Cell having intracellular transmission mechanism for extracellular chemical environment, and use of said cell

Abstract

The whole-cell sensor, which uses the whole microorganism as a sensor, is very promising technology but has the significant technical problem of a concentration difference between inside and outside the cell membrane.　In the present invention, it is confirmed that, in the cell system: a molecule recognition protein A is fused to an extracellular loop of a membrane protein B through a linker C, and the molecule recognition protein A is presented in the periplasmic space; and the function of the membrane protein B is improved by stabilisation due to recognition of a ligand D by the molecule recognition element A. As a result, it is possible to provide, as a whole-cell sensor, a cell having the function of transmitting molecule recognition information regarding the outside of the cell, such as the periplasmic space, into the cell.

Description

Cells having intracellular transmission mechanism of extracellular chemical environment and uses of said cells

[0001] The present invention relates to cells having the function of transmitting the extracellular chemical environment (particularly the abundance of chemical molecules) into the cell, and uses of the cells.

[0002] In the field of synthetic biology, a "whole cell sensor" that uses the entire microorganism as a sensor is considered promising (Non-Patent Document 1). The greatest features of the whole cell sensor are its portability and flexibility. -15 All the functions required for the sensor (detection → transmission → output) are contained in a bag with an internal volume of 1 L. Furthermore, the sensor does not require a power source to operate, and output formats such as fluorescence, chemiluminescence, and oxidation-reduction reactions coupled with electrode reactions can be freely selected. For this reason, vigorous efforts are being made to expand the lineup of whole cell sensors that do not require equipment for fieldwork and environmental monitoring (Non-Patent Documents 2, 3, and 4).

[0003] Many molecular machines that "transmit external information to the interior" are known in nature. Examples include G-protein coupled receptors (GPCRs) and histidine kinases (HKs) in two-component systems. Both are transmembrane proteins. The protruding portion of the membrane protein contains a domain that specifically recognizes target molecules. Interaction between this domain and the target molecule triggers an allosteric conformational change in the membrane protein. This then triggers a structural change in the domain on the opposite side of the membrane, the cytoplasmic side. In the case of GPCRs, changes in the interaction properties with G-proteins, arrestins, and other proteins trigger a cascade of intracellular gene expression. In two-component systems, target recognition on the exterior of the cell exposes a phosphorylation domain in the intracellular domain, which then triggers phosphorylation of the target molecule to a partner response regulator, thereby turning the signal transduction pathway on and off.

[0004] Eukaryotic organisms, such as yeast and mammalian cells, possess their own GPCR cascades. By overexpressing heterologous GPCR proteins in these organisms, the endogenous GPCR cascades are "hijacked" by the overexpressed GPCRs, allowing them to respond to the heterologous GPCR's target molecules. The resulting eukaryotic cells expressing heterologous GPCRs function as whole-cell sensors for specific target molecules (Non-Patent Documents 5 and 6). However, because this biosensor utilizes the endogenous cascades directly, crosstalk with endogenous GPCRs is inevitable.

[0005] Techniques for displaying / anchoring proteins such as antibodies to the periplasm of Escherichia coli via membrane proteins have been reported (Non-Patent Document 7, Patent Document 1). However, the membrane proteins in these examples were only used as scaffolds to display proteins on the cell surface, and lacked a mechanism for transmitting extracellular interactions into the cell.

[0006] Special Publication No. 2021-505675

[0007] Sensors, 17(7), 1623, (2017)Biosens. Bioelectron., 15, 70, 482-90 (2015)Biotechnol. J., 13(1), (2018)Analyst, 143, 630-634 (2018)Curr. Chem.Genom., 4, 84-91 (2010)Cell, 177, 782-796 (2019)PNAS, 101, 25, 9193-9198 (2004)

[0008] Whole cell sensors face a significant technical challenge: the concentration difference between the inside and outside of the cell membrane. Because most whole cell sensors use sensor proteins inside cells, the target substance is detected inside the cell. This type of sensor has difficulty detecting molecules that cannot pass through the cell membrane, such as metal ions and highly water-soluble molecules. Even if a target molecule can be forcibly drawn into the cell using a transporter, the concentration inside the cell will be far different from the original concentration in the measurement sample (extracellular). Furthermore, once inside the cell, the target molecule is diluted among thousands of similar contaminant molecules, making accurate detection of its concentration difficult.

[0009] To overcome the significant drawbacks of whole-cell sensors, the inventors focused on the periplasmic space of bacteria such as Escherichia coli. This space is surrounded by outer and inner membranes. The intracellular membrane has low molecular permeability, blocking the passage of highly water-soluble and ionic molecules (logP < 3). This space is isolated from the intracellular metabolic network. On the other hand, the outer membrane contains numerous porins, ensuring free movement of compounds with molecular weights of 600 or less (Reference 1: BiochimBiophys Acta. 2009 May;1794(5):808-16.). The periplasmic space is an environment similar to the extracellular environment.

[0010] The present inventors (1) fused a molecular recognition protein A to an extracellular loop of a membrane protein B via a linker C in a cell system, and confirmed that the molecular recognition protein A was presented in the periplasmic space. Furthermore, they confirmed that the function of the membrane protein B was improved by stabilization following recognition of a ligand D by the molecular recognition protein A. Furthermore, the present inventors (2) fused a molecular recognition protein A to an extracellular loop of a membrane protein B via a linker C and fused a reporter protein E to the membrane protein B in a cell system, and confirmed that the molecular recognition protein A was presented in the periplasmic space. Furthermore, they confirmed that the function of the reporter protein E was improved by stabilization following recognition of a ligand D by the molecular recognition protein A. Based on the above, the present inventors confirmed that a cell capable of transmitting information (particularly molecular recognition information) from outside the cell (particularly the periplasmic space) into the cell had been completed.

[0011] That is, the present invention is as follows: 1. A cell that expresses or is capable of expressing the following fusion protein, and that has the function of transmitting molecular recognition information related to the amount, concentration, or change in amount of a target chemical molecule outside the cell into the cell via the fusion protein, wherein the fusion protein comprises a molecular recognition protein A, a membrane protein B, and a linker C, wherein the molecular recognition protein A is fused to an extracellular loop of the membrane protein B via the linker C, and the molecular recognition protein A is displayed extracellularly, and the fusion protein is unstable when it does not recognize ligand D by the molecular recognition protein A, and the fusion protein is stable when it recognizes ligand D by the molecular recognition protein A, and this stabilization improves the protein folding ability of the molecular recognition protein A and the membrane protein B, and further improves the function of the membrane protein B. Note that the "cell" of the present invention is typically a genetically modified cell, but may also be a cell created using genome editing technology. Furthermore, "being in an unstable state when the molecular recognition protein A does not recognize ligand D" refers to a state in which the molecular recognition protein A is unable to perform its inherent function due to abnormal protein folding of molecular recognition protein A and / or membrane protein B, and "stable state" refers to a state in which the molecular recognition protein A is able to perform its inherent function. Furthermore, "improved function of membrane protein B" refers to a state in which the membrane protein B is able to perform its inherent function. For example, in the case of a typical membrane protein such as TetA, which has a function that can confer drug resistance to antibiotics or the like, the growth rate in the presence of a toxic drug serves as an indicator of functional improvement. In other words, the cells described here are cells in which a molecular recognition protein A such as an enzyme, antibody, or receptor is linked to the extracellular loop of membrane protein B via linker C to present the fusion protein, thereby utilizing the recognition event of molecular recognition protein A for ligand D to regulate the ON / OFF function of membrane protein B, and can be said to function as a "whole cell sensor."Examples of molecular recognition proteins A include enzymes (oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, and translocases), metal-binding proteins, small molecule-binding proteins, protein-binding proteins, and proteins with molecular recognition capabilities for receptors and multicomponent signal transduction systems, as well as proteins with small molecule or protein-binding motifs present in proteins and peptides with small molecule or protein-binding capabilities. Typical examples of enzymes include, but are not limited to, alkaline phosphatase, galactosidase, glucuronidase, peroxidase, lipase, acyl-CoA ligase, nuclease, luciferase, glucose oxidase, and urease. Metal-binding proteins include, but are not limited to, the zinc-binding protein ZinT, the lanthanide-binding protein Lanmodulin, the calcium-binding protein Calmodulin, the nickel-responsive transcription factor NikR, and the copper-responsive transcription factor CueR. Small molecule binding proteins and their motifs may include, but are not limited to, glutamate binding protein, maltose binding protein, ATP synthase subunit, arsenic binding peptide, retinoic acid receptor, etc.

[0012] Combinations of molecular recognition proteins A and ligands D will be obvious to those skilled in the art, but typical combinations may include, but are not limited to, phosphorylated compounds such as alkaline phosphatase and BCIP (5-bromo-4-chloro-3-indolyl-phosphate), as well as phosphate and phosphite, glucuronidase and glucuronic acid-modified compounds, acyl-CoA ligase and organic acids, luciferase and luciferin, glucose oxidase and glucose, urease and urea, ATP synthase subunits and ATP, glutamate-binding proteins and glutamate, arsenic-binding peptides and arsenic, Lanmodulin and NdCl3, and retinoic acid receptors and retinoic acid.

[0013] Furthermore, membrane protein B can include a group of membrane proteins that have a transmembrane site and their variants.Typically, membrane protein B can include, but is not limited to, transport proteins (uniporters, symporters, antiporters, ABC transporters), channel proteins, transmembrane enzymes, photoreceptor pump proteins, photoreceptor signaling proteins, scaffold-forming membrane proteins, stress-responsive membrane proteins, stress-receptor transmembrane enzymes, signal-receptor membrane proteins, transmembrane chaperone proteins, and transmembrane division control proteins.

[0014] The "linker C" is an amino acid or polyamino acid (peptide) used to bind to the extracellular loops of the molecular recognition protein A and the membrane protein B, and is generally a sequence of 1 to 12 amino acid residues, although the number of amino acids (length) is not limited thereto. It is preferably provided at the N-terminus and C-terminus of the molecular recognition protein A, in which case it is divided into linkers C1 and C2. C1 and C2 may have the same sequence or different sequences. Alternatively, a library can be prepared in which random DNA sequences are provided as linkers C1 and C2 at both ends of the DNA encoding the molecular recognition protein A, and optimal linker mutants can be selected, for example, by utilizing the selective pressure function of membrane protein B in the medium.

[0015] 2. A cell that expresses or is capable of expressing the following fusion protein, and that has the function of transmitting molecular recognition information related to the amount, concentration, or changes in the amount, of an extracellular target chemical molecule into the cell via the fusion protein, wherein the fusion protein comprises a molecular recognition protein A, a membrane protein B, and a linker C, wherein the molecular recognition protein A is fused to an extracellular loop of the membrane protein B via the linker C and the molecular recognition protein A is displayed extracellularly, and a reporter protein E is fused to the membrane protein B with or without a linker F, wherein the reporter protein E is displayed in the cytoplasm, and the fusion protein is unstable when it does not recognize ligand D by the molecular recognition protein A, and the fusion protein is stable when it recognizes ligand D by the molecular recognition protein A, and the stabilization improves the protein folding ability of the molecular recognition protein A, the membrane protein B, and the reporter protein E, and further improves the function of the reporter protein E. In other words, the cell here presents a fusion protein in which a molecular recognition protein A, such as an enzyme, antibody, or receptor, is linked via a linker C to the extracellular loop of a membrane protein B, and a reporter protein E is further fused to the membrane protein B. This allows the recognition event of the molecular recognition protein A with respect to a ligand D to be used to regulate the ON / OFF function of the membrane protein B, and is a cell that can function as a "whole cell sensor."

[0016] Representative reporter proteins E include one or more proteins selected from the group consisting of proteins that can be fused to membrane proteins as polyamino acids, such as transcription factors, enzymes, protein-binding proteins, and functional partial structures of membrane proteins and water-soluble proteins, or their mutants. Typical transcription factors include, but are not limited to, AraC, SigV, TetR, LacI, and T7RNAP. Enzymes include, but are not limited to, Cat, FDS, CrtM, LacZ, hsvTK, and Aph. Protein-binding proteins include, but are not limited to, partial structures of RsiV.

[0017] Here, "the function of reporter protein E is improved" refers to, for example, in the case of an enzyme or a fluorescent protein, an increase in the intensity of color development or fluorescence.

[0018] 3. The cell according to preceding paragraph 1 or 2, wherein the extracellular space is the periplasmic space. Typically, the cell according to preceding paragraph 3 is the periplasmic space of Escherichia coli. 4. The cell according to preceding paragraph 3, wherein the ligand D has the property of being able to pass through the outer membrane. 5. The cell according to preceding paragraph 1 or 2, wherein the function of the membrane protein B is regulated and / or controlled depending on the presence or absence of the ligand D. 6. The cell according to preceding paragraph 2, wherein the function of the reporter protein E is regulated and / or controlled depending on the presence or absence of the ligand D. 7. The cell according to preceding paragraph 6, wherein the function of the reporter protein E is cell growth, cell death, division, biosynthesis, signal transduction, gene expression regulation, or electron transport. 8. The cell according to preceding paragraph 6, wherein the function of the reporter protein E is cell growth, cell death, division, biosynthesis, signal transduction, gene expression regulation, or electron transport. 8. A biosensor comprising the cell according to preceding paragraph 1 or 2. 9. The biosensor according to preceding paragraph 8, wherein the biosensor is for detecting the ligand D outside the cell and / or quantifying the concentration of ligand D in the environment. 10. A method for monitoring ligand D in an environment, comprising the following steps: (1) introducing the cell described in item 1 or 2 above into an environment containing ligand D, and (2) monitoring the concentration of ligand D in the environment using the function of the reporter protein E as an indicator. 11. A method for screening ligand D for molecular recognition protein A, comprising the following steps: (1) introducing the cell described in item 1 or 2 above into an environment containing a candidate compound D' for ligand D, and (2) detecting the activity of candidate compound D' for ligand D using the function of the reporter protein E as an indicator. 12. The method described in item 11 above, wherein a competitor of ligand D is present in (1).13. A fusion protein comprising a molecular recognition protein A, a membrane protein B, and a linker C, wherein the molecular recognition protein A is fused to an extracellular loop of the membrane protein B via the linker C, and the fusion protein is unstable when it does not recognize ligand D by the molecular recognition protein A, and the fusion protein is stable when it recognizes ligand D by the molecular recognition protein A, and the stabilization improves the protein folding ability of the molecular recognition protein A and the membrane protein B, and further improves the function of the membrane protein B. 14. A fusion protein comprising a molecular recognition protein A, a membrane protein B, a linker C, and a reporter protein E, wherein the molecular recognition protein A is fused to an extracellular loop of the membrane protein B via the linker C, and the reporter protein E is fused to the membrane protein B with or without a linker F, and wherein the fusion protein is unstable when it does not recognize a ligand D by the molecular recognition protein A, and the fusion protein is stable when it recognizes a ligand D by the molecular recognition protein A, and the stabilization improves the protein folding ability of the molecular recognition protein A, the membrane protein B, and the reporter protein E, and further improves the function of the reporter protein E.

[0019] The present invention provides cells that have the function of transmitting extracellular chemical information (particularly the presence or absence of proteins, enzyme substrates, receptor ligands, coenzymes, etc.) into cells, as well as various uses of the cells.

[0020] (A): Cells expressing a fusion protein containing molecular recognition protein A, membrane protein B, and linker C. (B): Cells expressing a fusion protein containing molecular recognition protein A, membrane protein B, linker C, and reporter protein E. (A) Model structure of the sensor using the crystal structure of PhoA (PDB: 1HQA) and the predicted structure of TetA by αFold2. The area between PhoA and TetA indicated by a red circle X corresponds to the random mutation site. Green circles (TG, ST) correspond to restriction enzyme sites, and yellow circles (S, T) correspond to the amino acid at the insertion point of TetA. (B) Creation of a PhoA linker library. Restriction enzyme sites (green: GGTACC-, -ACTAGT) and randomized bases (red: -NNK NNKNNK, NNKNNKNNK-) were added to the N- and C-termini of the phoA gene, respectively, and inserted into the extracellular loop of the tetA gene. (C) Selection of linker mutants exhibiting TetA and PhoA functions. (D) Functional evaluation of linker mutants selected on plates in liquid culture. Red circles represent the mutants obtained. (E) Functional evaluation of the selected linker mutants. Amino acid sequence of the linker of the linker mutant. Results showing dose-response to Tc, the PhoA-specific ligand D. (A) Model structure of the sensor using the crystal structure of LanM (PDB:6MI5) and the predicted structure of TetA by αFold2. The area indicated by the red circle X between LanM and TetA corresponds to the random mutation site. (B) Selection of linker mutants that demonstrate TetA function. (C) Results of confirmation of metal specificity of the selected linker mutants. The vertical axis shows the rate of increase in turbidity in the presence of metal, relative to the metal-free condition, set as 1. From the left, the bars represent Lin-3, Lin-4, Lin-5, Lin-6, Lin-14, and Lin-15. Results showing dose-response to NdCl3, the LanM-specific ligand D. RsiV cyto Schematic diagram of the construction of a transmission mechanism from extracellular space (permeability space) to intracellular space using V1 as a reporter. (A) RsiV cyto (B) Schematic diagram of the plasmid construct for the extracellular-to-internal transfer mechanism using V1 as a reporter. (C) RsiV using the TI reporter system. cytoSchematic diagram of the mechanism of action of the sensor using V1 as a reporter. Conversion of enzyme substrate recognition in extracellular space (perpendicular space) into transcription factor output. (A) Dose-response curve of RsiV function for the PhoA substrate BCIP. (B) Dose-response curve of RsiV function for EDTA in the presence of BCIP. (C) Dose-response curve of RsiV function for metal ions in the presence of BCIP and EDTA. AraC DBD Overview of the construction of a transmission mechanism from extracellular space (peripheral space) to intracellular space using AraC as a reporter. (A) AraC DBD Schematic diagram of the plasmid construct for the extracellular-to-internal transfer mechanism using AraC as a reporter. (B) AraC DBD Schematic diagram of the mechanism of action of the sensor using AraC as a reporter. DBD Results of conversion of enzyme substrate recognition into transcription factor output outside the cell (permeability space) using RsiV. Structural diagram of the plasmid used in each example. Structural diagram of the plasmid used in Examples 3-4. Structural diagram of the plasmid used in Example 2. Structural diagram of the plasmid used in Example 3. Structural diagram of the plasmid used in Example 3. Structural diagram of the plasmid used in Example 4. Structural diagram of the plasmid used in Example 4. Construction of an extracellular molecular sensor using TetA as a reporter and evaluation of enzyme substrate specificity RsiV cyto Construction of extracellular → intracellular transfer mechanism using V1 as a reporter. Diagram of the structure of the plasmid used in Example 6. Linker sequences of Linker-1 to Linker-3 of TetA-ZintT-RsiVcytoV1

[0021] (Summary of the Invention) The present invention relates to a cell (hereinafter sometimes abbreviated as "the cell of the invention") that has the function of transmitting the extracellular chemical environment (particularly, molecular recognition information) into the cell, and uses of the cell of the invention. The chemical environment refers to the abundance or concentration of specific chemical molecules.

[0022] (Cells of the Present Invention) The cells of the present invention are not particularly limited as long as they have the function of transmitting the extracellular chemical environment into the cell, but are at least cells that express or are capable of expressing the fusion proteins described below. Fusion protein (1): Comprises a molecular recognition protein A, a membrane protein B, and a linker C, wherein the molecular recognition protein A is fused to the extracellular loop of membrane protein B via the linker C, and the molecular recognition protein A is displayed extracellularly. Fusion protein (2): Comprises a molecular recognition protein A, a membrane protein B, and a linker C, wherein the molecular recognition protein A is fused to the extracellular loop of membrane protein B via the linker C, and the molecular recognition protein A is displayed extracellularly, and reporter protein E is fused to membrane protein B with or without a linker F, and the reporter protein E is displayed in the cytoplasm. Note that the "extracellular" in the present invention refers to the periplasmic space in the case of Gram-negative bacteria including Escherichia coli, and further when membrane protein B is an inner membrane protein. Furthermore, the cells of the present invention are not particularly limited as long as they express or are capable of expressing the above-mentioned fusion protein, and also include, for example, genetically modified cells (including prokaryotic cells and eukaryotic cells), artificial cells (e.g., pseudocells containing transcription / translation systems derived from Escherichia coli, yeast, wheat, etc., liposomes), intracellular organelles capable of expressing membrane proteins (e.g., mitochondria, endoplasmic reticulum, chloroplasts), etc. Furthermore, when the cells of the present invention do not have an endogenous membrane protein expression system, a heterologous membrane protein B gene(s) can be introduced into them.

[0023] (Operational Principle of the Present Invention) The operational principle of the present invention is shown in Figure 1. The operational principle of the present invention is as follows. Fusion protein (1) is unstable when it does not recognize ligand D by molecular recognition protein A, and is stable when it recognizes ligand D by molecular recognition protein A. This stabilization improves the protein folding ability of molecular recognition protein A and membrane protein B, and further improves the function of membrane protein B. Fusion protein (2) is unstable when it does not recognize ligand D by molecular recognition protein A, and is stable when it recognizes ligand D by molecular recognition protein A. This stabilization improves the protein folding ability of molecular recognition protein A, membrane protein B, and reporter protein E, and further improves the function of reporter protein E.

[0024] (Method for Constructing the Cell of the Present Invention) An overview of the cell of the present invention is shown in Figure 1. The cell of the present invention can be produced by any method as long as it satisfies the above-mentioned operating principle. Examples include the following: (A) First, a molecular recognition protein A of your choice is genetically fused in-frame to the extracellular loop of membrane protein B, thereby displaying it extracellularly. If the molecular recognition protein A thus displayed is moderately destabilized by genetic mutation (and the associated introduction of amino acid substitutions), it can no longer fold (form a functional structure) on its own (i.e., its folding ability is reduced). In this case, the misfolding of molecular recognition protein A also prevents the linked membrane protein B from forming a folded structure, resulting in the loss (decreased) of membrane protein B's function. On the other hand, if protein A is stabilized by ligand D, an external factor that stabilizes molecular recognition protein A (through interaction with a partner protein or binding to a small molecule that should interact as a receptor), and its folded structure is restored, the linked membrane protein B can form a structure, improving the function of membrane protein B (Figure 1A). (B) A reporter protein E is fused to the membrane protein B of the fusion protein (1) described in (A) above so that it is oriented toward the cytoplasm. In this configuration, the molecular recognition protein A is stabilized by binding to the ligand D outside the cell (periplasmic space), which enhances the function of the reporter protein E on the cytoplasmic side via the membrane protein B (Figure 1B).

[0025] (Genetic Mutation) The "genetic mutation" in the present invention can be any known mutation method (e.g., random mutation, site-specific amino acid substitution, partial deletion of amino acid residues, or insertion of a peptide or other protein). In addition, a preferred example of the mutation in the present invention is destabilization of a protein (fusion mutant). Protein stability refers to the stability of the folded state, i.e., the free energy change (ΔG fold) that occurs when the polypeptide chain constituting the protein forms a functional structure (folds). The ΔG fold of a correctly folded protein has a large negative value. "Destabilization" refers to the reduction of the ΔG fold by the "genetic mutation" defined above, bringing it asymptotically to zero (or even slightly toward ΔG fold > 0). For example, if a certain amino acid substitution reduces the stability of the functional structure (fold), the amino acid substitution is a "destabilizing mutation (destabilizing mutation)."

[0026] The capture of ligand D, the target molecule of molecular recognition protein A placed extracellularly, leads to the stabilization of fusion protein (1) or fusion protein (2). By selecting a gene expression regulator such as a transcription factor as the intracellular (cytoplasmic) reporter protein E, molecular information in the extracellular environment (concentration of ligand D) can be translated into intracellular protein expression levels. Furthermore, by placing a fluorescent protein such as GFP downstream of the reporter protein E, a sensor can be constructed that reads the concentration of ligand D based on the cellular fluorescence level. Furthermore, by placing a gene set related to physiological functions such as proliferation, cell death, and biosynthesis under control instead of GFP, any physiological function of the cell can be externally manipulated with a high degree of flexibility. The function of reporter protein E is not limited to transcription factors. Examples of reporter protein E include phosphate signaling proteins, enzymes involved in biosynthesis and biodegradation, chaperones, and electron transport. The same applies to membrane protein B. In addition to selective and active transport of substances, reporter protein E can also function as a photosynthetic protein, an electron transport protein, an enzyme, or even a sensor itself.

[0027] A key feature of the cells (sensors) of the present invention is their high flexibility. The molecular recognition protein A used as an extracellular sensor element can be either a receptor or an enzyme. Therefore, all molecules known as receptor ligands and enzyme substrates can be targeted, making it possible to create biosensors that detect a variety of small molecules. Furthermore, because molecular recognition occurs outside the cell (in the case of E. coli, in the permeabilized space) where intracellular contaminants are absent, sensing based on the inherent binding affinity of intracellular proteins is possible. Furthermore, in the operating principle of the present invention, recognition of ligand D by molecular recognition protein A improves the folding ability of membrane protein B or reporter protein E, eliminating the need to redesign the so-called allosteric mechanism.

[0028] (Molecular Recognition Protein A) The "molecular recognition protein A" of the present invention is not particularly limited as long as it can bind to membrane protein B, which will be described below, via linker C, which will be described below, and can be presented extracellularly (particularly in the periplasmic space). Molecular recognition protein A is preferably a water-soluble protein. Examples of the protein include metal-binding peptides, arsenic-binding peptides, antibodies, enzymes, and receptors. Specific examples include alkaline phosphatase, glucuronidase, acyl-CoA ligase, ATP synthase subunits, glutamate-binding protein GluBP, maltose-binding protein MalE, zinc-binding protein ZinT, and lanthanoid-binding protein Lanmodulin. In addition, molecular recognition protein A also includes a peptide motif.

[0029] (Ligand D) The "ligand D" of the present invention is not particularly limited as long as it is recognized by (e.g., binds to) the molecular recognition protein A. For example, combinations of molecular recognition protein A and ligand D include an enzyme and a substrate, a peptide motif and its target, an antibody / miniantibody and an antigen, a nuclear receptor and its ligand, etc. Specific examples include alkaline phosphatase and BCIP (5-bromo-4-chloro-3-indolyl-phosphate), glucuronidase and glucuronic acid, acyl-CoA ligase and an organic acid, luciferase and luciferin, glucose oxidase and glucose, urease and urea, ATP synthase subunit and ATP, glutamate-binding protein and glutamate, arsenic-binding peptide and arsenic, Lanmodulin and NdCl3, and a retinoic acid receptor and retinoic acid. When the host cell is a Gram-negative bacterium (with double-membrane cells) such as Escherichia coli, and membrane protein B is an inner membrane-localized protein (e.g., TetA, LacY), it is preferable that ligand D has the property of being able to pass through the outer membrane. This is because the outer membrane contains porins, which allow water-soluble molecules with a molecular weight of approximately 650 to be freely delivered to the periplasmic space. Furthermore, by modifying this porin, even larger molecules can be used as ligand D. Furthermore, the outer membrane can be removed by the action of peptidoglycan-degrading enzymes, for example. This makes it possible to apply this method to the detection of antigen-antibody reactions, etc.

[0030] (Membrane Protein B) The "membrane protein B" of the present invention includes not only known wild-type membrane protein(s) but also genetically modified membrane protein(s). Examples include rhodopsin, two-component regulatory systems, transporters, and GPCRs. Specific examples include TetA, GR (Gloeobacter-derived rhodopsin), channelrhodopsin ChR, various histidine kinases (PhoQ, RcsC, EnvZ, ArcB), transport proteins TetA, LacY, COX, aquaporins, and various GPCRs (α1B-adrenergic receptor α1BAR, adenosine A2A receptor A2AR, serotonin 2A receptor (5H2TR), dopamine D2 receptor (D2R), etc.).

[0031] (Linker C) The sequence of the "linker C" of the present invention is not particularly limited as long as it can bind the molecular recognition protein A to the extracellular loop of the membrane protein B. Furthermore, the "linker C" of the present invention may have the role of maintaining the destabilized state of the fusion protein when the molecular recognition protein A is not recognizing the ligand D. Specifically, the sequence (structure) of the linker C inhibits the normal (usual) protein folding ability of the molecular recognition protein A, or the normal (usual) protein folding ability of the molecular recognition protein A and the membrane protein B.

[0032] The "linker C" is an amino acid or polyamino acid (peptide) used to bind to the extracellular loops of the molecular recognition protein A and the membrane protein B, and is generally a sequence of 1 to 12 amino acid residues, although the number of amino acids (length) is not limited thereto. It is preferably provided at the N-terminus and C-terminus of the molecular recognition protein A, in which case it is divided into linkers C1 and C2. C1 and C2 may have the same sequence or different sequences. Alternatively, a library can be prepared in which random DNA sequences are provided as linkers C1 and C2 at both ends of the DNA encoding the molecular recognition protein A, and optimal linker mutants can be selected, for example, by utilizing the selective pressure function of membrane protein B in the medium.

[0033] (Reporter Protein E) The "reporter protein E" of the present invention is not particularly limited as long as it is bound directly or indirectly to a membrane protein and is presented intracellularly (cytoplasmically). Examples include transcription factors, signal transduction proteins, enzymes, fluorescent proteins, etc. Specific examples include RsiV, AraC, SigV, TetR, LacI, T7RNAP, EnvZ, Cat, FDS, LacZ, GFP, etc. The function of reporter protein E is not particularly limited, and examples include physiological functions such as cell proliferation, cell death, division, biosynthesis, signal transduction, gene expression regulation, and electron transport.

[0034] (State of fusion protein) The "fusion protein" of the present invention is in an unstable state when the molecular recognition protein A does not recognize the ligand D. The unstable state of the fusion protein refers to a state in which the normal protein folding ability of the molecular recognition protein A and / or membrane protein B is inhibited. The "fusion protein" of the present invention is in a stable state when the molecular recognition protein A recognizes the ligand D. The stable state of the fusion protein refers to an improved (higher) protein folding ability of the molecular recognition protein A and / or membrane protein B compared to the unstable state. Specifically, the folding energy ΔGfold value increases by taking into account the binding energy when the molecular recognition protein A binds to the ligand D, and the folding of the molecular recognition protein A is stabilized. This stabilization of the molecular recognition protein A is also essential for the folding of the fusion partner membrane protein B, and therefore the function of the membrane protein B is conditional on the molecular recognition protein A interacting with the ligand D. The method for destabilizing the fusion protein is not particularly limited as long as it can maintain the above-mentioned state. However, as described in the Examples below, the sequence (structure) of linker C can inhibit the normal (usual) protein folding ability of molecular recognition protein A or the normal (usual) protein folding ability of molecular recognition protein A and membrane protein B. Alternatively, a molecular recognition protein A mutant and / or membrane protein B mutant that have been destabilized by prior amino acid mutations may be used. As is clear from the results of the Examples below, linker F linking membrane protein B to reporter protein E is not essential. Specifically, when molecular recognition protein A is stabilized by ligand D, membrane protein B can maintain its functional structure. Furthermore, reporter protein E tethered to it can maintain its functional structure (exhibit protein activity) under conditions (cases) in which membrane protein B maintains its functional structure. Furthermore, the fusion form of molecular recognition protein A-membrane protein B-reporter protein E does not necessarily have to be expressed as a single in-frame gene (translation product) as long as it is fused in an operational state.That is, A, B, and C may be expressed as separate translation products and then fused enzymatically after translation using an intein or sortase, or chemically fused using a click reagent or condensing agent.

[0035] (Uses of the Cells of the Present Invention) As will be apparent from the Examples below, the cells of the present invention can be used for various applications (particularly as sensors) by detecting and quantifying changes in the function of membrane protein B and / or reporter protein E. Examples are described below, but are not limited to these. (1) Detection and quantification of ligand D based on changes in the activity of membrane protein B and / or reporter protein E. Specifically, it is a biosensor for detecting ligand D outside the cell and / or quantifying the concentration of ligand D in the environment. (2) Regulation and control of the function of membrane protein B and / or reporter protein E based on the presence or absence of ligand D. Specific examples of regulation and control include regulation and control of physiological functions such as cell proliferation, cell death, division, biosynthesis, signal transduction, gene expression control, and electron transport.

[0036] A method for monitoring ligand D in the environment, comprising the following steps: (1) introducing the cells of the present invention into an environment containing ligand D; and (2) monitoring the concentration of ligand D in the environment using the function of reporter protein E as an indicator. The environment is not particularly limited as long as it allows the cells of the present invention to grow, and examples thereof include plant facilities.

[0037] A screening method for a ligand D of a molecular recognition protein A, comprising the following steps: (1) placing the cell of the present invention into an environment containing a candidate compound D' for the ligand D; and (2) detecting the activity of the candidate compound D' for the ligand D using the function of the reporter protein E as an indicator. In (1), a competitor of the ligand D may be present. The activity of the candidate compound D' for the ligand D is calculated based on the improvement or decrease in the function of the reporter protein E.

[0038] (Promoter) The promoter to be introduced into the cells of the present invention is appropriately selected depending on the species of host cell used. When using bacteria as the host, any promoter may be used, as long as it can be expressed in host cells such as E. coli. Examples include promoters derived from E. coli or phages, such as the λPR promoter, PL promoter, trp promoter, and lac promoter. Artificially engineered promoters, such as the tac promoter, may also be used. When using yeast as the host, any promoter may be used, as long as it can be expressed in yeast. Examples include the gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, and AOX1 promoter. When using animal cells as the host, it is preferable that the recombinant vector be capable of autonomous replication in the cell and comprise a promoter, RNA splice site, target gene, polyadenylation site, and transcription termination sequence. An origin of replication may also be included, if desired. As the promoter, an SRα promoter, an SV40 promoter, an LTR promoter, a CMV promoter, or the like can be used, and also an early gene promoter of a cytomegalovirus, or the like may be used.

[0039] (Expression Vector) An expression vector is DNA that carries an exogenous gene into a host cell, in other words, vector DNA, which is capable of expressing a target gene in the host cell. The vector DNA is not particularly limited as long as it is replicable in the host, and is appropriately selected depending on the type of host and the intended use. The vector DNA may be vector DNA obtained by extracting naturally occurring DNA, or vector DNA from which portions of DNA other than those necessary for replication are partially deleted. Representative examples of vector DNA include vector DNA derived from plasmids, bacteriophages, and viruses. Examples of plasmid DNA include plasmids derived from Escherichia coli, Bacillus subtilis, and yeast.

[0040] (Fusion Proteins of the Present Invention) The fusion proteins of the present invention are not particularly limited as long as they have the following configuration and properties. Fusion protein (1) comprises a molecular recognition protein A, a membrane protein B, and a linker C. Note that molecular recognition protein A is fused to an extracellular loop of membrane protein B via linker C. Fusion protein (1) is unstable when it does not recognize ligand D via molecular recognition protein A. Fusion protein (1) is stable when it recognizes ligand D via molecular recognition protein A, and this stabilization improves the protein folding ability of molecular recognition protein A and membrane protein B, and further improves the function of membrane protein B. Fusion protein (2) comprises a molecular recognition protein A, membrane protein B, linker C, and reporter protein E. Note that molecular recognition protein A is fused to an extracellular loop of membrane protein B via linker C, and reporter protein E is fused to membrane protein B with or without linker F. Fusion protein (2) is unstable when it does not recognize ligand D via molecular recognition protein A. The fusion protein (2) is stable when it recognizes the ligand D via the molecular recognition protein A, and this stabilization improves the protein folding ability of the molecular recognition protein A, the membrane protein B, and the reporter protein E, and further improves the function of the reporter protein E.

[0041] The present invention will be specifically described below using examples, but the present invention is not limited to these examples. Other terms and concepts used in the present invention are based on the meanings of terms commonly used in the relevant field, and the techniques used to carry out the present invention, except for those whose sources are specifically indicated, can be easily and reliably carried out by those skilled in the art based on known literature, etc. Various analyses were performed mutatis mutandis using methods described in the instruction manuals, catalogs, etc. of the analytical instruments, reagents, and kits used. The contents of the technical literature, patent publications, and patent application specifications cited in this specification are to be construed as the contents of the present invention.

[0042] Example 1: Construction Example 1 of a Cell Containing (Expressing) a Fusion Protein Comprising Molecular Recognition Protein A, Membrane Protein B, and Linker C In this example, cells capable of detecting (recognizing) ligand D, an extracellular (periplasmic) molecule, were constructed. The 12-span transmembrane protein tetracycline (Tc) transporter TetA (SEQ ID NO: 1) was selected as membrane protein B. TetA confers Tc resistance to Escherichia coli, and its function can be easily determined by monitoring the growth of E. coli in the presence of Tc. The molecular recognition protein A was Escherichia coli alkaline phosphatase PhoA (SEQ ID NO: 2). PhoA has two disulfide bonds and is functional only when expressed outside the cell (periplasmic space) in E. coli. Therefore, confirmation of its functional activity confirms that PhoA is presented outside the cell (periplasmic space) and interacts with a substrate. A linker library was constructed by inserting the phoA gene, which had a linker sequence encoding a linker with three random amino acid mutations at both the N- and C-termini, into the sixth of the six extracellular loops of the TetA protein (Fig. 2A-B). From this library, we searched for linker mutants that could detect the stabilization of PhoA by substrate binding using TetA function (Fig. 2B). Colonies were grown on plates containing 5 μg / ml Tc and 100 μM BCIP, a colorimetric substrate for PhoA that forms a blue pigment upon dephosphorylation. Of these, 353 Tc-resistant colonies were formed, and 48 colonies were selected that showed sufficient PhoA activity (forming a blue pigment) (Fig. 2C). Selected colonies were cultured in LB liquid medium in the presence of 5 μg / ml Tc in the presence or absence of 100 μM BCIP to screen for mutants that exhibited TetA function in response to the PhoA substrate. Eleven mutants exhibited a BCIP-dependent increase in turbidity of at least twofold (Fig. 2D). Plasmids from these mutants were recovered and re-functionalized under the same conditions. Four mutants, Lin-27, Lin-33, Lin-43, and Lin-46 (Fig. 3), exhibited a turbidity increase of at least twofold in the presence or absence of BCIP (Fig. 2E).We confirmed that cells containing any of these four linker mutants (fusion proteins (protein groups) containing molecular recognition protein A, membrane protein B, and linker C) function as sensors of TetA (membrane protein B) in response to a substrate of PhoA (molecular recognition protein A).

[0043] To evaluate the function of PhoA linker mutants Lin-27, 33, 43, and 46 as sensors for the extracellular (periplasmic) molecule ligand D, we examined their dose-response to their substrate, BCIP (Fig. 4). E. coli expressing each linker mutant was incubated in liquid media containing 3, 4, or 5 μg / ml Tc, with BCIP at a maximum concentration of 400 μM (1.6-fold serial dilution series), and growth was measured. Lin-3, a LanM mutant that responds to Nd, was used as a negative control (see Example 2 below). The PhoA linker mutant Lin-27 increased turbidity at 5 μg / ml Tc in a BCIP concentration-dependent manner, with a maximum signal-to-noise (S / N) ratio of 28.5 (Fig. 4). Lin-33, 43, and 46 showed a BCIP concentration-dependent increase in turbidity in the presence of 3 and 4 μg / ml Tc, with the maximum signal-to-noise ratios of 18.7-fold for Lin-33 at 3 μg / ml Tc, 30.5-fold for Lin-43 at 4 μg / ml Tc, and 32.6-fold for Lin-46 at 4 μg / ml Tc. The negative control, a linker mutant of LanM, showed no response to BCIP at any Tc concentration.

[0044] From the above results, we confirmed that cells expressing a fusion protein containing protein A (PhoA), membrane protein B (TetA), and linker C with a mutation that connects the two proteins together are dose-responsive biosensors for BCIP, a ligand D specific to PhoA.

[0045] Example 2: Construction of Cells Containing (Expressing) a Fusion Protein Comprising Molecular Recognition Protein A, Membrane Protein B, and Linker C. In this example, we attempted to construct a sensor using the lanthanoid-binding protein lanmodulin LanM (SEQ ID NO: 3) derived from Methylobacterium extorquens as molecular recognition protein A. A linker library was created in which LanM was inserted into the sixth periplasmic loop of TetA together with a library linker (Figure 5A). From this library, we searched for linker mutants that exhibit TetA function in response to the lanthanoid neodymium (Nd). On plates containing 3 μg / mL Tc and 100 μM NdCl3, 180 colonies exhibiting Tc resistance were formed, of which 96 were randomly selected. The selected colonies were cultured in LB liquid medium containing 3 μg / mL Tc with or without 100 μM NdCl3, and turbidity was compared. Nd ３＋ Six mutants (Lin-3, Lin-4, Lin-5, Lin-6, Lin-14, and Lin-15; Figure 3) were selected that showed a 1.2-fold or greater increase in turbidity depending on the linker (Fig. 5B). Plasmids from these mutants were recovered and retransfected into E. coli for colony formation. To confirm metal selectivity, colonies were picked and reanalyzed for Tet A function under five conditions: 3 μg / ml Tc, 100 μM NdCl3, 50 μM EuCl3, 100 μM YCl3, 100 μM CaCl2, and no metal. These results confirmed that cells containing any of the linker mutants (fusion proteins containing molecular recognition protein A, membrane protein B, and linker C) functioned as sensors of TetA (membrane protein B) in response to the substrate of LanM (molecular recognition protein A) (Fig. 5C).

[0046] To evaluate the sensor function of LanM's Lin-3, we examined its dose response to NdCl3 (Figure 6). E. coli expressing Lin-3 was grown in liquid media containing 2, 3, or 4 μg / ml Tc at eight dilutions (3-fold serial dilutions) with a maximum NdCl3 concentration of 1000 μM. PhoA's Linker-27, which responds to BCIP, was used as a negative control. Lin-3 increased turbidity in a NdCl3 concentration-dependent manner at each Tc concentration, with the maximum signal-to-noise ratio reaching 11.1-fold at 3 μg / ml Tc. The negative control, PhoA's Linker-27, did not exhibit NdCl3-dependent growth at any Tc concentration.

[0047] These results demonstrate that cells expressing a fusion protein containing LanM (protein A), TetA (membrane protein B), and a mutated linker C linking the two proteins together function as a biosensor that exhibits dose-responsiveness to Nd (NdCl), a LanM-specific ligand D (Figure 6). The amino acid sequences of the linker mutants obtained in Examples 1 and 2 are shown in Figure 3.

[0048] (Example 3) Example 1 of Construction of a Cell Containing (Expressing) a Fusion Protein Comprising a Molecular Recognition Protein A, a Membrane Protein B, a Linker C, and a Reporter Protein E (3-1) In this example, to confirm whether a molecule (ligand D) capture event by a receptor protein (molecular recognition protein A) outside the cell can be transmitted to the inside of the cell, an extracellular molecule detection sensor was constructed using Bacillus subtilis RsiV as the intracellular protein, reporter protein E. RsiV is an anti-sigma factor that specifically inhibits the function of SigV, one of the ECF sigma factors that interact with RNA polymerase and control gene expression. This SigV capture requires the intracellular domain of RsiV, RsiV cyto Only the RsiV (SEQ ID NO: 4) is required. cyto RsiV with destabilizing mutations (K13E, V15D) cytoA sensor plasmid carrying a protein gene in which V1 was fused to the C-terminus of the linker mutant Lin-27 (see Example 1) was constructed (Figure 7A). cyto To evaluate the function of V1, we constructed a reporter plasmid carrying stably expressed SigV, sfGFP expressed by SigV, and stably expressed mCherry in the opposite orientation to sfGFP (Fig. 7A). cyto When V1 is functional, intracellularly expressed SigV is captured and transcription of sfGFP from the SigV-regulated promoter is repressed, resulting in a decrease in the green fluorescent signal. This reporter system also employs a signal inversion circuit using transcriptional interference (TI). Briefly, mCherry is expressed from a constitutive promoter, but in the opposite direction to sfGFP. Therefore, when RsiV is inactive, transcription from the SigV-regulated promoter triggers TI, repressing mCherry transcription. On the other hand, when RsiV is functional, transcription from the SigV-regulated promoter is repressed, relieving TI. This results in increased expression of mCherry from the constitutive promoter, resulting in an increase in the red fluorescent signal. In this way, in the presence of a PhoA substrate (ON condition), the OFF signal, a decrease in sfGFP fluorescent signal, is reversed to the ON signal, an increase in mCherry fluorescent signal (Figure 7B).

[0049] To investigate whether substrate-mediated stabilization of PhoA could be interpreted as RsiV function, we examined the dose response of wild-type and mutant PhoA to BCIP, a substrate. The sensor and reporter plasmids shown in Figure 7A were introduced into E. coli and allowed to form colonies. Three colonies were selected from these and analyzed for RsiV function in liquid medium at eight dilutions (2-fold serial dilutions) with BCIP at a maximum concentration of 400 μM. In addition to the wild-type, we also analyzed the D153G mutant, which exhibits increased enzyme activity; the D153A mutant, which exhibits increased substrate affinity; and the R166S mutant, which exhibits decreased enzyme activity and substrate affinity. The wild-type mutant exhibited the best response, with mCherry / GFP levels more than doubled at 200 μM BCIP, and the signal-to-noise ratio was up to 4.3-fold higher than at 3.125 μM BCIP (Figure 8A). We also confirmed that the response curve changed with the introduction of mutations into PhoA. These results indicate that the binding event between the enzyme and the substrate that occurred extracellularly was transmitted to the intracellular domain via the membrane protein and converted into a change in gene expression. In addition, they also demonstrate that it is possible to read out changes in the profile of the enzyme itself, such as changes in stability due to mutations in the enzyme displayed extracellularly. More specifically, this means that the cells in which the fusion protein of the present invention is expressed or capable of expression are not limited to a specific molecular recognition protein A. Furthermore, R siV, a reporter protein E fused to the C-terminus of TetA, a membrane protein B, is expressed. cyto V1 is a protein that inhibits the activity of the transcription factor SigV, which is expressed in the cytoplasm. cyto V1 must be present in the cytoplasm in order for reporter protein E to function. In this example, the result that SigV activity was suppressed in a ligand D-dependent manner (GFP fluorescence decreased and mCherry fluorescence increased) indicates that reporter protein E, RsiV, is displayed in the cytoplasm.

[0050] (3-2) PhoA is a metalloprotein and requires metal ions (zinc and magnesium) for functional conformation. Therefore, chelating environmental metal ions with a chelating agent, thereby rendering PhoA unavailable, inhibits PhoA functional conformation. To confirm whether this could be read out by the function of the reporter protein E, RsiV, we examined the dose-response of the chelating agent EDTA. RsiV function was analyzed in a liquid medium containing 400 μM BCIP, with eight dilutions (2-fold serial dilutions) containing EDTA at a maximum concentration of 400 μM. The mCherry / GFP values ​​decreased in an EDTA concentration-dependent manner (Figure 8B). This indicates that the chelating agent reduced the environmental metal ion concentration, preventing PhoA from binding to its substrate, resulting in the inability to form a functional conformation and the loss of RsiV function.

[0051] (3-3) By adding excess metal ions in the presence of EDTA, TetA-linker-PhoA-RsiV was synthesized in a metal ion concentration-dependent manner. cyto Assuming that the formation of functional V1 structure can be read out by the function of the reporter protein E, RsiV, we examined the dose-response to metal ions in the presence of BCIP and EDTA. In a liquid medium containing 400 μM BCIP and 400 μM EDTA, ZnCl ２ and MgCl ２ We analyzed the function of reporter protein E, RsiV, using eight metal solutions with a 2:1 ratio (2-fold serial dilution series) with a maximum concentration of 1000 μM. We confirmed that the mCherry / GFP expression level increased depending on the metal ion concentration (Fig. 8C). This indicates that metal ion-dependent PhoA functional conformation can be read out as the function of reporter protein E, RsiV.

[0052] From the above results, it was confirmed that cells expressing a fusion protein containing PhoA (protein A), TetA (membrane protein B), a mutated linker linking the two proteins, and RsiV (reporter protein E) are biosensors that exhibit dose-responsiveness to BCIP (ligand D) specific to PhoA. In addition, in this example, RsiV (reporter protein E) cyto V1 and SigV are proteins derived from other organisms, and express fluorescent proteins from promoters specifically controlled by SigV. These proteins are not affected by other proteins in the host E. coli, so crosstalk does not occur.

[0053] Example 4: Construction Example 2 of a Cell Containing (Expressing) a Fusion Protein Containing Molecular Recognition Protein A, Membrane Protein B, Linker C, and Reporter Protein E In this example, a sensor was constructed using the transcription factor AraC as a reporter to confirm whether a protein other than RsiV can be used as the cytoplasmic reporter protein E. AraC is an intracellular transcription factor with a total length of 291 amino acids and consisting of two domains, a substrate-binding domain (LBD) and a DNA-binding domain (DBD). Upon activation, the DBD regulates (parameterizes) the transcription of the cytoplasmic reporter protein E. BAD ) promoter, transcription occurs, and the gene is expressed. A sensor plasmid carrying a protein gene was constructed in which only the DBD portion of AraC (from positions 168 to 291) was fused to the C-terminus of Lin-27 of TetA+PhoA (Fig. 9A). In addition, the reporter plasmid contained a protein gene encoding Para, which is positively regulated by AraC. BAD7 The sfGFP gene is under the control of the AraC promoter. DBD When AraC (SEQ ID NO: 5) is functionally expressed, sfGFP is expressed and emits green fluorescence (Figure 9A-B). DBDHowever, to investigate whether stabilization by binding of PhoA to the substrate could be read out, we examined the dose response to BCIP. Using the plasmid shown in Figure 9A, E. coli JW0063, which lacks the endogenous araC gene, was transformed and colonies were formed. Three colonies were picked from the transformed cells and diluted in liquid medium with BCIP at a maximum concentration of 400 μM (2-fold serial dilution series) to form eight colonies. DBD As a result, both the wild type (WT) and the mutant (Mut: SEQ ID NO: 6) that was adjusted to improve TetA function showed a BCIP concentration-dependent increase in sfGFP fluorescence (Figure 10). DBD It was also shown that the binding events between PhoA and substrate occurring outside the cell can be read out in this system.

[0054] From the above results, we confirmed that cells expressing a fusion protein containing protein A PhoA, membrane protein B TetA, linker C with a mutation that connects the two proteins, and reporter protein E AraC are dose-responsive biosensors for PhoA-specific ligand D BCIP.

[0055] RsiV used as reporter protein E of the fusion protein in Example 3 and this Example cyto V1 and SigV are characterized by their ability to function as biosensors without introducing mutations into the linker C that connects membrane protein B (without being limited by the linker sequence). Specifically, membrane protein B can maintain its functional structure when stabilized by the recognition of ligand D by molecular recognition protein A. Furthermore, we confirmed that reporter protein E tethered to it can maintain its functional structure (exhibit protein activity) under conditions (cases) in which membrane protein B maintains its functional structure.

[0056] As described above, cells expressing or capable of expressing the fusion protein of the present invention can be used to search (screen) for ligand D that binds to molecular recognition protein A, to search for molecular recognition protein A mutants that bind more strongly to ligand D, and to search for molecular recognition protein A mutants that bind to ligand D even in the presence of a competing substance.

[0057] In this example, the reporter protein E is RsiV. cyto V1 and SigV are proteins derived from other organisms, and express fluorescent proteins from promoters specifically controlled by SigV. These proteins are not affected by other proteins in the host E. coli, so crosstalk does not occur.

[0058] Example 5: Evaluation of the substrate specificity of E. coli cells containing the linker mutant Lin-27. In this example, we evaluated the substrate specificity of E. coli cells expressing Lin-27, one of the TetA::PhoA linker mutants selected in Example 1. To evaluate the substrate specificity, the PhoA phosphorylation substrates BCIP, p-nitrophenylphosphate (pNPP), and pyridoxal phosphate (PLP) were added to liquid media containing the Lin-27 mutant, and the dose response to each phosphorylation substrate was compared (Figure 18). Specifically, E. coli cells expressing the Lin-27 mutant were diluted with 5, 7.5, or 10 μg / ml of tetracycline (Tc) at eight points (1.6-fold serial dilutions) for each substrate, with a maximum PhoA concentration of 500 μM. Growth of the E. coli cells was measured.

[0059] As a result, BCIP induced a concentration-dependent increase in turbidity at all Tc concentrations, with a maximum S / N ratio of 12-fold at a Tc concentration of 7.5 μl / ml. At 5 μg / ml of Tc, a concentration-dependent increase in turbidity was also observed in response to pNPP, with a S / N ratio of 6-fold. However, no response was observed to PLP. Thus, we demonstrated that by using a membrane protein with cytotoxicity resistance, such as TetA, as membrane protein A, we can provide a "whole cell sensor" that can indicate changes in the extracellular environment using the cell growth level as an indicator.

[0060] Example 6: Construction of a metal sensor using ZinT as molecular recognition protein A: Extracellular-to-intracellular transfer mechanism of Cd using ZinT as a sensor element In this example, we attempted to construct a metal sensor using the zinc-binding protein ZinT (SEQ ID NO: 8) derived from Escherichia coli as water-soluble protein A. ZinT is a periplasmic protein involved in the uptake of zinc present in the external environment, but is known to bind more strongly to cadmium (Reference 2: BBRC, 364: 66-71 (2007)). The zinT gene was inserted in-frame into the sixth periplasmic loop of TetA using a library linker. This TetA contained an RsiV site at its C-terminus, similar to the TetA::PhoA used above (Example 1). cyto The library was then transformed into E. coli carrying a reporter plasmid, and mutants exhibiting RsiV function in response to zinc were screened. Colonies were grown in LB solid medium containing 300 μM ZnCl2, and 88 colonies exhibiting yellow to red fluorescence were randomly selected using the "TI-type" reporter system. The selected colonies were then cultured in LB liquid medium with or without 300 μM ZnCl2, and RsiV activity (PSigV transcriptional repression ability, i.e., enhanced mCherry / GFP fluorescence ratio (Figure 19)) was compared. Four linker mutants were found to exhibit ZnCl2-dependent RsiV activity increased by more than 1.2-fold (Figure 19B).

[0061] Plasmids from three of these linker mutants (Figure 21) were recovered and reintroduced into E. coli carrying the TI reporter system to allow colony formation. Grown colonies were picked and their metal responsiveness was retested. ZnCdCl2, CuCl2, and CdCl2 were diluted at eight points (1.6-fold serial dilutions) with a maximum concentration of 500 μM. Linker-2 and Linker-3 showed a slight dose-dependent increase in RsiV activity in response to ZnCl2, with a maximum S / N ratio of 1.2-fold. None of the linker mutants responded to CuCl2. Furthermore, all linker mutants showed a dose-dependent increase in RsiV activity in response to CdCl2. In particular, Linker-3 showed a maximum S / N ratio of 1.8-fold at 300 μM (Figure 19C-E).

[0062] As described above, we have demonstrated that by using ZinT, a zinc-binding protein known to bind strongly to Cd, as a sensor element, we can construct a metal sensor that, upon binding to an extracellular metal (Cd), activates an intracellular signaling mechanism, activating the drug release function of TetA and the RsiV function (which captures SigV, which is necessary for PSigV transcription initiation). In other words, we have provided a "whole cell sensor" in which ZinT is the sensor element displayed extracellularly and RsiV is the output.

[0063] The present invention can provide cells that have the function of transmitting extracellular information (particularly the presence or absence of proteins, enzyme substrates or ligands, coenzymes, etc.) into cells, as well as various uses of the cells.

Claims

1. A cell in which the following fusion protein is expressed or capable of being expressed, and which has the function of transmitting molecular recognition information related to the amount / concentration or change in the amount / concentration of a target chemical molecule outside the cell into the cell via the fusion protein, wherein the fusion protein comprises a molecular recognition protein A, a membrane protein B, and a linker C, wherein the molecular recognition protein A is fused to an extracellular loop of the membrane protein B via the linker C, and the molecular recognition protein A is presented outside the cell, and the fusion protein is in an unstable state when it does not recognize ligand D by the molecular recognition protein A, and the fusion protein is in a stable state when it recognizes ligand D by the molecular recognition protein A, and the stabilization improves the protein folding ability of the molecular recognition protein A and the membrane protein B, and further improves the function of the membrane protein B.

2. A cell expressing or capable of expressing the following fusion protein, the cell having the function of transmitting molecular recognition information related to the amount / concentration or change in the amount / concentration of a target chemical molecule outside the cell into the cell via the fusion protein, the fusion protein comprising a molecular recognition protein A, a membrane protein B and a linker C, wherein the molecular recognition protein A is fused to an extracellular loop of the membrane protein B via the linker C and the molecular recognition protein A is presented outside the cell, and a reporter protein E is fused to the membrane protein B with or without a linker F, wherein the reporter protein E is presented in the cytoplasm, and the fusion protein is in an unstable state when it does not recognize ligand D by the molecular recognition protein A, and the fusion protein is in a stable state when it recognizes ligand D by the molecular recognition protein A, the stabilization of which improves the protein folding ability of the molecular recognition protein A, the membrane protein B and the reporter protein E, and further improves the function of the reporter protein E.

3. The cell described in claim 1 or 2, wherein the extracellular space is the periplasmic space.

4. The cell according to claim 3, wherein said ligand D has the property of being capable of passing through the outer membrane.

5. A cell according to claim 1 or 2, wherein the function of said membrane protein B is regulated and / or controlled depending on the presence or absence of said ligand D.

6. The cell described in claim 2, wherein the function of the reporter protein E is regulated and / or controlled depending on the presence or absence of the ligand D.

7. The cell according to claim 6, wherein the function of the reporter protein E is cell proliferation, cell death, division, biosynthesis, signal transduction, gene expression control or electron transport.

8. A biosensor comprising the cells according to claim 1 or 2.

9. The biosensor according to claim 8, which is a biosensor for detecting said ligand D outside a cell and / or for quantifying the concentration of ligand D in the environment.

10. A method for monitoring ligand D in an environment, comprising the steps of: (1) introducing a cell described in claim 1 or 2 into an environment containing ligand D; and (2) monitoring the concentration of ligand D in the environment using the function of the reporter protein E as an indicator.

11. A method for screening a ligand D of a molecular recognition protein A, comprising the steps of: (1) introducing the cell according to claim 1 or 2 into an environment containing a candidate compound D' for ligand D; and (2) detecting the activity of the candidate compound D' for ligand D using the function of the reporter protein E as an indicator.

12. The method of claim 11, wherein in step (1), a competitor of ligand D is present.

13. A fusion protein comprising a molecular recognition protein A, a membrane protein B and a linker C, wherein the molecular recognition protein A is fused to an extracellular loop of the membrane protein B via the linker C, and the fusion protein is in an unstable state when it does not recognize ligand D by the molecular recognition protein A, and the fusion protein is in a stable state when it recognizes ligand D by the molecular recognition protein A, and the stabilization improves the protein folding ability of the molecular recognition protein A and the membrane protein B, and further improves the function of the membrane protein B.

14. A fusion protein comprising a molecular recognition protein A, a membrane protein B, a linker C, and a reporter protein E, wherein the molecular recognition protein A is fused to an extracellular loop of the membrane protein B via the linker C, and the reporter protein E is fused to the membrane protein B with or without a linker F, and wherein the fusion protein is in an unstable state when it does not recognize ligand D by the molecular recognition protein A, and wherein the fusion protein is in a stable state when it recognizes ligand D by the molecular recognition protein A, and the stabilization improves the protein folding ability of the molecular recognition protein A, the membrane protein B, and the reporter protein E, and further improves the function of the reporter protein E.

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