Electron-accepting proteins

Abstract

To provide a novel protein (multiheme cytochrome C) that has a high ability to deliver an electron from an electrode into a cell, and can improve the production efficiency of useful substance by microbial electrosynthesis (MES).SOLUTION: A protein comprises an amino acid sequence having at least 90% sequence identity to an amino acid sequence represented by SEQ ID NO: 1 and has multiheme cytochrome C activity.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to proteins. More specifically, the present invention relates to electron-accepting proteins. Even more specifically, the present invention relates to novel multiheme cytochrome C. [Background technology]

[0002] It is known that electromicrobial organisms such as Shewanella oneidensis possess an extracellular electron transport pathway for releasing electrons outside the cell and can transfer electrons to an electrode via the extracellular electron transport pathway. It has also been shown that they can also transfer electrons from an electrode into the cell via the extracellular electron transport pathway. Microbial electrosynthesis (MES), which utilizes the latter electron transfer, is a method for producing useful substances within microbial cells (e.g., Non-Patent Documents 1 and 2).

[0003] It has been shown that by introducing a gene cassette (MtrA-MtrB-MtrC-OmcA genes) containing the multiheme cytochrome C of Shewanella oneidensis into Escherichia coli, which does not originally have electron-accepting activity, it is possible to introduce electron-accepting activity into Escherichia coli (Non-patent Document 3).

[0004] Furthermore, Patent Document 1 discloses a method for improving the electron transfer reaction rate of a heme protein, characterized in that a specific amino acid that is located between heme 1 and heme 2 of cytochrome c3 and controls electron transfer between heme 1 and heme 2 is substituted from a polar, uncharged amino acid to a nonpolar amino acid. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2001-178477 [Non-patent literature]

[0006] [Non-Patent Document 1] Biotechnol. Biofuels(2016)9:11 [Non-patent document 2] mBiO,2018,Vol.9,No.1,e02203-17 [Non-patent document 3] PNAS,2010,Vol.107,No.45,pp.19213-19218 Summary of the Invention [Problem to be solved by the invention]

[0007] The gene cassette (MtrA-MtrB-MtrC-OmcA genes) used in Non-Patent Document 3 is primarily used in the pathway for transferring electrons to the electrode. However, the electron transfer rate in the reverse pathway, which is the pathway for receiving electrons from the electrode, is slow, and the production efficiency is insufficient when used to produce useful substances by MES.

[0008] Therefore, an object of the present invention is to provide a novel protein (multiheme cytochrome C) that has a high ability to transfer electrons from an electrode into cells and can improve the efficiency of useful substance production by microbial electrosynthesis (MES). Another object of the present invention is to provide a microorganism transformed with the protein and a method for producing useful substances using the microorganism. [Means for solving the problem]

[0009] The present inventors have found that a microorganism (Shewanella oneidensis) transformed to express a novel electron-accepting protein (multihemocytochrome c (MmcX original)) discovered from metagenomic information of a hyper-reducing environment has an improved ability to transfer electrons from an electrode into the cell, thereby improving the efficiency of production of useful substances through microbial electrosynthesis (MES). The present invention is based on this novel finding.

[0010] The present invention relates to a protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 and having multiheme cytochrome C activity.

[0011] The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence obtained by deleting the signal peptide sequence from multihemocytochrome C (MmcX original). Because the protein of the present invention has a sufficiently high sequence identity with the amino acid sequence shown in SEQ ID NO: 1, it has a high ability to transfer electrons from an electrode into cells, and can improve the production efficiency of useful substances through microbial electrosynthesis (MES).

[0012] The present invention also relates to nucleic acids encoding the proteins according to the invention described above.

[0013] The present invention also relates to an expression vector having the base sequence of a nucleic acid encoding the protein according to the present invention described above and one or more regulatory sequences operably linked to the base sequence.

[0014] The present invention also relates to a microorganism transformed with the above-mentioned expression vector. The microorganism according to the present invention has an improved electron transfer rate in the pathway through which electrons are received from the electrode into the cell, and is therefore highly efficient in producing useful substances through microbial electrosynthesis (MES).

[0015] The microorganism preferably lacks or has reduced multihemocytochrome C activity, which is inherent in the microorganism. This improves the electron transfer rate in the pathway through which electrons are received from the electrode into the cell, and also improves the efficiency of producing useful substances through microbial electrosynthesis (MES).

[0016] The present invention also relates to a method for producing useful substances, which includes the steps of preparing a microbial electrolysis cell having at least one pair of electrodes, with a microorganism transformed with the above-mentioned expression vector placed at the cathode, and a solution in which the electrodes are immersed; setting the cathode to a low potential; and supplying electrons from the cathode to the microorganism, causing a reduction reaction within the microorganism to biosynthesize the useful substance. [Effects of the Invention]

[0017] The present invention provides a novel protein (multiheme cytochrome C) that has a high ability to transfer electrons from an electrode into cells and can improve the efficiency of useful substance production by microbial electrosynthesis (MES). The present invention also provides a microorganism transformed with the protein and a method for producing useful substances using the microorganism. [Brief explanation of the drawings]

[0018] [Figure 1] 1 shows a vector map of the expression vector (pHSG-mmcX). [Figure 2] (A) Schematic diagram showing the electron transfer pathway from the electrode to the cell in a wild-type Shewanella oneidensis strain (MR-1 strain). (B) Schematic diagram showing the electron transfer pathway from the electrode to the cell in a Shewanella oneidensis transformant (ΔomcAΔmtrC(pHSGmmcX)) lacking OmcA and MtrC and carrying MmcX. [Figure 3] 1 is a graph showing the current density measurements obtained when a Shewanella oneidensis wild-type strain (MR-1(pHSG298)), a Shewanella oneidensis deletion strain in which OmcA and MtrC have been deleted (ΔomcAΔmtrC(pHSG298)), and a Shewanella oneidensis transformant strain in which OmcA and MtrC have been deleted and MmcX has been introduced (ΔomcAΔmtrC(pHSGmmcX)) were cultured in an inorganic salt medium in an electrochemical cell. [Figure 4] 1 is a graph showing the results of measuring the amount of succinic acid produced by a wild-type Shewanella oneidensis strain (MR-1(pHSG298)) and a Shewanella oneidensis transformant strain (ΔomcAΔmtrC(pHSGmmcX)) in which OmcA and MtrC have been deleted and MmcX has been introduced. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0020] 〔protein〕 The protein according to this embodiment comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and has multiheme cytochrome C activity.

[0021] The amino acid sequence shown in SEQ ID NO: 1 is an amino acid sequence obtained by deleting the signal peptide sequence from a novel electron-accepting protein (multiheme cytochrome C (MmcX original)) discovered from metagenomic information on a super-reducing environment. The protein of this embodiment may have a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 1. The sequence identity is preferably 92% or more, more preferably 94% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. The sequence identity may even be 100%. Furthermore, the amino acid sequence shown in SEQ ID NO: 1 contains four heme-binding regions (amino acid sequence: Cys-XX-Cys-His, where X represents any amino acid residue). The protein of this embodiment preferably retains the amino acid sequences of these heme-binding regions.

[0022] The protein according to this embodiment has multiheme cytochrome C activity. In this embodiment, the multiheme cytochrome C activity may be at least the activity of transferring electrons. That is, the protein according to this embodiment maintains the activity of transferring electrons from an electrode into a cell, which is possessed by a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0023] The above-described protein may contain an amino acid sequence (signal peptide sequence) involved in protein transport and localization. Because the protein of the present invention has a high electron transfer rate in the pathway for receiving electrons from the electrode into the cell, it is preferably localized on the outermost membrane of the microorganism (e.g., outer membrane, cell membrane). Therefore, a signal peptide sequence that directs transport and localization on the outermost membrane of the microorganism is preferred. Such a signal peptide sequence can be appropriately selected depending on the type of microorganism (host) to be transformed with the protein of the present invention. For example, when Shewanella oneidensis is used as the host, a signal peptide sequence having the amino acid sequence shown in SEQ ID NO: 5 can be used. The amino acid sequence shown in SEQ ID NO: 5 is the signal peptide sequence of the MtrC protein of the Shewanella oneidensis strain MR-1.

[0024] One embodiment of the present invention is a protein comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 1 and having multiheme cytochrome C activity, to which the amino acid sequence set forth in SEQ ID NO: 5 has been added at the N-terminus. This protein is localized to the outer membrane of a microorganism belonging to the genus Shewanella, preferably a microorganism belonging to Shewanella oneidensis, due to the function of the signal peptide sequence, and can therefore be suitably used for transforming a microorganism belonging to the genus Shewanella, preferably a microorganism belonging to Shewanella oneidensis. A specific example of this protein is one having the amino acid sequence set forth in SEQ ID NO: 2 (also referred to herein as "MmcX"). MmcX is the amino acid sequence set forth in SEQ ID NO: 1 to which the amino acid sequence set forth in SEQ ID NO: 5 has been added at the N-terminus.

[0025] [Nucleic acid] The nucleic acid according to this embodiment encodes the protein according to the present invention. A specific example of the nucleic acid is one having the nucleotide sequence shown in SEQ ID NO: 3. The nucleotide sequence shown in SEQ ID NO: 3 is a nucleotide sequence that encodes the amino acid sequence shown in SEQ ID NO: 2.

[0026] The nucleic acid according to this embodiment is not particularly limited except that it encodes the protein according to the present invention, and may be, for example, a nucleic acid whose codons are optimized according to the type of microorganism (host) to be transformed with the nucleic acid.

[0027] [Expression vector] The expression vector of this embodiment has the base sequence of the nucleic acid of this embodiment and one or more regulatory sequences operably linked to the base sequence. The regulatory sequences are sequences that control the expression of proteins in a host (e.g., promoters, enhancers, ribosomal binding sequences, transcription termination sequences, etc.), and can be selected appropriately depending on the type of host.

[0028] The type of expression vector can be appropriately selected depending on the type of host, and examples include plasmid vectors, viral vectors, and artificial chromosome vectors.

[0029] The expression vector according to this embodiment may be capable of autonomous replication within the host cells, or may be integrated into the host chromosome and replicate along with chromosomal replication.

[0030] The expression vector according to this embodiment may contain a selection marker gene for selecting a transformant. Examples of the selection marker gene include genes that confer resistance to antibiotics.

[0031] [Microorganisms] The microorganism according to this embodiment is transformed with the expression vector according to this embodiment (herein, the transformed microorganism is also particularly referred to as a "transformed strain"). The transformed strain can be obtained by introducing the expression vector according to this embodiment into a microorganism (host) and transforming it. The method for introducing the expression vector can be any method known in the art, depending on the type of expression vector, the type of host, etc.

[0032] The microorganism (host) to be transformed is not particularly limited and may be, for example, a bacterium, a fungus, a unicellular alga, a protozoan, or an animal or plant cell. When a microorganism without an extracellular electron transport pathway is used as the microorganism (host), an electron transfer pathway from the electrode to the cell can be formed by transforming the microorganism by introducing MtrA and MtrB in addition to the protein of the present invention.

[0033] Microorganisms possessing an extracellular electron transport pathway (electromicroorganisms) can be used as hosts. Examples of electromicroorganisms include those belonging to the genera Geobacter, Shewanella, Aeromonas, Tenderia, and Mariprofundus. More specific examples of electromicroorganisms include those belonging to Geobacter sulfurreducens, Shewanella oneidensis, Aeromonas hydrophila, Tenderia electrophaga, and Mariprofundus ferrooxidans.

[0034] When a microorganism (electromicrobe) having an extracellular electron transport pathway is used as a host, the microorganism may be transformed by introducing an expression vector according to the present embodiment. Alternatively, the microorganism may be genetically modified to eliminate or reduce the inherent multihemocytochrome C activity of the microorganism, and then the genetically modified microorganism may be transformed by introducing an expression vector according to the present embodiment. By eliminating or reducing the inherent multihemocytochrome C activity of the host microorganism, the transformant strain improves the electron transfer rate in the pathway through which electrons are received from the electrode into the cell and improves the efficiency of producing useful substances through microbial electrosynthesis (MES). The inherent multihemocytochrome C activity of the host microorganism can be eliminated or reduced by genetically modifying the microorganism to eliminate or reduce the transcriptional activity of the inherent multihemocytochrome C gene.

[0035] [Method for producing useful substances] The method for producing a useful substance according to this embodiment includes the steps of: preparing a microbial electrolysis cell having at least one pair of electrodes, with the microorganism (transformed strain) according to this embodiment placed at the cathode, and a solution in which the electrodes are immersed (preparation step); setting the cathode to a low potential (setting step); and supplying electrons from the cathode to the microorganism (transformed strain) to carry out a reduction reaction within the microorganism (transformed strain) to biosynthesize a useful substance (biosynthesis step).

[0036] In the preparation step, a microbial electrolysis cell is prepared, which includes at least one pair of electrodes, with the microorganism (transformed strain) according to this embodiment disposed at the cathode, and a solution in which the electrodes are immersed. The cathode on which the transformant is disposed may be, for example, an electrode on which the transformant is immobilized in advance, or the transformant may be added to a solution in the microbial electrolysis cell and cultured as necessary, thereby disposing the transformant on the cathode.

[0037] The solution in the microbial electrolysis cell preferably has a composition that allows the transformant to survive and grow. Specifically, an inorganic salt medium (e.g., M9 medium) appropriate for the type of microorganism used to produce the transformant can be used. Depending on the type of useful substance to be produced, a substance that serves as a raw material (substrate) for the useful substance may be added to the solution.

[0038] A pair of electrodes in the microbial electrolysis cell is connected to an external power source (voltage application device). The material forming the electrodes is not particularly limited as long as it functions as an electrode, and examples thereof include carbon, stainless steel, platinum, and titanium. The shape of the electrodes is also not particularly limited, but the cathode is preferably formed in a plate shape, which makes it easier to arrange the transformed strain.

[0039] In the setting step, the cathode on which the microorganism (transformed strain) is placed is set to a low potential. The low potential is set to facilitate the transfer of electrons from the electrode to the extracellular electron transport pathway of the transformant. Specifically, the potential to be set is −0.4 V or less, preferably −0.5 V or less, and more preferably −0.6 V or less, relative to the standard hydrogen electrode (SHE).

[0040] In the biosynthesis step, electrons are supplied from the cathode to the microorganism (transformant), and a reduction reaction occurs within the microorganism (transformant), biosynthesizing useful substances. By supplying electrons from the cathode to the transformant, useful substances are biosynthesized according to the biosynthetic pathway possessed by the transformant.

[0041] The type of useful substance depends on the biosynthetic pathway possessed by the transformed strain, but specific examples include hydrocarbons such as methane, organic acids such as acetic acid and succinic acid, and biodegradable polymers such as polyhydroxyalkanoic acid (PHA). Furthermore, a desired useful substance can be produced by using a transformed strain into which a gene that biosynthesizes the desired useful substance has been introduced by genetic recombination in addition to the protein according to this embodiment. [Example]

[0042] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0043] [Design of a novel multiheme cytochrome C (MmcX) and construction of its expression vector] A putative multihemocytochrome C (MmcX original) gene was searched for from metagenomic information on super-reducing environments (see non-patent literature: The ISME Journal, 2017, vol. 11, pp. 2584-2598). The amino acid sequence encoded by the putative multihemocytochrome C gene was the amino acid sequence shown in SEQ ID NO: 4. A signal peptide region was deduced from the amino acid sequence shown in SEQ ID NO: 4, and an amino acid sequence (SEQ ID NO: 1) with the signal peptide sequence deleted was identified. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence obtained by deleting the signal peptide sequence from the amino acid sequence of the putative multihemocytochrome C (MmcX original) (SEQ ID NO: 4).

[0044] An amino acid sequence was designed in which the signal peptide sequence of the MtrC protein of Shewanella oneidensis strain MR-1 (SEQ ID NO: 5) was added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 (SEQ ID NO: 2). A nucleotide sequence was designed that encodes the amino acid sequence shown in SEQ ID NO: 2 and was codon-optimized to optimize expression in Shewanella oneidensis strain MR-1 (SEQ ID NO: 3). The nucleic acid having the nucleotide sequence shown in SEQ ID NO: 3 was obtained by chemical synthesis. Restriction enzyme (EcoRI and BamHI) recognition sites were added to the 5' and 3' ends of the sequence for incorporation into an expression vector.

[0045] The synthesized nucleic acid and an expression vector (pHSG298, manufactured by Takara Bio Inc.) were each digested with restriction enzymes (EcoRI and BamHI) and ligated to prepare an expression vector (pHSG-mmcX) incorporating the base sequence shown in SEQ ID NO: 3. Figure 1 shows the vector map of the expression vector (pHSG-mmcX).

[0046] [Preparation of transformed strains] The omcA and mtrC genes of Shewanella oneidensis strain MR-1 were deleted by double crossover to generate the omcA and mtrC deletion strain (ΔomcAΔmtrC). The deletion of the omcA and mtrC genes was confirmed by PCR amplification fragment length.

[0047] An expression vector (pHSG-mmcX) containing the base sequence shown in SEQ ID NO: 3 was electroporated into the omcA and mtrC deletion strain (ΔomcAΔmtrC) to obtain a transformed strain expressing MmcX instead of omcA and mtrC (referred to as the ΔomcAΔmtrC(pHSGmmcX) strain). As a control, an empty vector (pHSG298) was electroporated into the Shewanella oneidensis MR-1 strain and the omcA and mtrC deletion strain (ΔomcAΔmtrC), respectively, to obtain strains carrying the empty vector (referred to as the MR-1(pHSG298) strain and the ΔomcAΔmtrC(pHSG298) strain, respectively).

[0048] Figure 2(A) is a schematic diagram showing the electron transfer pathway from the electrode into the cell in a wild-type Shewanella oneidensis strain (MR-1 strain). Electrons transferred from the electrode are received by OmcA and MtrC on the outer membrane surface, and then transported into the periplasm by MtrB and MtrA. Figure 2(B) is a schematic diagram showing the electron transfer pathway from the electrode into the cell in a transformed strain (ΔomcAΔmtrC(pHSGmmcX) strain). Because the ΔomcAΔmtrC(pHSGmmcX) strain lacks OmcA and MtrC on the outer membrane surface, the pathway that works in the wild-type strain does not function. Instead, electrons are received from the electrode by MmcX on the outer membrane surface, and then transported into the periplasm by MtrB and MtrA.

[0049] [Evaluation of transformed strains] A pair of electrodes (working electrode: graphite felt (2.25 cm)) connected via a potentiostat 2 The transformed strains were evaluated using a microbial electrolysis cell consisting of a primary electrode (a platinum wire (10 cm)), a counter electrode (a platinum wire), and a reference electrode (Ag / AgCl (saturated KCl)) immersed in an electrolyte (inorganic salt medium (pH 7.4)).

[0050] First, the ΔomcAΔmtrC (pHSGmmcX) strain was analyzed at OD 600The microbial cells were inoculated into the electrolyte so that the ratio of the microbial activity to the total microbial activity was 0.1. The current density was then measured over time at an applied potential of -0.4 V vs. SHE. Two hours after the start of the measurements, approximately 20 mM fumaric acid was added dropwise as an electron acceptor. Fumaric acid was converted to succinic acid through a microbial electrosynthesis reaction (MES) using Shewanella oneidensis. The current density was measured for 20 hours after the start of the measurements. The amount of succinic acid produced was also measured 20 hours after the start of the measurements.

[0051] The MR-1(pHSG298) strain and the ΔomcAΔmtrC(pHSG298) strain were also evaluated using the same procedures.

[0052] Figure 3 shows the current density measurements of a Shewanella oneidensis wild-type strain (MR-1(pHSG298)), a Shewanella oneidensis mutant strain lacking OmcA and MtrC (ΔomcAΔmtrC(pHSG298)), and a Shewanella oneidensis transformant lacking OmcA and MtrC and carrying MmcX (ΔomcAΔmtrC(pHSGmmcX)). As shown in Figure 3, the current density increased significantly when OmcA and MtrC were replaced with MmcX.

[0053] Figure 4 is a graph showing the results of measuring the amount of succinic acid produced by a wild-type Shewanella oneidensis strain (MR-1(pHSG298)) and a Shewanella oneidensis transformant (ΔomcAΔmtrC(pHSGmmcX)) in which OmcA and MtrC were deleted and MmcX was introduced. As shown in Figure 4, the amount of succinic acid produced increased in response to an increase in current density. [Sequence List Free Text]

[0054] SEQ ID NO: 1: Amino acid sequence of multihemocytochrome C (MmcX original) with the signal peptide sequence deleted SEQ ID NO: 2: Amino acid sequence in which the signal peptide sequence of the MtrC protein of Shewanella oneidensis MR-1 strain has been added to the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 (MmcX) SEQ ID NO: 3: A base sequence encoding the amino acid sequence shown in SEQ ID NO: 2 SEQ ID NO: 4: Amino acid sequence of multihemocytochrome C (MmcX original) SEQ ID NO: 5: Signal peptide sequence of MtrC protein of Shewanella oneidensis strain MR-1

Claims

1. An expression vector having a base sequence of a nucleic acid encoding a protein having an amino acid sequence with 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and having multi-heme cytochrome C activity, and one or more regulatory sequences operably linked to the base sequence.

2. A microorganism transformed with the expression vector of claim 1.

3. The microorganism according to claim 2, wherein the multiheme cytochrome C activity inherent in the microorganism is deleted or reduced.

4. Providing a microbial electrolysis cell having at least one pair of electrodes with the microorganism of claim 2 or 3 disposed on the cathode and a solution in which the electrodes are immersed; setting the cathode to a low potential; supplying electrons from the cathode to the microorganism to carry out a reduction reaction within the microorganism to biosynthesize a useful substance.

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