPP subunits in Bacillus subtilis, for example, the pHT1469 vector with an inducible Pgrac promoter or the pHT2133 vector with a mutated and constitutive Pgrac promoter can be employed. For heterologous production in B. megaterium, inducible vectors such as pMGBm19 and pSTOP1622 or pSTOP1623 with an inducible PxylA promoter can be used, as well as the pPconst1326 vector with a constitutive PpdhABCD promoter. For heterologous production in Escherichia coli, pACYC(Duet) or pET(Duet) vectors with a strong and inducible 77 promoter can be employed, or vectors like pSF-OXB11 with a very weak mutated PrecA promoter can be used, enabling constitutive production of the MEPP subunits.

[0112] In a preferred embodiment a vector comprises a nucleic acid that encodes a protein having about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 6, or is said nucleic acid and a nucleic acid that encodes a protein having about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 7, or is said nucleic acid. Host cells

[0113] Host cells are prokaryotic or eukaryotic cells that can be genetically transformed. They have one or two membranes, but in the case of two membranes, a permeation of the outer membrane by the electron mediators must be warranted. This can be, for example, by passive diffusion through the membranes (e.g., for hydrogen) or diffusion facilitators (pores) or by active transport (e.g., cobalamin-transporter).

[0114] The host cell must be amenable to genetic transformation to be able to incorporate genes encoding the MEPP and biosynthetic pathways of choice. Preferably the host cells are well- described production organisms such as E. coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, Clostridium acetobutylicum or Saccharomyces cerevisiae. The host cells will then transcribe and translate the incorporated genes into proteins which assemble in the host cell to a functional MEPP. For that, the host cells need the machinery to maturate the cloned proteins, including the TAT-system for protein export, a SUF or ICS system for [Fe-S] cluster biosynthesis, hydrogenase maturation genes for the assembly of the [Ni-Fe] cluster of the hydrogenase, cofactor biosynthesis pathways (for corrinoids and b / s-MGD) or transporter and further chaperones such as GroEL.

[0115] The host cell comprises the genetic material requisite for the E_IN, CM and E_OUT modules. The genetic material encoding for the subunits of the E_IN, CM and E_OUT modules can be either integrated into the host cell’s genome or located extra-chromosomally. The procedure to introduce MEPP's genetic material into host cells involves genetic manipulation techniques. Microorganisms hosting either one (monoderm, gram-negative) or two cell membranes (diderm, gram-positive) can be used as suitable host cells. Transformation of host cells with the genetic material encoding the E_IN, CM, and E_OUT modules can be achieved through diverse methodologies. These methods encompass electric or thermic shock, particularly suited for electrically or chemically competent cells, as well as natural competence - a state wherein the host cell demonstrates the capability to adsorb, bind, and assimilate exogenous DNA. Alternatively, the protoplast transformation method can be employed, involving the digestion of the peptidoglycan or proteinogenic surface layer cell wall and the subsequent natural uptake of exogenous DNA.

[0116] According to one embodiment a host cell comprises the vector which comprises a nucleic acid that encodes a protein having about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 6, or is said nucleic acid and a nucleic acid that encodes a protein having about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 7, or is said nucleic acid.

[0117] Uses and applications

[0118] In a preferred embodiment, the redox-active system comprising a MEPP may be used to be transferred to nitrogen-fixing Bacillus megaterium or subtilis strains that could be trimmed to catalyse the energy intensive fixation of nitrogen from N2 and H2 to produce ammonium (NF ). The activation of one molecule N2 in natural settings via nitrogenase requires the energy from hydrolysis of 16 molecules of ATP. This high amount of energy is needed mostly for the activation of the triple bond in N2. The biotechnological fixation of N2 could substitute the Haber-Bosch- Process, a technical chemical process with outstanding importance for fertilizer production and with it, the nutrition of humanity. However, the Haber-Bosch-Process is also responsible for massive CO2 emissions contributing significantly to the climate crisis. The use of regenerative energy in the redox-active system according to the present invention for ATP regeneration as a source for energy for N2 fixation by nitrogenase could make fertilizer production almost CO2- emission free. For this application, the redox-active system according to the present invention may be cloned into an already nitrogen-fixing organism, as outlined above, or may be cloned together with the nitrogenase complex and the nitrogenase maturation system into another host strain.

[0119] In an alternative embodiment, the MEPP is cloned into a host organism together with genes for endergonic CO2 fixation to convert CO2 and H2 to fuels such as ethanol, butanol or higher alkanes. Here the ATP supplied by the MEPP is used for the activation of CO2 and its subsequent endergonic reduction to fuel compounds.

[0120] In yet another embodiment, the MEPP complex could be cloned into a production organism converting cheap basic chemicals such as glycerol or methanol plus N2 to value chemicals such as glutamate or other amino acids. In such a use case the biosynthetic pathways explored by hundreds of research groups working in biotechnology could be overexpressed in host cells and powered by almost unlimited amounts of ATP from the MEPP. There are many ATP-dependent biosynthesis reactions but for illustration we here give a short list of examples of such reactions to illustrate the biotechnological importance of ATP:

[0121] Table 1 Example enzymes that are dependent on ATP in the cytoplasm of biotechnologically relevant microorganisms, their catalysed reactions, the Uniprot-ID number and a possible source of the biological material.

[0122] In summary, the invention uses electric current to provide biologically useable energy for the production of platform chemicals, speciality chemicals, fuels and fertilizer.

[0123] Gene sequences SEQ ID NO. 1 (EJNL): HupL CbdbA130

[0124] MQKIVIDPITRIEGHLKIEATVDGGEVKDAKCVGTLFRGFEIFMKDRDPRDAVHITQRICGVCPTS

[0125] HGTTAALNLDAAFGVADKIPNNGRILRNLIQGANYIASHIVHFYHLAALDYVDVTEVADYDGTDP

[0126] ELLKVKDFISRALAAGDMSMLAPFYPRYEGDYRLPKKVAQAAVAHYVEALNMRRLAHELSAIYS

[0127] GRMPHSVAVVAGGVTSHPSIDSISSFMSKLNTLRHFIDNVYLPDVIAVAETYPDYFGIGVGCGNL LSYGVFDLEANGTNLATRQRLFTQGTVSASDLTLHTFDPNKITEDIKYGWFKGETNEYPLNEVT

[0128] EPDHTKAEGYSWLKAPRYENTPYEVGPLARMVVNYVAGDPVVQKMVNDILAHFGAGPAALFS

[0129] TLGRHAARALECKIVADEMVKWVMELKIGEPVCADYEIPESAEGMGLWEAPRGALGHWIKIEN HKISNYQCWPTTWNCSPKDGQGVYGPVEQALIGTKVRDNDNPFELVRIVRSFDPCLACAVHLI

[0130] SPTGNEISRFRVY

[0131] SEQ ID NO. 2 (E_INS): HupS CbdbA129

[0132] MFNTKLTRRDFVQLAAGSTAALSLGALKLPEFEKMFAEALKEIPVIWLQGAGCNGCTISTLNVVS

[0133] PTIQDLLLTSWPGTHVSMQFHPTIMAAQGDLAMNTITDTAAKGPFVLVIEGSVPLKDGGIYAEV

[0134] GEKDGEGITLLEHVLNLAPKALAVVAAGTCSAFGGITAAAPNPTGAKAVSEILKDHNITTPVVNL

[0135] PGCPPHPDWVVGTLATVLMSGLDALDLDDMGRPKAYYGKLLHDQCPRRGHYEKGLFATKLSE

[0136] PYCLFLVGCKGPVTYADCSDRLWNNKTRWCVEADSPCIGCAHPGFPDAVSPMFEAPPVINST

[0137] DKLAIGIAGTAVVLTAGVAAVELAKKAKRNAAKKG

[0138] SEQ ID NO. 3 (E_OUTA): RdhA CbdbA84

[0139] MSNFHSTVSRRDFMKALGLAGVGLGAAGAATPVFHDLDELISSKPEVRESPWWVKEREFEDP

[0140] TVEIDWSLKTHFDYNLVHSWVSKETAQEWQEKQAELIREAVANNTPGNTLKDMALANMGLHY

[0141] AGSDLFNYSQLSRPEYSTVILDTFDVSGQIDNKYGLTRTQLGIPKWQGTPEENSAMVAAVLHFL

[0142] GSTRVGYVSINENNKKVWFSPDKANRIISWGDVEEPVNTPGILWPGNKLGSLVLPNKCNSLISF

[0143] VIPQSGISKYHHTALSRAATFLGYAESTIISARLQIFLKTLGYDGVGSDASANNVGFGVLAGNGE

[0144] LGRLNYLVNPWHGALIRKADFMLTDLPLAPTRPIDSGITRFCATCKKCAEMCPGSALSLADGPS

[0145] WDTLSAQNGLGVKNYTNDWHKCRPWAWPPSPNTVGSCGVCQAVCVFSKLEESSVHDIIKPV

[0146] VSQTPLFNRFFKRMDDMFNYNNPENPEEWWSRDYKNYPYSRAVPGN

[0147] SEQ ID NO. 4 (E_OUTB): RdhB CbdbA85

[0148] MWFTIGMIFGLLISVIFWLLKRNNLNLNWFEWIIGLVGFGLLIFTVQNFMGSFAELEPTA

[0149] AYFFLLITGLPSLI LLAI AWQLAI RRTKKA

[0150] SEQ ID NO. 5 (CLS): OmeA CbdbA195

[0151] MRSQFSRRDFLKISGGTAGLFATAGLFRGPIKGVEPKMVDTRPRWVKETTTICPFDASGCGFIC

[0152] YTDSAGNLTNMEGDPDNPVNRGSACSKGASLIQLHNNKHRLEKVLYRAAGANDWEEKSWDW

[0153] AFGEIAKKVKASRDSGFIAKNAAGKTVNRTEAIASLGGATLDNEECYLLSKMLRALGIVYLDSQA

[0154] RLSTASSLEALAASFGRNAMTNSWTDVSNANVILDMGGNPAENYPACFSHLGEAMGKGAELIS

[0155] VDCRLTRTAAKAGTFVAARSGSEIAFIGGLIKYVISDIEAHPANYNLTYITEYTTAGMWNSNFKG

[0156] PADLDGMFVGYNTASHSYDQSNWNFTKASNGEPVIDKTLTNPNCVFQLLKKQYARYTPEMVS

[0157] ATCGCSQADFAKVCAFFAATGKADKAGAIVYAMSSTQHTSGAQTVRAYAVLQMLLANIGVSGG

[0158] GLFALGGESNVQGAADNGVSWNKLPGYLSAPNEDDTTFAAYASKAGDQSAASLLKAWYGSN

[0159] ASSSNGFGFGFLPKTDNNTNYSYVNAIKQMAAGKIKGAFAWGANPWESAAAGQATQALAKLD

[0160] WLVVTDMFESETAAFWKKDGVTSQTEVFLLPTACSYEKEGSVTNADRWIQWRNKAVSASGEA KSELEVISSLLSRLQTLYQGESGLNAEAITKLSWTYSTNPSAADVAKEMNGYKISSLQQLESPED LRADGTVACGNSLYIGSFTNAGNQMARREQTGAFHAGWAWSWPMNTRIMNNRASVDLSGKP FNAAKPVISWNGMSWTGDNVDGKNAPFNQSGGLPFKGTNGNTAQLFAAMADGPMPEHYEP WESPVDNALSGTQNNPTAMVFEGDTQGSAGEYPVICTTIRTVEHSLGGQLTRNMPFLVELAPA AYVEISEQLASEKGIKAGDIVKLTSARGSVSVAAAVTKRLKPFSIGGKTVHQVAIPNHWGFMGIA QGDSANVLAPNTADSNSSTPEFKAFLVKVEKAADGTVPTITGRYKVLED

[0161] SEQ ID NO. 6 (FCP): HupX CbdbA131

[0162] MPNGMLIDTTRCTGCRGCQVACKQWNENPAVKTTFSPEVTNPLEQNAFNYNLLECREIEEDG KLQWIFAHKRCLHCYEPACVSVCPVGALHKRPNGAVVWDQDKCFGCRYCQNACPFEIPKFEW DDNWAKITKCTMCWNRVDEGMEPACAATCPTGAIKFGERADLLAEAYERLSNNPDKYYPYVY GEKEAGGTHVLYLSSVHPEKLGFDMHEHEFFPSFTREFLGNIPLEISIVASLMVGVWYFRGRRI QAQAHDAEHAENKAGKH

[0163] SEQ ID NO. 7 (MAP): OmeB CbdbA193

[0164] MFSAIKGWVNKLEQTGNPNVPLFGKWGLLQIILGLGALTVAALKLAYGLGPITNLSDNWPWGL WVAFDVGAYIASAAGGFTMAAIVYIFGYEKFRPLVRPAILIGALCYTIGAIGIMIDLARSIRIVHPIW MWNHESIMFEVGWCVMMYLTVLYLEFSNNIFERIGWKKLGNLQHAFIIPLVIFGIMLSFLHQSSL GALFLINPAQQPLWHGPLMGHLFLVSAMALGLSVLTYFSVILSRSWKMTLRMDILSKLGLIIAWI MLVYLAARGVDYAITGNLATGLNGDWYTVLWVAEVGIGMLLPMILLFIKKIRSSRSGLLWSTGL

[0165] MIMGWLNRINTLVISQAPNREGSYFPHILEFVFTFGLIAGAMYVFRLCAKYLPLYTDHMKGLTP EQVKKAPAVIPAD

[0166] This example represents the MEPP of D. mccartyi strain CBDB1 (see Figure 3). Similar MEPPs are encoded in bacteria of other microorganisms of the class Dehalococcoidia and are available in public databases (NCBI, EBI, UniProt, KEGG).

[0167] The actual DNA is available for several Dehalococcoides strains from ATCC number BAA-2098, BAA-2099 and BAA-2100.

[0168] In addition to the genes encoding for the MEPP and its maturation, genes will be included in the production strain that provide information to express a biosynthetic pathway to produce a value chemical.

[0169] Examples

[0170] The present invention is further illustrated by the following example, from which further features, embodiments, aspects and advantages of the present invention can be seen. The OHR-complex of D. mccartyi strain CBDB1 is used as a MEPP for a microbial cell in an electrochemical set-up to provide energy (ATP) for biosynthetic reactions. The first part contains the cloning of the seven OHR genes into a vector and the heterologous expression in the host strain E. coli. We then describe two methods for the identification and characterization of the different components of the electrochemical system and then the construction of the electrochemical device itself. Finally, we propose two examples that would be interesting for biotechnological applications of our system. These are two synthesis pathways of value products that can be produced by ATP-dependent enzymes in a host cell.

[0171] Expression of the energy module in E. coli as a production strain

[0172] This example illustrates the use of the MEPP of D. mccartyi strain CBDB1 , heterologously expressed in E. coli for energy conservation in the electrode mode.

[0173] First, the seven subunits of the OHR complex of strain CBDB1 are amplified by PCR using CloneAmp HiFi PCR premix (Takara Bio USA, Inc.) and the primers SEQ ID NO. 8: 5’-ACC ACA GCC AGG ATC TGA GTA ACT TTC ATT CAA CAG TAA G-3’ (forward) and SEQ ID NO. 9: 5’- TAA TCT ATT TAG GCT TTT TTG GTT CTC CG-3’ (reverse) for the genes rdhAB (loci cbdbA84 and cbdbA85), SEQ ID NO. 10: 5’-CTT TAG CAG ACT CGA TTA GTC TGC CGG TAT AAC COS’ (forward) and SEQ ID NO. 11 : 5’-AAG GAG ATA TAG ATA TGA GGT CTC AGT TTA GTA GGC G-3’ (reverse) for the genes omeAB (loci cbdbA195 and cbdbA193), and SEQ ID NO. 12: 5’- GCT GCA GGC GCG CCG TTA GTA GAG GCG GAA GCG GC-3’ (forward) and SEQ ID NO. 13: 5’- AGC CTA AAT AGA TTA AAG CAG AAG GGG GGC-3’ (reverse) for the genes hupSLX (loci cbdbA129, cbdbA130 and cbdbA131). The primers are obtained from Eurofins Genomics (Germany, Ebersberg). The amplicons are cloned in the vector pACYCDuet™-1 (Sigma-Aldrich, Germany) with In-Fusion® cloning kit (Takara Bio USA, Inc.) and transformed in chemically- competent E. coli Stellar™ cells (Takara Bio USA, Inc.) as described by the manufacturer (1 min at 42 °C). For the cloning, the vector is linearized with BamHI and Sacl restriction enzymes (Thermo Fisher Scientific) beforehand, and the PCR-amplified genes rdhAB and hupSLX are inserted in one step. Then, the obtained vector is again linearized with Xhol and Ndel and the omeAB genes are cloned into the same vector. In addition to the obtained production vector termed as pACYC:RdhAB-HupSLX-OmeAB, an auxiliary plasmid pBAD:BtuB-F containing btuC, btuE, btuD, btuF and btuB genes, which encode for proteins responsible for cobalamin uptake (Fang, H., Kang, J. & Zhang, D. (2017), “Microbial production of vitamin B12: a review and future perspectives” Microbial Cell Factories 16: 15), is co-expressed.

[0174] Precultures of positive clones are prepared and plasmids containing all the seven OHR genes are purified by NucleoSpin Plasmid Kit (Macherey-Nagel GmbH & Co. KG, Germany). Subsequently, the purified production vector pACYC:RdhA-HupSLX-OmeAB is incorporated in E. coli BL21 (DE3) or E. coli BL21 (DE3) Suf++strains by electroporation by applying an electric shock at 1 ,800 V. Cells containing the pACYC:RdhA-HupSLX-OmeAB production plasmid are selected on lysogeny broth (LB) agar containing 30 pg mL'1amphenicol. Positive clones are used to set up an overnight culture in LB liquid medium, which is shaken at 37 °C and 160 rpm. On the next day, 1-2% (v / v) of the preculture is used to inoculate fresh LB medium. The cells are grown at 37 °C and 160 rpm until the optical density (OD) at 600 nm reaches 0.4-0.6. Then, the expression of the genes from the auxiliary plasmid pBAD:42BtuB-F is induced by 0.05% L-arabinose and 5 pM of B^ are added, and the shaking at 37 °C is continued for 1 hour. After this, the production of the OHR subunits from the pACYC:RdhA-HupSLX-OmeAB vector is induced by the addition of 100 pM IPTG. The heterologous production is then carried out at 20 °C and 160 rpm for at least 18 hours. Additionally, 1 mM L-cysteine and 1 mM iron(lll)ammonium citrate, 1 mM sodium molybdate and 1 mM nickel sulphate are added. After the incubation time, the cells are harvested at 7,000 rpm and 4 °C for 30 min. For cell disruption, protein purification and subsequent processes, cell pellets are resuspended in 50 mL tris / HCI buffer (pH 8.0), containing 10% glycerol.

[0175] Production of proteins is verified by SDS polyacrylamide gel electrophoresis followed by an in-gel trypsin digestion for nLC-MS / MS analysis. Identification of proteins from gel pieces by protein mass spectrometry is described for example in Kublik, A., Deobald, D., Hartwig, S., Schiffmann, C.L., Andrades, A., von Bergen, M. et al. (2016), “Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement”. Environmental Microbiology 18: 3044-3056.

[0176] Electron donor used by the OHR complex

[0177] It was previously shown in detail that hydrogen is an appropriate electron donor for the OHR complex of strain CBDB1 , for example by Jayachandran, G., Gdrisch, H. & Adrian, L. (2004), “Studies on hydrogenase activity and chlorobenzene respiration in Dehalococcoides sp strain CBDB1”. Archives of Microbiology 182: 498-504 or Hartwig, S., Dragomirova, N., Kublik, A., Turkowsky, D., von Bergen, M., Lechner, U. et al. (2017), “A H2-oxidizing, 1 ,2,3-trichlorobenzene- reducing multienzyme complex isolated from the obligately organohalide-respiring bacterium Dehalococcoides mccartyi strain CBDB1”. Environmental Microbiology Reports 9: 618-625, and it has also been shown that this hydrogenase can be functionally heterologously expressed in E. coli, for example by Hartwig, S., Thomas, C., Krumova, N., Quitzke, V., Turkowsky, D., Jehmlich, N. et al. (2015), “Heterologous complementation studies in Escherichia coli with the Hyp accessory protein machinery from Chloroflexi provide insight into [NiFe]-hydrogenase large subunit recognition by the HypC protein family”. Microbiology 161 : 2204-2219. Transfer of electrons from the OHR complex to an anode mediator is assayed with a methyl viologen based activity assay

[0178] Methyl viologen (MV) is an artificial dye that is often used in biochemistry as a redox dye (see Fehler! Verweisquelle konnte nicht gefunden werden.4a). It can be used as an artificial electron donor for RdhA for the reduction of organohalides, e.g., chlorobenzenes. Reduced MV is blue, while the oxidized form is colourless (see Figure 4b). To yield the reduced form of MV, the strong reducing agent titanium(l I l)citrate is added to the oxidized MV under anoxic conditions in an anaerobic chamber (Coy Laboratory, USA). The activity of the RdhA can be assayed either by determination of electron acceptor concentrations (substrate and product determination via gas chromatography or high-performance liquid chromatography) or by monitoring the oxidation of MV photometrically at around 600 nm (see Figure 4b and Figure 7). The latter method allows for kinetic studies.

[0179] Figures 4a and 4b show the use of viologens for monitoring of electron transfer processes. Fig. 4a: Viologens are bipyridium salts (shown here methyl- (MV), ethyl- (EV), and benzyl viologen (BV)) that are colourless in their oxidized state and can take up one electron developing a strong blue colour. In their reduced state they have one unpaired electron (radical) and are therefore strong reducing agents at a redox potential between approximately -340 and -450 mV.

[0180] Fig. 4b: In the activity test of the example, MV was reduced with titanium(l I l)citrate to get the blue reduced form of MV. This reduced MV interacts with the reductive dehalogenase of the MEPP protein complex and works as an artificial electron donor. Activity can be quantified by calculating the decrease in the absorption at 600 nm or by quantifying dehalogenated products (here monobromobenzene) by gas chromatography.

[0181] To run the MV-based activity test, activity assay buffer was mixed with enzymes or cells resulting in a final concentration of 100 mM potassium acetate buffer (pH 5.8), 1 mM MV reduced with 1 mM titanium(lll)citrate, and 200 pM organohalide as an electron acceptor in the set-up. No- substrate-controls (NSC) and no-enzyme-controls (NEC) were prepared, containing buffer instead of substrate and cells or crude extract, respectively. The dehalogenase enzyme activity was monitored photometrically at 600 nm.

[0182] Transfer of electrons from an anode mediator to an anode is determined by cyclic voltammetry

[0183] To determine if the putative anode mediators can reversibly exchange electrons with the anode, cyclic voltammetry experiments are performed. Cyclic voltammetry is an electrochemical technique used to study the redox properties of electroactive molecules in electrolytic solution. Three-electrode setup containing the working, counter and reference electrode is a common setup used in cyclic voltammetry. The working electrode serves as the site where the electrochemical reaction of interest occurs. The potential applied to the working electrode is swept linearly over time and subsequently reversed, causing redox reactions to take place, which manifest as a current flowing in or out of the electrode. The reference electrode is crucial for establishing a known potential against which the potential of the working electrode is measured. It remains constant throughout the experiment, providing a stable reference point for the measured potential at the working electrode. The counter electrode ensures that any current generated at the working electrode is balanced. It essentially completes the circuit for the current to flow in the electrochemical cell. This helps maintain the overall charge balance in the system during the experiment. In our experiments, anode mediators in a concentration of 300 pM are diluted in an electrolytic solution of 0.5 M potassium chloride. Titanium is used as the material for the counter electrode, gold or indium tin oxide as material for the working electrode and platinum or silver / silver chloride as reference electrode material. External voltage is applied by a multichannel potentiostat (MultiEmStat 4 LR; PalmSens BV, Netherlands), which is controlled by MultiTrace software (PalmSens BV, Netherlands). Potentials are set to a range from -1.5 to +1.5 V at a scan rate of 0.05 V / s. This broad scan range is used to observe all possible oxidation and reduction peaks.

[0184] Figure 5 depicts a schematic setup designed for cyclic voltammetry experiment aimed to test for the reversible redox reaction between an anode mediator and the working electrode, which will subsequently function as the anode. The potential is measured between the working electrode and the reference electrode, with a linear sweep over time, oscillating in cycles. During this process, when redox reactions take place at the working electrode, involving either the oxidation or reduction of the substance, a current is flowing between the working electrode and the counter electrode. At a specific potential, electrons are transferred from the negatively charged working electrode to the tested anode mediator, resulting in a distinct flow of reduction current (see Fig. 5, dashed line). Subsequently, as the potential of the working electrode is reversed, the reduced anode mediator undergoes oxidation at a particular potential, leading to the flow of an oxidation current (see Fig. 5, solid line).

[0185] Electrochemical device

[0186] In this example, the electrochemical device is a 100 mL customized borosilicate gastight bottle (Schott Duran, Germany). The system contains a titanium-cathode and a gold-anode electrode, which are inserted via a gastight rubber stopper. The titanium-cathode is shielded by a Nation™ 117 perfluorinated membrane (Sigma Aldrich, Germany) that functions as a proton-exchange- membrane (“PEM”) and leads to a two-compartment set-up. A reference electrode composed of platinum is placed into the anode compartment. Additionally, the E. coli strain containing all seven OHR-subunits (as described above) referred to as a host cell is also located in the anode compartment in a mineral medium, e.g., M9 minimal medium supplemented with nitrate. The system works under anoxic conditions and therefore the head space is flushed with 300 mbar overpressure N2 and then pressurized with 400 mBar H2. An external voltage is applied via a multichannel potentiostat (Multi EmStat 4 R; PalmSens BV, Netherlands). The potential of the cathode is set to -360 mV resulting in the reduction of protons to hydrogen, while the potential of the anode is set to +500 mV favouring the oxidation of cyanocobalamin (Sigma Aldrich, Germany) that is added in a concentration of 300 pM as an anode mediator. Due to the reduction of protons at the cathode, hydrogen is formed and diffuses through the PEM into the anode compartment and through the outer membrane of the host cell to the OHR complex located on the outside of the cytoplasmic membrane. Here, hydrogen is oxidized by the [NiFe] hydrogenase HupL and the electrons are shuttled through the OHR complex, while the generated protons diffuse back through the outer membrane of the host cell and through the PEM to the cathode where it is reduced to hydrogen again. The electrons are conducted through the OHR-complex and generate an electric current. In detail, electrons travel from the [Ni-Fe] centre of the HupL subunit across the subunit boundaries to one of the [Fe-S] centre of the HupS subunit and pass all three [Fe-S] centres of the HupS subunit. Then, they are transferred to HupX and travel through the four [Fe- S] centres to then reach the RdhA subunit. In the RdhA subunit, electrons are transferred through two cubane [Fe-S] clusters to the active site binding corrinoid. This electron flux to the active site of the RdhA subunit is coupled to a proton transport from the cytoplasm through the OmeB subunit (Fehler! Verweisquelle konnte nicht gefunden werden.6). From the corrinoid in the active centre of RdhA the electrons are transferred to anode mediator cyanocobalamin that carries the electrons to the anode. Cyanocobalamin is oxidized on the surface of the anode and can be used again as electron acceptor by RdhA. The special interaction of HupX and OmeB is responsible for the tight coupling of electron flux with proton export. The proton export is electrogenic and leads to a pmf that is exploited by the ATPase of the host strain.

[0187] Similar electrochemical devices were previously described for example by Kracke et al. 2019, “Robust and biocompatible catalysts for efficient hydrogen-driven microbial electrosynthesis”. Communications Chemistry 2 or Deutzmann et al. 2023, “Microbial electromethanogenesis powered by curtailed renewable electricity”, Cell Reports Physical Science 4 or Luo et al. 2023, “ATP production from electricity with a new-to-nature electrobiological module”, Joule 7: 1745- 1758.

[0188] Fig. 6 relates to experiments showing that the electron flux though the OHR complex are coupled to proton export. The protons are channelled from the cytoplasm onto the external halogenated substrate when electron flux is present. A) Results of an experiment with normal water in the cell and with an activity buffer set up with deuterated water (D2O). A very low initial deuterium integration into the extracellular substrate (Deuterium Degree of the reduced substrate) demonstrates that the protons come from the inside of the cell. After some minutes an equilibrium is reached due to diffusion of H2O and D2O through the cell membrane. B) Detail of the OHR complex showing a potential proton path though the membrane-integrated OmeB subunit (bottom) to the cobalamin active centre of RdhA.

[0189] Anode mediator

[0190] In this example cyanocobalamin is used as the anode mediator. The reduction of cyanocobalamin by the OHR complex is demonstrated in Figure 7. In Figure 7, the decrease in absorption at 600 nm in a methyl viologen-based enzymatic activity test with cyanocobalamin as electron mediator is shown. The line shows the decrease of the absorption of the electron donor (MV) in the presence of the OHR-complex and 300 pM of cyanocobalamin, demonstrating that electrons were transferred to cyanocobalamin. The other two curves are controls: dashed line: positive control with the halogenated electron acceptor 3,5-dibromo-L-tyrosine. This compound can be irreversibly reduced by the OHR complex but cannot reduce an anode. Dotted line: negative control with cyanocobalamin but without OHR-complex. Activity was calculated from the data as the concentration reduced in mediator per hour and 1 x 108cells.

[0191] For this example, we demonstrated that cyanocobalamin is reversibly exchanging electrons with an indium-tin-oxide electrode and a gold electrode meaning that it can be reduced as well as oxidized by both anode materials (see Figure 8).

[0192] Figure 8 relates to cyclic voltammetry and redox characteristics for cyanocobalamin redox reaction with an indium-tin oxide electrode or gold electrode. The graphs show that cyanocobalamin can reversibly exchange electrons with an electrode. It also shows that the mid redox potential against the normal hydrogen electrode (NHE) is at +0.42 V, meaning that a large share of the cyanocobalamin is oxidized at an anode potential of +0.5 V.

[0193] Biosynthetic genes

[0194] As mentioned in the description the biosynthetic genes are not part of this invention. The invention only delivers the ATP necessary for ATP-dependent biosynthesis. For completeness we could propose the heterologous expression of ATP-dependent glutamine synthetase here as a simple example. Then the E. coli host would be amended with a plasmid carrying a modified glutamine synthetase that is deregulated from product inhibition. If actively expressed the protein would take the ATP from the invented system to convert glutamate and ammonium to glutamine. In a more complex example, the host would carry nitrogenase and nitrogenase reductase to be able to produce ammonium from nitrogen gas (N2) and hydrogen gas (H2). Both would be supplied as substrates. The energy for this reaction would be delivered via the expressed MEPP in an electrochemical device as described above using externally applied voltage.

Claims

CLAIMS1. A redox-active system comprising a protein complex as microbial electricity-driven proton pump (MEPP) for a microbial cell comprisingA) a central module (CM) comprising(i) at least one electron-transfer subunit (FCP) containing at least two ironsulfur [Fe-S] clusters;(ii) at least one membrane-anchor subunit (MAP) linking the central module (CM) to the cytoplasmic membrane of the cell;B) at least one electron input module (E_IN) and at least one electron output module (E_OUT) comprising one or more redox-active reaction centres andC) at least one extracellular electron donor (ED) and at least one extracellular electron acceptor (EA), wherein the metal clusters of the subunit (i) and the module B) are oriented towards the outside of the cytoplasmic membrane of the cell.

2. The redox-active system according to claim 1 , wherein the central module (CM) further comprises(iii) at least one large subunit (CLS) comprising at least one transition metal cofactor located outside of the cytoplasmic membrane of the cell.

3. The redox-active system according to claim 1 or 2, wherein the electron-transfer subunit (FCP) comprises two or more cubane-like iron-sulfur [Fe-S] clusters; preferably the electron-transfer subunit (FCP) comprises or consists of a protein with an amino acid sequence of SEQ ID NO. 6 or with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO. 6.

4. The redox-active system according to any one of claims 1 to 3, wherein the membraneanchor subunit (MAP) comprises at least eight transmembrane helices; preferably themembrane-anchor subunit (MAP) comprises or consists of a protein with an amino acid sequence of SEQ ID NO. 7 or with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO. 7.

5. The redox-active system according to any one of claims 1 to 3, wherein the membraneanchor subunit (MAP) is a cytochrome b and comprises a quinone binding side at the inside of the cell.

6. The redox-active system according to any one of claims 1 to 5, wherein the transition metal cofactor of the large subunit (CLS) is selected of heme, [Ni-Fe]-centres, corrinoids or molybdopterin-cofactors; preferably the transition metal cofactor of the large subunit (CLS) comprises or consists of a protein with an amino acid sequence of SEQ ID NO. 5 or with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO. 5.

7. The redox-active system according to any one of claims 1 to 6, wherein the subunit (CLS) comprises at least one cubane iron-sulfur [Fe-S] cluster.

8. The redox-active system according to any one of claims 1 to 7, wherein the electron output module (E_OUT) comprises at least one subunit comprising a corrinoid as cofactor; preferably the electron output module (E_OUT) comprises or consists of a protein with an amino acid sequence of SEQ ID NO. 3 and / or 4 or with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO. 3 and / or 4.

9. The redox-active system according to any one of claims 1 to 8, wherein the electron input module (E_IN) comprises a [NiFe] hydrogenase enzyme; preferably the electron input module (E_IN) comprises or consists of a protein with an amino acid sequence of SEQ ID NO. 1 and / or 2 or with an amino acid sequence with at least 50%, 60%, 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO. 1 and / or 2.

10. The redox-active system according to any one of claims 1 to 9, wherein the electron donor (ED) and electron acceptor (EA) are water-soluble molecules that reversibly take up electrons.

11. An electrochemical reactor comprising the redox-active system based on a protein complex as microbial electricity-driven proton pump (MEPP) according to any one of claims 1 to 10 and an anode and cathode.

12. A method for producing a protein complex as microbial electricity-driven proton pump (MEPP) for a cell expressing a polynucleotide comprising a nucleic acid sequence which encodes a protein with amino acid sequence identity of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to an amino acid sequence selected of the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7 or is an amino acid sequence selected of the group consisting of SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7.

13. A vector comprising a nucleic acid that encodes a protein having about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 6, or is said nucleic acid and a nucleic acid that encodes a protein having about or at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to or homology with SEQ ID NO. 7, or is said nucleic acid.

14. A host cell comprising the vector according to claim 13.

15. Use of the redox-active system according to any one of claims 1 to 10 for the production of fuels, fertilizer, chemical building blocks and specialty chemicals.

Images