Antibody mimetic molecules that can activate phosphoenolpyruvate carboxylase

Abstract

This invention provides a monobody that increases succinic acid production by microorganisms, and a method for producing succinic acid using this monobody. [Solution] The disclosure includes an antibody mimetic molecule comprising an amino acid sequence in which at least one amino acid in the amino acid sequence of the fibronectin type III domain (FN3) is modified, and which can bind to phosphoenolpyruvate carboxylase (PEPC) and activate PEPC; a polynucleotide comprising a sequence encoding the antibody mimetic molecule; a vector comprising the polynucleotide; a succinic acid-producing microorganism comprising the antibody mimetic molecule, polynucleotide, or vector; a method for producing succinic acid comprising culturing the succinic acid-producing microorganism; and a method for activating PEPC comprising acting on PEPC in the presence of the antibody mimetic molecule, polynucleotide, or vector.

Description

Technical Field

[0001] The present disclosure relates to the production of succinic acid by microorganisms.

Background Art

[0002] Recently, the production of useful substances by microorganisms has attracted attention as an environmentally friendly production process. Since succinic acid is useful as a raw material for bioplastics, flavor components, and acidulants, the production of succinic acid using various bacteria has been carried out, and in Europe and the United States, the synthesis of succinic acid using Escherichia coli has been industrialized (Non-Patent Document 1).

[0003] Phosphoenolpyruvate carboxylase (PEPC) is an enzyme present in many microorganisms, protozoa, and all plants, and irreversibly catalyzes the reaction that produces oxaloacetic acid (OAA) and inorganic phosphate from phosphoenolpyruvate (PEP) and bicarbonate ions. PEPC is an allosteric enzyme whose activity is regulated by various effectors. The inactivating effectors of PEPC are L-aspartic acid and L-malate, and the activating effectors are fructose 1,6-bisphosphate and acetyl-CoA. The physiological role of PEPC in succinic acid-producing microorganisms is to fix bicarbonate ions to PEP generated in the glycolysis system and supply OAA necessary for the citric acid cycle. When the enzymes of the glycolysis system and the citric acid cycle function actively, L-malate and L-aspartic acid accumulate in the cell, so PEPC is subject to feedback inhibition.

[0004] Escherichia coli-derived PEPC (hereinafter, EcPEPC) is a key enzyme in the synthesis of succinic acid in Escherichia coli. In 2018, it was reported that the production of succinic acid by Escherichia coli was improved due to amino acid mutations (R849S or I829S) in EcPEPC (Non-Patent Document 2). In this report, it was found that the EcPEPC mutant (R849 or I829S) is less susceptible to feedback inhibition by L-aspartic acid, and as a result, the production of succinic acid by Escherichia coli is enhanced.

[0005] A monobody is an artificially bound protein consisting of approximately 90 amino acids, using the fibronectin type III domain (FN3) as a template. The FN3 domain has an extremely stable immunoglobulin fold (antibody-like structure) and can withstand numerous amino acid mutations in the regions exposed on its structural surface (solvent-exposed regions of loops and β-strands). From a combinatorial library with diversified amino acid sequences on its structural surface, it is possible to efficiently create monobodies that specifically bind to targets through selection using phage display and yeast display (Non-Patent Literature 3). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Li C, et al., J Hazardous Materials, 401, 123414, (2021) [Non-Patent Document 2] Tokuyama K, et al., Biotech. Bioeng., 115, 1542, (2018) [Non-Patent Document 3] Koide A, et al., J Mol Biol., 415, 393, (2012) [Overview of the project] [Problems that the invention aims to solve]

[0007] This disclosure aims to provide a monobody that increases succinic acid production by microorganisms, and a method for producing succinic acid using the same. [Means for solving the problem]

[0008] In one embodiment, the present disclosure relates to an antibody-mimicking molecule comprising an amino acid sequence in which at least one amino acid in the amino acid sequence of the fibronectin type III domain (FN3) is modified, and which can bind to and activate phosphoenolpyruvate carboxylase (PEPC).

[0009] In one embodiment, the present disclosure relates to a polynucleotide comprising a sequence encoding the antibody-mimicking molecule.

[0010] In one embodiment, this disclosure relates to a vector comprising the polynucleotide.

[0011] In one embodiment, the present disclosure relates to the antibody mimetic molecule, the polynucleotide, or a succinate-producing microorganism containing the polynucleotide.

[0012] In one embodiment, the present disclosure relates to a method for producing succinic acid, which includes culturing the succinic acid-producing microorganism.

[0013] In one aspect, the present disclosure relates to a method for activating phosphoenolpyruvate carboxylase, comprising acting on phosphoenolpyruvate carboxylase in the presence of one or more antibody-mimicking molecules, one or more polynucleotides, or one or more vectors. [Effects of the Invention]

[0014] The antibody-mimicking molecules of this disclosure can bind to and activate PEPC, thereby increasing succinate production in succinate-producing microorganisms. This disclosure makes it possible to increase the yield during succinate production by microorganisms and improve the efficiency of succinate production. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows the structure of the pHBT2-EcPEPC plasmid vector used in the example. [Figure 2A]Figure 2A shows the amino acid sequence (SEQ ID NO: 24) and structure of the monobody mutant library (loop library). X indicates the introduction of random amino acids encoded by NNC mixed base codons, where N contains equal amounts of A, C, G, and T (25%). The subscript number for X indicates the length range of the introduced sequence. U indicates the introduction of random amino acids encoded by TMT mixed base codons, where M contains equal amounts of A and C (50%). [Figure 2B] Figure 2B shows the amino acid sequence (SEQ ID NO: 25) and structure of the monobody mutant library (side library). X indicates the introduction of random amino acids encoded by a mixed base codon in NNC, where N contains equal amounts of A, C, G, and T (25%). The subscript number for X indicates the range of the length of the introduced sequence. J indicates the introduction of random amino acids encoded by a mixed base codon in NWT, where W contains equal amounts of A and T (50%). Z indicates the introduction of random amino acids encoded by a mixed base codon in YWT, where Y contains equal amounts of C and T (50%), and W contains equal amounts of A and T (50%). O indicates the introduction of random amino acids encoded by a mixed base codon in RMG, where R contains equal amounts of A and G (50%), and M contains equal amounts of A and C (50%). [Figure 3] Figure 3 shows the effect of monobody Mb(PEPC_L3) on the enzyme activity of wild-type EcPEPC. The enzyme activity (specific activity) in the presence of L-aspartic acid (L-Asp) is shown. [Figure 4A] Figure 4A shows the results of epitope analysis of Mb(PEPC_L5) (indicated as Mb(L5) in the figure, and the same in Figure 4B) and Mb(PEPC_L13) (indicated as Mb(L13) in the figure) using wild-type EcPEPC (WT) or the EcPEPC mutant (N113R). Flow cytometry results showing the binding of Mb(PEPC_L5) or Mb(PEPC_L13) to wild-type EcPEPC or the N113R mutant are shown. [Figure 4B]Figure 4B shows the results of the competition analysis between Mb(PEPC_L5) and Mb(PEPC_L13). Flow cytometry results (left) showing the binding of wild-type EcPEPC to yeast presenting Mb(PEPC_L13) in the absence (WT) or presence (WT + Mb(PEPC_L5)) of Mb(PEPC_L5), and relative mean fluorescence intensity (relative MFI) (right) of WT+Mb(L5) relative to WT in the absence of Mb(PEPC_L5). [Figure 5] Figure 5 shows the effects of various single antibodies (left) or combinations of single antibodies (right) on the enzyme activity of wild-type EcPEPC. The enzyme activity in the presence of L-aspartic acid (L-Asp) is shown. The vertical axis represents the relative activity (%) with the activity at 0 mM L-Asp under each condition set as 100%. In the left graph, Sh, L5, L13, L14, L15, and L16 represent the results of Mb(Shaved)+Mb(Shaved), Mb(PEPC_L5)+Mb(Shaved), Mb(PEPC_L13)+Mb(Shaved), Mb(PEPC_L14)+Mb(Shaved), Mb(PEPC_L15)+Mb(Shaved), and Mb(PEPC_L16)+Mb(Shaved), respectively. In the right graph, Sh, L5, L13, L14, L15, and L16 represent the results of Mb(Shaved)+Mb(Shaved), Mb(PEPC_L5)+Mb(Shaved), Mb(PEPC_L13)+Mb(PEPC_L5), Mb(PEPC_L14)+Mb(PEPC_L5), Mb(PEPC_L15)+Mb(PEPC_L5), and Mb(PEPC_L16)+Mb(PEPC_L5), respectively. [Figure 6A] Figure 6A shows the effects of various single antibodies or combinations of single antibodies on the enzyme activity of wild-type EcPEPC. The enzyme activity (specific activity) in the presence of 1 mM L-aspartic acid (L-Asp) is shown. [Figure 6B] Figure 6B shows the effects of various single antibodies or combinations of single antibodies on the enzyme activity of wild-type EcPEPC. The enzyme activity (specific activity) in the presence of 10 mM L-aspartic acid (L-Asp) is shown.

Best Mode for Carrying Out the Invention

[0016] Unless otherwise specifically defined, the terms used in this specification have the meanings generally understood by those skilled in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, etc. Definitions of some terms used in this specification are described below, and these definitions take precedence over the general understanding in this specification.

[0017] Succinic acid is produced as a metabolite of the citric acid cycle. In succinic acid-producing microorganisms, oxaloacetic acid (OAA) is produced from phosphoenolpyruvate (PEP) and bicarbonate ions generated in the glycolysis pathway by the action of phosphoenolpyruvate carboxylase (PEPC). In the citric acid cycle, succinic acid is produced via L-malate and fumaric acid. PEPC is a key enzyme for succinic acid production by succinic acid-producing microorganisms and is regulated in activity by various effectors. Inhibitory effectors of PEPC are L-aspartic acid and L-malate, and activating effectors are fructose 1,6-bisphosphate and acetyl-CoA. When the enzymes of the glycolysis pathway and the citric acid cycle function actively, L-malate and L-aspartic acid accumulate intracellularly, so PEPC is subject to feedback inhibition. The antibody-mimicking molecule of the present disclosure can increase the production of succinic acid by succinic acid-producing microorganisms by activating PEPC, an allosteric enzyme that is easily inactivated.

[0018] The antibody-mimetic molecules of this disclosure include an amino acid sequence in which at least one amino acid in the amino acid sequence of the fibronectin type III domain (FN3) is modified, and can also be called monobodies. A monobody is a polypeptide consisting of approximately 90 amino acids, with FN3 as the template. The monobody consists of seven β-strands (A, B, C, D, E, F, G from the N-terminus) and six loops (AB, BC, CD, DE, EF, FG) that connect each β-strand. The BC, DE, and FG loops are located on the top side of the monobody, while the AB, CD, and EF loops are located on the bottom side. β-strands A, B, and E, and β-strands C, D, F, and G, each form a β-sheet structure by hydrogen bonding. The amino acid sequence of human FN3 is shown in SEQ ID NO: 1.

[0019] The number of amino acids modified in the amino acid sequence of FN3 is not particularly limited, as long as the antibody mimetic molecule can bind to PEPC and activate PEPC, but it is at least one and may be two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty

[0020] In one embodiment, the antibody mimetic molecule of the present disclosure (1) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 2, (2) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 3, (3) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 4, (4) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 5. (5) A polypeptide comprising or consisting of an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 6, and (6) Polypeptides comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 7 Selected from.

[0021] Polypeptides comprising or consisting of amino acid sequences having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with any of the amino acid sequences of SEQ ID NOs: 2-7, and polypeptides comprising or consisting of amino acid sequences in which 1-20, 1-15, 1-10, 1-5, 1-3, or 1-2 amino acid residues are deleted, substituted, inserted, or added to any of the amino acid sequences of SEQ ID NOs: 2-7 include polypeptides in which the amino acids in the region of SEQ ID NOs: 2-7 corresponding to the monobody loop have not been modified. A person skilled in the art can modify the amino acids in the region of SEQ ID NOs: 2-7 corresponding to the β-strand of the monobody so as to maintain the structure of the monobody.

[0022] In one embodiment, the antibody mimetic molecule of the present disclosure (1') A polypeptide comprising, or consisting of, an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 2 are the amino acid sequences of SEQ ID NO: 14, 15, and 16, respectively. (2') A polypeptide comprising, or consisting of, an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-82 of SEQ ID NO: 3 are the amino acid sequences of SEQ ID NO: 17, 18, and 19, respectively. (3') A polypeptide comprising, or consisting of, an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 4, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 4 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively. (4') A polypeptide comprising, or consisting of, an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 5, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 5 are the amino acid sequences of SEQ ID NOs: 20, 18, and 22, respectively. (5') A polypeptide comprising, or consisting of, an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 6, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 6 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively, and (6') A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 7, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 7 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively. Selected from. The 25th to 28th, 53rd to 55th, and 75th to 81st or 75th to 82nd elements of sequence numbers 2 to 7 correspond to monobody BC loops, DE loops, and FG loops, respectively.

[0023] In one embodiment, the antibody mimetic molecules of (1) to (6) and (1') to (6') are polypeptides in which the amino acid corresponding to the 30th position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is either D or S. In one embodiment, the antibody mimetic molecules of (1) to (6) and (1') to (6') are polypeptides in which the amino acid corresponding to the 30th position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is either S. In one embodiment, the antibody mimetic molecules of (1) to (6) and (1') to (6') are polypeptides in which the amino acid corresponding to the 51st position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is either P, R, or T. In one embodiment, the antibody mimetic molecules of (1) to (6) and (1') to (6') are polypeptides in which the amino acid corresponding to the 51st position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is either R.

[0024] In this specification, an amino acid or amino acid sequence of a polypeptide corresponding to a predetermined position in a predetermined sequence number means the amino acid or amino acid sequence of the polypeptide that is located at the position corresponding to that predetermined position in the predetermined sequence number when the polypeptide is aligned to the amino acid sequence of the predetermined sequence number in an optimal state (a state in which the match is maximized).

[0025] In one embodiment, the antibody-mimicking molecule of this disclosure is a polypeptide comprising or consisting of any of the amino acid sequences of SEQ ID NOs: 2 to 7.

[0026] The amino acid lengths of the antibody mimetic molecules of this disclosure may be 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 amino acids or more, or 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, or 90 amino acids or less (the upper and lower limits are independently selected). In one embodiment, the amino acid lengths of the antibody mimetic molecules of this disclosure are 70-110, 80-100, or 85-95 amino acids.

[0027] The polynucleotides of this disclosure include sequences encoding the antibody-mimicking molecules of this disclosure. In one embodiment, the polynucleotides of this disclosure are (1'') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (1) above, (2'') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (2) above, (3'') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (3) above, (4'') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (4) above, (5'') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (5) above, and (6'') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (6) above. Selected from.

[0028] In one embodiment, the polynucleotides of the present disclosure are (1''') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (1') above, (2''') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (2') above, (3''') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (3') above, (4''') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (4') above, (5''') A polynucleotide comprising or consisting of a sequence encoding the polypeptide of (5') above, and (6''') A polynucleotide comprising or consisting of a sequence encoding the polypeptide (6') above. Selected from.

[0029] In one embodiment, the polynucleotides (1'')~(6'') and (1''')~(6''') include or consist of a sequence encoding a polypeptide in which the amino acid corresponding to the 30th position of any of the amino acid sequences of SEQ ID NOs. 2~7 is either D or S. In one embodiment, the polynucleotides (1'')~(6'') and (1''')~(6''') include or consist of a sequence encoding a polypeptide in which the amino acid corresponding to the 30th position of any of the amino acid sequences of SEQ ID NOs. 2~7 is either S. In one embodiment, the polynucleotides (1'')~(6'') and (1''')~(6''') include or consist of a sequence encoding a polypeptide in which the amino acid corresponding to the 51st position of any of the amino acid sequences of SEQ ID NOs. 2~7 is either P, R or T. In one embodiment, the polynucleotides (1'')~(6'') and (1''')~(6''') include or consist of a sequence encoding a polypeptide in which the amino acid corresponding to the 51st position of any of the amino acid sequences of SEQ ID NOs. 2~7 is either R.

[0030] In one embodiment, the polynucleotides of the Disclosure include or consist of sequences having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with any of the sequences of SEQ ID NOs: 8-13, or sequences in which 1-60, 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-5, 1-3, or 1-2 bases are deleted, substituted, inserted, or added to any of the sequences of SEQ ID NOs: 8-13. In one embodiment, the polynucleotides of the Disclosure include or consist of sequences of any of the sequences of SEQ ID NOs: 8-13. The polynucleotides of the Disclosure may have sequences different from any of the sequences of SEQ ID NOs: 8-13 due to degeneracy of the genetic code.

[0031] In this specification, the term "a sequence containing" a predetermined sequence is used to mean a sequence to which one or more amino acids (in the case of an amino acid sequence) or bases (in the case of a base sequence) are added, and a sequence consisting of that predetermined sequence.

[0032] Regarding amino acid sequences or nucleotide sequences, sequence identity refers to the percentage of matching amino acid residues or nucleotides between two sequences that are optimally aligned (to the state where the match is maximized). Sequence identity is calculated using the following formula: Sequence Identity (%) = [(matching amino acid residues or nucleotides between the two sequences) / (alignment length)] × 100. Sequence identity can be calculated using programs such as FASTA and BLAST.

[0033] Modifications of amino acids and bases include deletions, substitutions, insertions, and additions of amino acids and bases. When two or more amino acids or bases are modified, each modification of an amino acid or base is independently selected from deletions, substitutions, insertions, and additions.

[0034] The antibody mimetic molecules of this disclosure can be produced by methods commonly used in polypeptide production, such as genetic engineering techniques or chemical synthesis. The binding of the antibody mimetic molecules to PEPC can be investigated by the method described in the Examples. The antibody mimetic molecules of this disclosure, when measured under the conditions described in the Examples, have a binding-dissociation constant (K) of, for example, 0.1 nM to 100 nM, or 1 nM to 100 nM. D ) may have. The ability of the antibody mimetic molecule to activate PEPC can also be investigated by the method described in the examples. Specifically, this can be investigated by measuring the enzymatic activity of PEPC using a coupling reaction catalyzed by malate dehydrogenase in or out of the presence of the antibody mimetic molecule of the disclosure. If the enzymatic activity increases in the presence of the antibody mimetic molecule of the disclosure compared to its absence, it can be said that the antibody mimetic molecule of the disclosure can activate PEPC. In some embodiments, the antibody mimetic molecule of the disclosure increases the enzymatic activity of PEPC by 1.2 to 10 times or more, for example, 1.2, 1.5, 1.8, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times or more, preferably 1.5 times or more, compared to the absence of the antibody mimetic molecule. The enzymatic activity of PEPC may be measured in the presence of a PEPC inactivating effector (e.g., L-aspartic acid). In one embodiment, the antibody-mimetic molecule of this disclosure can activate PEPC in the presence of a PEPC inactivating effector (e.g., L-aspartic acid).

[0035] Succinate production in succinate-producing microorganisms can be enhanced by expressing the antibody mimetic molecule of the present disclosure in such microorganisms and treating them with phosphoenolpyruvate carboxylase in the presence of the antibody mimetic molecule of the present disclosure. The antibody mimetic molecule of the present disclosure can be expressed in such microorganisms by introducing the polynucleotide of the present disclosure into such microorganisms. The polynucleotide of the present disclosure can be introduced into succinate-producing microorganisms by a vector containing the polynucleotide. That is, the present disclosure includes a vector containing the polynucleotide of the present disclosure; a succinate-producing microorganism containing the antibody mimetic molecule, polynucleotide, or vector of the present disclosure; and a method for activating phosphoenolpyruvate carboxylase, which includes treating the phosphoenolpyruvate carboxylase in the presence of the antibody mimetic molecule, polynucleotide, or vector of the present disclosure.

[0036] The polynucleotides and vectors of this disclosure can be manufactured by conventional methods, such as genetic engineering techniques. The vectors of this disclosure are not particularly limited, as long as they can deliver the polynucleotides of this disclosure to a succinate-producing microorganism that is a host, so as to express the antibody-mimicking molecule of this disclosure in the said microorganism. Examples of vectors include plasmid vectors, viral vectors, and phage vectors. In one embodiment, the vector is a plasmid vector. In addition to the polynucleotides of this disclosure, the vector may include other elements such as an origin of replication for vector replication in the host, regulatory sequences such as a promoter, and sequences encoding selection markers for selection of transformants. The regulatory sequences are selected to be suitable for expression in succinate-producing microorganisms. The vectors can be introduced into succinate-producing microorganisms by heat shock, electroporation, etc. Those skilled in the art can select an appropriate vector and method of introduction based on the type of succinate-producing microorganism. The polynucleotides of this disclosure may be incorporated into the chromosome of the succinate-producing microorganism by homologous recombination or genome editing techniques, or they may exist without being incorporated into the chromosome (e.g., in the cytoplasm in a plasmid).

[0037] As succinate-producing microorganisms, those conventionally used for succinate production can be used. Examples of succinate-producing microorganisms include Escherichia coli, yeast, Corynebacteria, Bacillus subtilis, filamentous fungi, and cyanobacteria. In one embodiment, the succinate-producing microorganism is Escherichia coli. The succinate-producing organism into which the polynucleotide of this disclosure is introduced may be wild-type or genetically modified.

[0038] To enhance succinate production, one or more antibody-mimicking molecules of the present disclosure can be used. Therefore, the succinate-producing organism of the present disclosure may contain one antibody-mimicking molecule, polynucleotide, or vector of the present disclosure, or two or more antibody-mimicking molecules, polynucleotides, or vectors of the present disclosure. Using two or more antibody-mimicking molecules may further enhance succinate production than using one antibody-mimicking molecule. When using two or more antibody-mimicking molecules, it is preferable to use antibody-mimicking molecules that bind to different epitopes of PEPC, and therefore, when the succinate-producing organism contains two or more polynucleotides or vectors, it is preferable to include a polynucleotide encoding an antibody-mimicking molecule that binds to different epitopes of PEPC, or a vector containing the same.

[0039] In one embodiment, the succinic acid-producing organism of the Disclosure comprises the polypeptide of (2) and any polypeptide of (3) to (6). In one embodiment, the succinic acid-producing organism of the Disclosure comprises the polypeptide of (2) and the polypeptide of (4). In one embodiment, the succinic acid-producing organism of the Disclosure comprises the polypeptide of (2') and any polypeptide of (3') to (6'). In one embodiment, the succinic acid-producing organism of the Disclosure comprises the polypeptide of (2') and the polypeptide of (4'). In one embodiment, the succinic acid-producing organism of the Disclosure comprises a polypeptide comprising or consisting of the sequence of SEQ ID NO: 3 and a polypeptide comprising or consisting of any sequence of SEQ ID NO: 4 to 7. In one embodiment, the succinic acid-producing organism of the Disclosure comprises a polypeptide comprising or consisting of the sequence of SEQ ID NO: 3 and a polypeptide comprising or consisting of the sequence of SEQ ID NO: 5.

[0040] In one embodiment, the succinic acid-producing organism of the Disclosure comprises the polynucleotide of (2'') and any of the polynucleotides of (3'') to (6''). In one embodiment, the succinic acid-producing organism of the Disclosure comprises the polynucleotide of (2'') and the polynucleotide of (4''). In one embodiment, the succinic acid-producing organism of the Disclosure comprises the polynucleotide of (2''') and any of the polynucleotides of (3''') to (6''). In one embodiment, the succinic acid-producing organism of the Disclosure comprises the polynucleotide of (2''') and the polynucleotide of (4'''). In one embodiment, the succinic acid-producing organism of the Disclosure comprises a polynucleotide comprising or consisting of the sequence of SEQ ID NO: 9 and a polynucleotide comprising or consisting of any of the sequences of SEQ ID NO: 10 to 13. In one embodiment, the succinic acid-producing organism of the Disclosure comprises a polynucleotide comprising or consisting of the sequence of SEQ ID NO: 9 and a polynucleotide comprising or consisting of the sequence of SEQ ID NO: 11.

[0041] In one embodiment, the succinic acid-producing organism of the Disclosure comprises a vector comprising the polynucleotide of (2'') and a vector comprising any of the polynucleotides of (3'') to (6''). In one embodiment, the succinic acid-producing organism of the Disclosure comprises a vector comprising the polynucleotide of (2'') and a vector comprising the polynucleotide of (4''). In one embodiment, the succinic acid-producing organism of the Disclosure comprises a vector comprising the polynucleotide of (2''') and a vector comprising any of the polynucleotides of (3''') to (6'''). In one embodiment, the succinic acid-producing organism of the Disclosure comprises a vector comprising the polynucleotide of (2''') and a vector comprising the polynucleotide of (4'''). In one embodiment, the succinic acid-producing organism of the Disclosure comprises a vector comprising a polynucleotide comprising or consisting of the sequence of SEQ ID NO: 9 and a vector comprising a polynucleotide comprising or consisting of the sequence of SEQ ID NOs: 10 to 13. In one embodiment, the succinic acid-producing organism of the present disclosure includes a vector comprising a polynucleotide comprising or consisting of the sequence of SEQ ID NO: 9, and a vector comprising a polynucleotide comprising or consisting of the sequence of SEQ ID NO: 11.

[0042] In one embodiment, the present disclosure relates to a method for producing succinic acid, comprising culturing a succinic acid-producing microorganism containing the antibody mimetic molecule, polynucleotide, or vector of the present disclosure. The polynucleotide or vector of the present disclosure can express the antibody mimetic molecule of the present disclosure within the succinic acid-producing microorganism. By culturing the succinic acid-producing microorganism containing the antibody mimetic molecule, polynucleotide, or vector of the present disclosure under conditions suitable for succinic acid production, succinic acid can be obtained in the presence of the antibody mimetic molecule.

[0043] Those skilled in the art can select appropriate culture conditions, such as culture medium, culture temperature, and culture time, depending on the type of succinic acid-producing microorganism. The culture medium can be one commonly used for culturing succinic acid-producing microorganisms. The culture medium typically contains a carbon source, a nitrogen source, and inorganic salts, and may optionally contain other components such as amino acids and vitamins. Examples of carbon sources include glucose, fructose, galactose, lactose, sucrose, and glycerol. Examples of nitrogen sources include inorganic nitrogen sources such as ammonium salts and nitrates, and organic nitrogen sources such as soybean hydrolysates, yeast extracts, and amino acids. Examples of inorganic salts include phosphates, magnesium salts, and calcium salts. The carbon source is preferably derived from plants such as corn and sugarcane. The culture temperature is, for example, in the range of 30 to 45°C. The incubation period can be, for example, several hours (e.g., 1 to 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours), several days (e.g., 1 to 7 days, such as 1, 2, 3, 4, 5, 6, or 7 days), several weeks (e.g., 1 to 7 weeks, such as 1, 2, 3, 4, 5, 6, or 7 weeks), or longer.

[0044] Culturing can be carried out under microaerophilic or anaerobic conditions. Those skilled in the art can create microaerophilic or anaerobic conditions as appropriate. Culturing under microaerophilic conditions can be carried out, for example, by culturing while gently agitating the culture medium. Culturing under anaerobic conditions can be carried out, for example, by reducing the dissolved oxygen in the culture medium by introducing carbon dioxide or nitrogen into the medium. Culturing under aerobic conditions may be carried out prior to culturing under microaerophilic or anaerobic conditions. Culturing under aerobic conditions can increase the growth of succinic acid-producing microorganisms and increase the yield of succinic acid. In one embodiment, the method of the present disclosure includes culturing under aerobic conditions for several hours (e.g., 1 to 24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 24 hours) and culturing under microaerobic or anaerobic conditions for several days (e.g., 1 to 7 days, such as 1, 2, 3, 4, 5, 6, or 7 days), several weeks (e.g., 1 to 7 weeks, such as 1, 2, 3, 4, 5, 6, or 7 weeks), or longer.

[0045] The succinic acid produced can be recovered from the culture medium by conventional methods. Succinic acid can be separated from the culture medium by methods such as filtration, centrifugation, dialysis, and ion-exchange chromatography.

[0046] Exemplary embodiments of this disclosure are described below. [1] An antibody-mimicking molecule containing an amino acid sequence in which at least one amino acid in the fibronectin type III domain (FN3) is modified, and capable of binding to and activating phosphoenolpyruvate carboxylase (PEPC). [2] (1) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 2. (2) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 3. (3) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 4. (4) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 5. (5) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 6, and (6) A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 7. An antibody-mimicking molecule selected from the above, according to 1. [3] (1') A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 2 are the amino acid sequences of SEQ ID NO: 14, 15, and 16, respectively. (2') A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-82 of SEQ ID NO: 3 are the amino acid sequences of SEQ ID NO: 17, 18, and 19, respectively. (3') A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 4, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 4 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively. (4') A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 5, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 5 are the amino acid sequences of SEQ ID NOs: 20, 18, and 22, respectively. (5') A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 6, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 6 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively, and (6') A polypeptide comprising an amino acid sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 7, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 7 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively. An antibody mimetic molecule selected from the above, according to 1 or 2. [4] An antibody-mimicking molecule according to any one of 1 to 3, wherein the polypeptide is a polypeptide in which the amino acid corresponding to the 30th position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is D or S. [5] An antibody-mimicking molecule according to any one of 1 to 4 above, which is a polypeptide in which the amino acid corresponding to the 30th position of the amino acid sequence of any of sequence numbers 2 to 7 is S. [6] An antibody-mimicking molecule according to any one of 1 to 5, wherein the polypeptide is a polypeptide in which the amino acid corresponding to the 51st position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is P, R, or T. [7] An antibody-mimicking molecule according to any one of 1 to 6 above, which is a polypeptide in which the amino acid corresponding to the 51st position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is R. [8] An antibody-mimicking molecule according to any one of 1 to 7, comprising the amino acid sequence of any one of sequence numbers 2 to 7. [9] A polynucleotide comprising a sequence encoding an antibody-mimicking molecule as described in any of items 1 to 8 above.

[10] (1'') A polynucleotide containing a sequence encoding the polypeptide described in (1), (2'') A polynucleotide containing a sequence encoding the polypeptide of (2) above, (3'') A polynucleotide containing a sequence encoding the polypeptide of (3) above, (4'') A polynucleotide containing a sequence encoding the polypeptide of (4) above, (5'') A polynucleotide comprising a sequence encoding the polypeptide of (5) above, and (6'') A polynucleotide containing a sequence encoding the polypeptide of (6) above A polynucleotide selected from the above, according to item 9.

[11] (1''') A polynucleotide containing a sequence encoding the polypeptide of (1') above, (2''') A polynucleotide containing a sequence encoding the polypeptide of (2') above, (3''') A polynucleotide containing the sequence encoding the polypeptide (3') above, (4''') A polynucleotide containing the sequence encoding the polypeptide of (4') above, (5''') A polynucleotide comprising a sequence encoding the polypeptide of (5') above, and (6''') A polynucleotide containing the sequence encoding the polypeptide (6') above A polynucleotide according to 9 or 10, selected from the above.

[12] The polynucleotide according to any one of 9 to 11, wherein the polypeptide is a polypeptide in which the amino acid corresponding to the 30th position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is D or S.

[13] The polynucleotide according to any one of 9 to 12, wherein the polypeptide is a polypeptide in which the amino acid corresponding to the 30th position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is S.

[14] The polynucleotide according to any one of 9 to 13, wherein the polypeptide is a polypeptide in which the amino acid corresponding to the 51st position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is P, R, or T.

[15] The polynucleotide according to any one of 9 to 14, wherein the polypeptide is a polypeptide in which the amino acid corresponding to the 51st position of any of the amino acid sequences of SEQ ID NOs. 2 to 7 is R.

[16] A sequence having 80, 85, 90, 95, 96, 97, 98, or 99% or more sequence identity with any of sequence numbers 8-13, or Sequences in which 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 to 2 bases are deleted, substituted, inserted, or added in any of the sequences of sequence numbers 8 to 13. A polynucleotide according to any one of the above 9 to 15, including the above.

[17] A polynucleotide according to any one of the above 9 to 16, comprising any sequence of sequence numbers 8 to 13.

[18] A vector comprising a polynucleotide as described in any of items 9 to 17 above.

[19] A plasmid vector, as described in item 18 above.

[20] A succinate-producing microorganism comprising one or more antibody-mimicking molecules described in any of items 1 to 8 above, one or more polynucleotides described in any of items 9 to 17 above, or one or more vectors described in item 18 or 19 above. [twenty one] The aforementioned one or more antibody-mimicking molecules The polypeptides in (2) above, and Any of the polypeptides (3) to (6) above The succinic acid-producing microorganism described in item 20 above. [twenty two] The aforementioned one or more antibody-mimicking molecules The polypeptides in (2) above, and The polypeptide described in (4) above The succinic acid-producing microorganism described in item 20 above. [twenty three] The aforementioned one or more antibody-mimicking molecules The polypeptide (2') and Any of the polypeptides (3') to (6') above The succinic acid-producing microorganism described in item 20 above. [twenty four] The aforementioned one or more antibody-mimicking molecules The polypeptide (2') and The polypeptide (4') described above The succinic acid-producing microorganism described in item 20 above. [twenty five] The succinic acid-producing microorganism according to 20, wherein the one or more antibody-mimicking molecules are a polypeptide containing the sequence of SEQ ID NO: 3 and a polypeptide containing any of the sequences of SEQ ID NOs: 4 to 7.

[26] The succinic acid-producing microorganism according to 20, wherein the one or more antibody-mimicking molecules are a polypeptide containing the sequence of SEQ ID NO: 3 and a polypeptide containing the sequence of SEQ ID NO: 5.

[27] The one or more of the aforementioned polynucleotides The polynucleotides of (2'') and any of the polynucleotides (3'') to (6'') above The succinic acid-producing microorganism described in item 20 above.

[28] The one or more of the aforementioned polynucleotides The polynucleotides of (2'') and The polynucleotides of (4'') The succinic acid-producing microorganism described in item 20 above.

[29] The one or more of the aforementioned polynucleotides The (2'') polynucleotides, and Any of the polynucleotides (3''') to (6''') mentioned above The succinic acid-producing microorganism described in item 20 above.

[30] The one or more of the aforementioned polynucleotides The (2'') polynucleotides, and The polynucleotides of (4''') mentioned above The succinic acid-producing microorganism described in item 20 above.

[31] The succinate-producing microorganism according to 20, wherein the one or more polynucleotides are a polynucleotide containing the sequence of SEQ ID NO: 9 and a polynucleotide containing any of the sequences of SEQ ID NOs: 10 to 13.

[32] The succinate-producing microorganism according to 20, wherein the one or more polynucleotides are a polynucleotide containing the sequence of SEQ ID NO: 9 and a polynucleotide containing the sequence of SEQ ID NO: 11.

[33] The aforementioned one or more types of vectors A vector containing the polynucleotide (2'') described above, and A vector containing any of the polynucleotides described in (3'') to (6'') above. The succinic acid-producing microorganism described in item 20 above.

[34] The aforementioned one or more types of vectors A vector containing the polynucleotide (2'') described above, and Vector containing the polynucleotide (4'') described above The succinic acid-producing microorganism described in item 20 above.

[35] The aforementioned one or more types of vectors A vector containing the (2'') polynucleotide, and A vector containing any of the polynucleotides (3''') to (6''') above. The succinic acid-producing microorganism described in item 20 above.

[36] The aforementioned one or more types of vectors A vector containing the (2'') polynucleotide, and Vector containing the polynucleotide (4''') described above The succinic acid-producing microorganism described in item 20 above.

[37] The succinic acid-producing microorganism according to 20, wherein the one or more vectors include a vector containing a polynucleotide containing the sequence of SEQ ID NO: 9, and a vector containing a polynucleotide containing any of the sequences of SEQ ID NOs: 10 to 13.

[38] The succinic acid-producing microorganism according to 20, wherein the one or more vectors include a vector containing a polynucleotide containing the sequence of SEQ ID NO: 9 and a vector containing a polynucleotide containing the sequence of SEQ ID NO: 11.

[39] A succinic acid-producing microorganism, which is Escherichia coli, as described in any of items 20 to 38 above.

[40] A method for producing succinic acid, comprising culturing a succinic acid-producing microorganism described in any of items 20 to 39 above.

[41] A method for activating phosphoenolpyruvate carboxylase, comprising acting on phosphoenolpyruvate carboxylase in the presence of one or more antibody-mimicking molecules described in any of items 1 to 8, one or more polynucleotides described in any of items 9 to 17, or one or more vectors described in item 18 or 19.

[42] The aforementioned one or more antibody-mimicking molecules The polypeptides in (2) above, and Any of the polypeptides (3) to (6) above The method according to 41 above.

[43] The aforementioned one or more antibody-mimicking molecules The polypeptides in (2) above, and The polypeptide described in (4) above The method according to 41 above.

[44] The aforementioned one or more antibody-mimicking molecules The polypeptide (2') and Any of the polypeptides (3') to (6') above The method according to 41 above.

[45] The aforementioned one or more antibody-mimicking molecules The polypeptide (2') and The polypeptide (4') described above The method according to 41 above.

[46] The method according to 41, wherein the one or more antibody-mimetic molecules are a polypeptide containing the sequence of SEQ ID NO: 3 and a polypeptide containing any of the sequences of SEQ ID NOs: 4 to 7.

[47] The method according to 41, wherein the one or more antibody-mimicking molecules are a polypeptide containing the sequence of SEQ ID NO: 3 and a polypeptide containing the sequence of SEQ ID NO: 5.

[48] The one or more of the aforementioned polynucleotides The polynucleotides of (2'') and any of the polynucleotides (3'') to (6'') above The method according to 41 above.

[49] The one or more of the aforementioned polynucleotides The polynucleotides of (2'') and The polynucleotides of (4'') The method according to 41 above.

[50] The one or more of the aforementioned polynucleotides The (2'') polynucleotides, and Any of the polynucleotides (3''') to (6''') mentioned above The method according to 41 above.

[51] The one or more of the aforementioned polynucleotides The (2'') polynucleotides, and The polynucleotides of (4''') mentioned above The method according to 41 above.

[52] The method according to 41, wherein the one or more polynucleotides are a polynucleotide containing the sequence of SEQ ID NO: 9 and a polynucleotide containing any of the sequences of SEQ ID NOs: 10 to 13.

[53] The method according to 41, wherein the one or more polynucleotides are a polynucleotide containing the sequence of SEQ ID NO: 9 and a polynucleotide containing the sequence of SEQ ID NO: 11.

[54] The aforementioned one or more types of vectors A vector containing the polynucleotide (2'') described above, and A vector containing any of the polynucleotides described in (3'') to (6'') above. The method according to 41 above.

[55] The aforementioned one or more types of vectors A vector containing the polynucleotide (2'') described above, and Vector containing the polynucleotide (4'') described above The method according to 41 above.

[56] The aforementioned one or more types of vectors A vector containing the (2'') polynucleotide, and A vector containing any of the polynucleotides (3''') to (6''') above. The method according to 41 above.

[57] The aforementioned one or more types of vectors A vector containing the (2'') polynucleotide, and Vector containing the polynucleotide (4''') described above The method according to 41 above.

[58] The method according to 41, wherein the one or more vectors are a vector containing a polynucleotide containing the sequence of SEQ ID NO: 9, and a vector containing a polynucleotide containing any of the sequences of SEQ ID NOs: 10 to 13.

[59] The method according to 41, wherein the one or more vectors are a vector containing a polynucleotide containing the sequence of SEQ ID NO: 9, and a vector containing a polynucleotide containing the sequence of SEQ ID NO: 11.

[60] Use of one or more antibody-mimicking molecules described in any of items 1 to 8 above, one or more polynucleotides described in any of items 9 to 17 above, or one or more vectors described in item 18 or 19 above, for activating phosphoenolpyruvate carboxylase.

[61] The aforementioned one or more antibody-mimicking molecules The polypeptides in (2) above, and Any of the polypeptides (3) to (6) above The use described in 60 above.

[62] The aforementioned one or more antibody-mimicking molecules The polypeptides in (2) above, and The polypeptide described in (4) above The use described in 60 above.

[63] The aforementioned one or more antibody-mimicking molecules The polypeptide (2') and Any of the polypeptides (3') to (6') above The use described in 60 above.

[64] The aforementioned one or more antibody-mimicking molecules The polypeptide (2') and The polypeptide (4') described above The use described in 60 above.

[65] The use according to 60, wherein the one or more antibody-mimicking molecules are a polypeptide containing the sequence of SEQ ID NO: 3 and a polypeptide containing any of the sequences of SEQ ID NOs: 4 to 7.

[66] The use according to 60, wherein the one or more antibody-mimicking molecules are a polypeptide containing the sequence of SEQ ID NO: 3 and a polypeptide containing the sequence of SEQ ID NO: 5.

[67] The one or more of the aforementioned polynucleotides The polynucleotides of (2'') and any of the polynucleotides (3'') to (6'') above The use described in 60 above.

[68] The one or more of the aforementioned polynucleotides The polynucleotides of (2'') and The polynucleotides of (4'') The use described in 60 above.

[69] The one or more of the aforementioned polynucleotides The (2'') polynucleotides, and Any of the polynucleotides (3''') to (6''') mentioned above The use described in 60 above.

[70] The one or more of the aforementioned polynucleotides The (2'') polynucleotides, and The polynucleotides of (4''') mentioned above The use described in 60 above.

[71] The use according to 60, wherein the one or more polynucleotides are a polynucleotide containing the sequence of SEQ ID NO: 9 and a polynucleotide containing any of the sequences of SEQ ID NOs: 10 to 13.

[72] The use according to 60, wherein the one or more polynucleotides are a polynucleotide containing the sequence of SEQ ID NO: 9 and a polynucleotide containing the sequence of SEQ ID NO: 11.

[73] The aforementioned one or more types of vectors A vector containing the polynucleotide (2'') described above, and A vector containing any of the polynucleotides described in (3'') to (6'') above. The use described in 60 above.

[74] The aforementioned one or more types of vectors A vector containing the polynucleotide (2'') described above, and Vector containing the polynucleotide (4'') described above The use described in 60 above.

[75] The aforementioned one or more types of vectors A vector containing the (2'') polynucleotide, and A vector containing any of the polynucleotides (3''') to (6''') above. The use described in 60 above.

[76] The aforementioned one or more types of vectors A vector containing the (2'') polynucleotide, and Vector containing the polynucleotide (4''') described above The use described in 60 above.

[77] The use according to 60, wherein the one or more vectors are a vector containing a polynucleotide containing the sequence of SEQ ID NO: 9, and a vector containing a polynucleotide containing any of the sequences of SEQ ID NOs: 10 to 13.

[78] The use according to 60, wherein the one or more vectors are a vector containing a polynucleotide containing the sequence of SEQ ID NO: 9, and a vector containing a polynucleotide containing the sequence of SEQ ID NO: 11.

[0047] The present invention will be further described by the following embodiments, but the present invention is not limited in any sense to these embodiments. [Examples]

[0048] Experiment 1 It has been reported that amino acid mutations (R849S or I829S) in E. coli-derived PEPC (EcPEPC) enhance succinate production by E. coli (Tokuyama K, et al., Biotech. Bioeng. 115, 1542, (2018)). This report revealed that EcPEPC mutants (R849 or I829S) are less susceptible to feedback inhibition by L-aspartate, resulting in enhanced succinate production by E. coli. Therefore, we attempted to obtain an artificial binding protein monobody that can maintain high activity of EcPEPC.

[0049] 1. Obtaining an artificial binding protein monobody that binds to EcPEPC. 1-1 Preparation of the plasmid vector pHBT2-EcPEPC for expressing biotinylated EcPEPC Biotinylated EcPEPC was prepared for monobody screening. The common method for preparing biotinylated EcPEPC involves adding the AviTag (GLNDIFEAQKIEWHE) (SEQ ID NO: 26) to the N-terminus of EcPEPC and then biotinylating the AviTag site with a biotin ligase. Therefore, a plasmid vector expressing AviTag-EcPEPC was constructed.

[0050] An original vector pHBT2-GST (with AviTag (GLNDIFEAQKIEWHE) (SEQ ID NO: 26) inserted between the HisTag and TEV protease cleavage sequence) based on pET28a (kanamycin (Kan) resistant, Addgene), and an expression plasmid containing the EcPEPC sequence (Kai Y, et al., Proc. Natl. Acad. Sci. USA, 96(3), 823-828 (1999), pT3-EcPEPC) were used as templates to perform PCR using Q5 High-Fidelity DNA Polymerase (M0491L) to prepare vector DNA fragments and insert DNA fragments, respectively. After PCR, 12 μl of the reaction solution was mixed with 3 μl of Midori Green Direct and subjected to agarose electrophoresis at 150V for 20 minutes to confirm the reaction. Then, 1 μl of DpnI was added to the remaining reaction solution and incubated at 37°C for 1 hour. The vector and insert were purified according to the PCR product purification protocol of the Gel / PCR Extraction Kit (FastGene). Subsequently, each DNA fragment was assembled using the NEBuilder® HiFi DNA Assembly Master Mix (E2621L) kit (New England BioLabs). This product is a kit that recognizes and clones approximately 15 homologous bases at the ends of PCR-prepared DNA fragments. The NEBuilder assembly reaction was performed according to the NEBuilder HiFi DNA Assembly Master Mix protocol, and the pHBT2-EcPEPC plasmid vector was prepared by incubation at 50°C for 30 minutes (Figure 1).

[0051] 1-2 Transformation and cultivation of E. coli using pHBT2-EcPEPC After the NEBuilder assembly reaction, 20 ng of pHBT2-EcPPC was added to 50 μl of competent cell DH5α (turbidity OD600=0.4, Takara Bio), left to stand on ice for 30 minutes, and then heated at 42°C for 1 minute. After standing on ice for several minutes, 1 mL of SOC medium was added and cultured with shaking at 37°C for 1 hour. The entire volume was spread onto LB agar medium containing 50 μg / ml Kan in a clean bench and incubated at 37°C for 16 hours. The formed colonies were picked and added to 4 ml of LB liquid medium (50 μg / ml Kan) and incubated at 37°C for 16 hours. Subsequently, plasmid DNA was purified from the culture medium according to the Plasmid Mini Kit (FastGene) Standard protocol, and DNA sequencing confirmed that the target plasmid vector pHBT2-EcPEPC had been successfully produced.

[0052] 100 μl of competent cell BL21 (DE3) (turbidity OD600 = 0.4, New England BioLabs) was mixed with 100 μg each of pHBT2-EcPEPC plasmid vector and the biotin ligase-encoding plasmid vector pBirAcm (chloramphenicol (Cm) resistant, Avidity). The mixture was left to stand on ice for 10 minutes, then subjected to a heat shock at 42°C for 45 seconds, and then left to stand on ice again for 3 minutes. Subsequently, 1 mL of SOC medium was added and the mixture was cultured with shaking at 37°C for 1 hour. The mixture was then spread onto LB agar containing 50 μg / ml Kan and 20 μg / ml Cm, which had been previously incubated at 37°C, and incubated at 37°C for 16 hours.

[0053] The formed colonies were picked and added to 3 mL of LB liquid medium containing 50 μg / ml Kan and 20 μg / ml Cm, and incubated for 1 day at 37°C and 140 rpm using a BR-40LF (TAITEC). Subsequently, this 3 mL of LB liquid medium was added to 1 L of LB liquid medium containing 50 μg / ml Kan and 20 μg / ml Cm, and incubated at 37°C and 140 rpm using a BR-300LF (TAITEC). The culture was terminated when the OD600 exceeded 0.4. Then, IPTG was added at a final concentration of 0.5 mM and biotin at a final concentration of 100 μM, and induction was performed for 18 hours using a BR-300LF (TAITEC) set to 17°C and 110 rpm. The bacterial cells were collected by centrifugation at 4°C for 20 minutes at 5000 rpm (JLA 8,100) using an Avanti J-26S XPI (BECKMAN COULTER) and recovered. The cells were suspended in 150 mM NaCl and then centrifuged at 4°C for 15 minutes at 5000 rpm (HIGHConic III) using a Sorvall ST 8R (Thermo Scientific). After discarding the supernatant, the remaining cells were divided into 5 g portions, flash-frozen in liquid nitrogen, and stored at -80°C.

[0054] 1-3 Purification of AviTag-EcPEPC The bacterial cells were lysed in 20 ml of Ni affinity column chromatography binding buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM imidazole, 10 mM L-aspartic acid) per g of bacterial cells. The bacterial suspension was stirred and kept in an ice bath, and subjected to 10 sets of sonication disruption using a Qsonica Q500 ultrasonic homogenizer (Time: 1 min, Pulse: 01:01, Ampl: 40%). A 3-minute interval was taken after each set to maintain the temperature of the bacterial suspension. After disruption, the bacterial suspension was centrifuged in a small ultracentrifuge CS100FNX (himac) at 4°C for 30 minutes at 39,000 rpm (S50A), and the supernatant was collected. The supernatant was then filtered using a syringe filter (SLNY2545N, filter diameter: 25 mm, pore size: 0.45 μm) (Hawach Scientific).

[0055] The extract after filtration was purified by Ni affinity column chromatography. A 5 ml His Trap HP (Cytiva) was connected to an AeKTA Start (Cytiva), and the column was equilibrated with binding buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM imidazole, 10 mM L-aspartic acid). The extract was applied to the equilibrated column, and the sample was eluted using elution buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 500 mM imidazole, 10 mM L-aspartic acid). The eluted fraction was collected and concentrated to approximately 2 mL. The concentrated sample was applied to a HiLoad 16 / 60 Superdex200 (Cytiva) column equilibrated with gel filtration buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 10 mM L-aspartic acid). The solution was eluted with gel filtration buffer and collected in a test tube. The eluted fraction was concentrated to approximately 10 mg / mL at 4 °C and 3,500 rpm.

[0056] 1-4 Obtaining artificial binding protein monobodies that bind to EcPEPC Phage display is a technique that displays the outer shell protein of a phage fused with an external protein molecule E on the surface of a phage particle. Using this technique, a phage library (1.32-2.20 × 10¹⁶) displaying various artificially bound protein monobodies has been created. 10Phages were prepared (100 cells / mL), and phages presenting monobodies that specifically recognized biotinylated EcPEPC were selected from this library. The preparation and selection of the phage library were basically carried out according to previously reported methods (Koide A et al. J. Mol. Biol. 415, 393-405, 2012). The amino acid sequences and structures of the monobodies prepared in this study are shown in Figures 2A and 2B. Of the two libraries, the loop library is a library in which the amino acid sequences of the three loops (BC loop, DE loop, FG loop) located at one end of the FN3 template structure have been mutated and diversified. On the other hand, the side library is a library in which the amino acid sequences of the FG loop and the CD loop located at the opposite end, as well as the amino acid sequences of the two β strands (C strand and D strand) between these two loops, have been mutated and diversified. The selection process was carried out a total of four times, with the biotin EcPEPC concentration set to 200 nM for the first and second selections, and 100 nM for the third and fourth selections.

[0057] The BC-DE loop and FG loop genes of each monobody in the pooled population after the above selection were amplified by PCR. Subsequently, a new monobody mutant library was prepared by shuffling the BC-DE loop and FG loop genes using the principle of homologous recombination in yeast cells. From this yeast surface display library, which presented these monobody mutants on the cell surface, monobodies that specifically recognized biotinylated EcPEPC were selected. The amplification and shuffling of the BC-DE loop and FG loop genes, presentation on the yeast surface, and selection by flow cytometry were basically carried out according to the previously reported method (Tanaka Si et al., Nature Chem. Biol. 11(10), 762-764, 2015). Selection was performed a total of two times, and the biotinylated EcPEPC concentration in each step was 100 nM. Table 1 shows the amino acid sequence of Mb(PEPC_L3), a monobody that showed strong binding affinity at a biotinylated EcPEPC concentration of 50 nM, obtained from monoclonalized yeast libraries after selection.

[0058] For Mb(PEPC_L3), the binding-dissociation constant K was determined according to the previously reported method (Tanaka Si et al., Nature Chem. Biol. 11(10), 762-764, 2015). D The following was determined. Specifically, flow cytometry was performed with varying concentrations of biotinylated EcPEPC, and the median value of the fluorescence intensity, which increased with increasing monobody binding affinity, was plotted against the concentration of biotinylated EcPEPC. A binding curve was drawn from the plot using SigmaPlot, and K D The following values ​​were calculated (as shown in Table 1). Mb(Shaved) consisted mainly of serine residues in the loop region and had no binding affinity to PEPC; therefore, it was used as a negative control for subsequent activity measurements. Mb(PEPC_L3) and Mb(Shaved) were obtained as described in 2-1 below.

[0059] 2. Activity measurement of EcPEPC 2-1 Expression and Purification of EcPEPC and Monobodies 100 μg of expression plasmid containing the EcPEPC sequence (Tokuyama K, et al., Biotech. Bioeng. 115, 1542, (2018), pET28a-EcPEPC) was added to 100 μl of competent cell BL21 (DE3) (turbidity OD600 = 0.5, New England BioLabs). The cells were left to stand on ice for 10 minutes, then subjected to a heat shock at 42°C for 1 minute, and then left to stand on ice again for 2 minutes. Subsequently, 300 μL of SOC medium was added and cultured with shaking at 37°C for 1 hour. The cells were then spread onto LB agar medium containing 50 μg / ml Kan, which had been previously incubated at 37°C, and incubated at 37°C for 16 hours. The amino acid sequence of EcPEPC is shown in SEQ ID NO: 27.

[0060] Colonies formed on LB agar were picked and added to 3 mL of LB liquid medium containing 50 μg / ml Kan. The cells were incubated for 1 day at 37°C and 140 rpm using a BR-40LF (TAITEC) incubator. Subsequently, this 3 mL of LB liquid medium was added to 2 L of LB liquid medium containing 50 μg / ml Kan, and incubated at 37°C and 130 rpm using a BR-300LF (TAITEC) incubator. The culture was terminated when the OD600 exceeded 0.5. Then, IPTG at a final concentration of 0.5 mM and biotin at a final concentration of 100 μM were added. Induction was performed for 18 hours using a BR-300LF (TAITEC) incubator set to 17°C and 130 rpm. Cell collection was performed by centrifugation using an Avanti J-26S XPI (BECKMAN COULTER) at 4°C for 20 minutes at 5000 rpm (JLA 8,100). The bacterial cells were suspended in 150 mM NaCl and centrifuged at 4°C for 15 minutes at 5000 rpm (HIGHConic III) using a Sorvall ST 8R (Thermo Scientific). After discarding the supernatant, the remaining cells were divided into 5 g portions, flash-frozen in liquid nitrogen, and stored at -80°C.

[0061] The bacterial cells were lysed in 20 ml of Ni affinity column chromatography binding buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, 10 mM L-aspartic acid) per g of bacterial cells. The bacterial cell suspension was stirred and kept in an ice bath, and subjected to 10 sets of sonication disruption using a Qsonica Q500 ultrasonic homogenizer (Time: 1 min, Pulse: 01:01, Ampl: 60%). A 3-minute interval was taken after each set to maintain the temperature of the bacterial cell suspension. After disruption, the bacterial cell suspension was centrifuged in a small ultracentrifuge CS100FNX (himac) at 4°C for 30 minutes at 39,000 rpm (rotor: S50A), and the supernatant was collected. The supernatant was then filtered using a syringe filter (SLNY2545N, filter diameter: 25 mm, pore size: 0.45 μm) (Hawach Scientific).

[0062] The extract, after filtration, was purified by Ni affinity column chromatography. A 5 ml His Trap HP (Cytiva) was connected to an AeKTA Start (Cytiva), and the column was equilibrated with binding buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, 10 mM L-aspartic acid). The extract was applied to the equilibrated column, and the sample was eluted using elution buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 500 mM imidazole, 10 mM L-aspartic acid). The eluted fraction was collected, and thrombin (SIGMA T7513) was added at a ratio of 0.5 U thrombin / 1 mg. The HisTag region was cleaved by dialyzing overnight with gel filtration buffer (20 mM Tris-HCl pH8.0, 50 mM NaCl, 10 mM L-aspartic acid), and the total volume was concentrated to approximately 2 mL. The concentrated sample was applied to a HiLoad 16 / 60 Superdex200 (Cytiva) equilibrated with gel filtration buffer (20 mM Tris-HCl pH8.0, 50 mM NaCl, 10 mM L-aspartic acid). Elution was performed with gel filtration buffer and collected in a test tube. The eluted fraction was concentrated to approximately 10 mg / mL at 4 °C and 3,500 rpm.

[0063] For the monobodies, the same protocol was used for purification up to Ni affinity column chromatography, similar to EcPEPC (however, the monobodies were divided into 2.5 g portions and stored at -80°C, and dissolved in 30 ml of Ni affinity column chromatography binding buffer per g). Then, 1 / 10 of the sample volume of 2 mg / ml TEV protease (stored in 50% glycerol) was added, and dithiothreitol was added to a final concentration of 1 mM. The HisTag region was cleaved by dialyzing overnight with gel filtration buffer (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 10 mM L-aspartic acid). After that, gel filtration chromatography was performed using the same protocol, and the concentration was increased to approximately 4 mg / mL.

[0064] 2-2 Activity measurement The enzymatic activity of EcPEPC at 30°C was measured using a coupling reaction catalyzed by malate dehydrogenase (yeast-derived, manufacturer code 46610022, Fujifilm Wako Pure Chemical Industries, hereinafter referred to as MDH) in the presence and absence of the monobody.

[0065] The concentration of phosphoenolpyruvic acid (manufacturer code 160-14763, Fujifilm Wako Pure Chemical Industries, hereinafter referred to as PEP) was fixed at 4.0 mM, and activity measurements were performed. 30 mL of measurement buffer (100 mM Tris-HCl pH 8.0, 20 mM MgSO4, 10 mM NaHCO3, 5.0 mM PEP, 0.2 mM NADH, 1.5 U / ml MDH) was prepared. 97 μl of the measurement buffer was placed in an absorbance cell, followed by the addition of 3 μL of the purified sample PEPC protein solution, and the measurement was started. The decrease in NADH absorbance at 340 nm was measured using a V-750 (JASCO). Activity measurements were performed in the presence or absence of L-aspartic acid, and in and without the presence of monobodies. The concentrations of EcPEPC and monobody were measured using EcPEPC: 1.00 nM and monobody: 12.00 μM for both the EcPEPC / Mb(PEPC_L3) and EcPEPC / Mb(Shaved) combinations. The summarized results are shown in Figure 3.

[0066] In the presence of L-aspartic acid, an inactivating effector of EcPEPC, Mb(PEPC_L3) reduced the sensitivity of EcPEPC to L-aspartic acid-mediated inhibition of its activity (Figure 3). In other words, Mb(PEPC_L3) enhanced the activity of EcPEPC in the presence of L-aspartic acid (Figure 3).

[0067] Experiment 2 1. Obtaining an artificial binding protein monobody that binds to EcPEPC. In the same manner as in Experiment 1, monobodies that bind to EcPEPC, Mb(PEPC_L5) and Mb(PEPC_L13), were obtained by phage display and yeast surface display library methods. Furthermore, mutations were introduced into Mb(PEPC_L13) by Error-Prone PCR, and the mutants were selected by yeast surface display library methods to obtain monobodies that bind to EcPEPC, Mb(PEPC_L14), Mb(PEPC_L15), and Mb(PEPC_L16). Table 1 shows the amino acid sequences (sequence codes 1-7 and 23, respectively) of wild-type human FN3, Mb(PEPC_L3) obtained in Experiment 1, Mb(PEPC_L5), Mb(PEPC_L13), Mb(PEPC_L14), Mb(PEPC_L15), and Mb(PEPC_L16) obtained in Experiment 2, and the negative control Mb(Shaved).

[0068] [Table 1]

[0069] 2. Epitope analysis of Mb(PEPC_L5) and Mb(PEPC_L13) Epitope analysis of Mb(PEPC_L5) and Mb(PEPC_L13) was performed using the yeast surface display method. Monobodies were presented on yeast via V5-tags. The amount of monobodies presented on yeast was fluorescently labeled with PerCP Goat anti-mouse IgG (BioLegend) using Anti-V5-tag mAb (IgG / Mouse, MBL) (Figure 4A, 4B, Red Log). In addition, EcPEPC, which was biotinylated by biotin ligase using Avi-tag, was used to bind to the monobodies on yeast. The biotinylated EcPEPC bound to yeast was fluorescently labeled with Native Streptavidin protein (Abcam) (Figure 4A, 4B, Yellow Log). When protein-to-protein binding occurred, the fluorescence intensity of the biotinylated protein (Figure 4A, 4B, Yellow Log) increased with the amount of presentation on yeast (Figure 4A, 4B, Red Log). Regarding Figure 4A, to confirm whether the obtained Mb(PEPC_L5) and Mb(PEPC_L13) bind to the vicinity of the inhibitor binding site of EcPEPC, epitope analysis was performed using an EcPEPC mutant (mutant N113R) with a mutation in the inhibitor binding site. As a result, it was confirmed that Mb(PEPC_L5) binds to mutant N113R as well, indicating that it binds at a site other than the vicinity of the inhibitor binding site. On the other hand, Mb(PEPC_L13) does not bind to mutant N113R, confirming that Mb(PEPC_L13) binds to the vicinity of the inhibitor binding site. Next, to confirm that the binding sites of Mb(PEPC_L5) and Mb(PEPC_L13) are different, competitive binding analysis of two types of monobodies was performed (Figure 4B). In this case, Mb(PEPC_L13) was presented on yeast, and Mb(PEPC_L5) was expressed and purified using the pHFT2 vector. In this analysis, biotinylated EcPEPC was first bound to Mb(PEPC_L5), and then Mb(PEPC_L13) presented on yeast was bound to it, followed by fluorescent labeling in the same manner as described above. As a result, it was confirmed that the binding sites of Mb(PEPC_L5) and Mb(PEPC_L13) were different.Furthermore, it was confirmed that Mb(EcPEPC_L13) binds more strongly to EcPEPC in the presence of Mb(PEPC_L5) (Figure 4B), K. D The value was 16.9 nM, which is an improvement in affinity compared to 35.0 nM for Mb(PEPC_L13) alone.

[0070] 3. Activity measurement Using the acquired monobodies, the sensitivity of EcPEPC to inhibition in the presence of the inactivating effector L-aspartic acid (0, 1, 2, 5, 10 mM) was measured. Combinations of Mb(PEPC_L5), Mb(PEPC_L13), Mb(PEPC_L14), Mb(PEPC_L15), or Mb(PEPC_L16) and Mb(Shaved) (Figure 5, left), or Mb(PEPC_L13), Mb(PEPC_L14), Mb(PEPC_L15), or Mb(PEPC_L16) and Mb(PEPC_L5) (Figure 5, right) were tested.

[0071] The enzymatic activity of EcPEPC at 30°C was measured using an enzyme coupling reaction catalyzed by malate dehydrogenase (yeast-derived, manufacturer code 46610022, Fujifilm Wako Pure Chemical Industries, hereinafter referred to as MDH) in the presence of a monobody. An enzyme solution (15 nM PEPC, 15 μM Mb1, 15 μM Mb2, 25 mM FBP, 0-10 mM Asp, 100 mM Tris-HCl pH 8.0, 10 mM NaHCO3) was prepared, 20 μL was applied to one well, and incubated at 30°C for 2 minutes. This procedure allows for the measurement of PEPC after the transition. Next, a measurement buffer (100 mM Tris-HCl pH 8.0, 25 mM MgSO4, 10 mM NaHCO3, 6.25 mM PEP, 0.25 mM NADH, 0.1875 IU / ml MDH, 0-10 mM L-Asp) was prepared. 80 μL of the measurement buffer was added to 20 μL of enzyme solution in the incubated wells, and the measurement was started. The decrease in NADH absorbance at 340 nm was measured using a SpectraMax i3x multimode microplate reader (Molecular Devices). Activity measurements were performed in the presence of monobodies with L-aspartic acid concentrations of 0 mM, 1 mM, 2 mM, 5 mM, or 10 mM. The final concentrations of EcPEPC, Mb1, and Mb2 were all EcPEPC: 3.00 nM, Mb1: 3.00 μM, and Mb2: 3.00 μM. The control set consisted of Mb1 and Mb2, both designated as Mb(Shaved). When measuring the effect of a single type of Mb, the target monobody was used for Mb1 and Mb(Shaved) for Mb2.

[0072] First, to measure the effect of individual monobodies, we investigated the effects when Mb1 was the target monobody and Mb2 was Mb(Shaved). From the changes in activity inhibition sensitivity under each condition, it was confirmed that Mb(PEPC_L5), Mb(PEPC_L13), Mb(PEPC_L14), Mb(PEPC_L15), and Mb(PEPC_L16) mitigated sensitivity, with Mb(PEPC_L5) and Mb(PEPC_L14) being particularly effective (Figure 5, left).

[0073] Next, we investigated the effects of two types of monobodies by using Mb(PEPC_L5) for Mb1 and Mb(PEPC_L13), Mb(PEPC_L14), Mb(PEPC_L15), or Mb(PEPC_L16) with different epitopes for Mb2. As a result, we found that enzyme activity was maintained in all combinations compared to Mb(PEPC_L5) alone (Figure 5, right). In particular, under the condition of Mb(PEPC_L5) + Mb(PEPC_L14), we were able to maintain as much as 80% of the enzyme activity even in the presence of 10 mM L-aspartic acid.

[0074] Regarding specific activity, a comparison was made under the presence of 1 mM L-aspartic acid and 10 mM L-aspartic acid (Figures 6A and 6B). Similar to Figure 5, Mb(PEPC_L5), Mb(PEPC_L13), Mb(PEPC_L14), Mb(PEPC_L15), and Mb(PEPC_L16) mitigated the sensitivity to activity inhibition, and it was confirmed that Mb(PEPC_L5) and Mb(PEPC_L14) were particularly effective (Figure 6A). The combination of Mb(PEPC_L5) + Mb(PEPC_L14) showed a specific activity of 81.57 U / mg even under the harsh condition of 10 mM L-aspartic acid (Figure 6B).

[0075] These results demonstrate that the monobody of this disclosure can activate PEPC and improve the productivity of succinic acid.

[0076] [Table 2-1] Table 2-2

Claims

1. An antibody-mimicking molecule containing an amino acid sequence in which at least one amino acid in the amino acid sequence of the fibronectin type III domain (FN3) is modified, and which can bind to and activate phosphoenolpyruvate carboxylase (PEPC).

2. (1) A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO:

2. (2) A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO:

3. (3) A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO:

4. (4) A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO:

5. (5) A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 6, and (6) Polypeptides containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO:

7. An antibody-mimicking molecule according to claim 1, selected from the above.

3. (1') A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 2 are the amino acid sequences of SEQ ID NO: 14, 15, and 16, respectively. (2') A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 3, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-82 of SEQ ID NO: 3 are the amino acid sequences of SEQ ID NO: 17, 18, and 19, respectively. (3') A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 4, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 4 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively. (4') A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 5, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 5 are the amino acid sequences of SEQ ID NO: 20, 18, and 22, respectively. (5') A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 6, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 6 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively, and (6') A polypeptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 to 10 amino acid residues are deleted, substituted, inserted, or added to the amino acid sequence of SEQ ID NO: 7, wherein the amino acid sequences corresponding to positions 25-28, 53-55, and 75-81 of SEQ ID NO: 7 are the amino acid sequences of SEQ ID NO: 20, 18, and 21, respectively. An antibody-mimicking molecule according to claim 1, selected from the above.

4. The antibody-mimicking molecule according to claim 1, comprising any of the amino acid sequences of SEQ ID NOs: 2 to 7.

5. A polynucleotide comprising a sequence encoding the antibody-mimicking molecule described in claim 1.

6. (1'') A polynucleotide containing a sequence encoding the polypeptide of (1) above, (2'') A polynucleotide containing a sequence encoding the polypeptide of (2) above, (3'') A polynucleotide containing a sequence encoding the polypeptide of (3) above, (4'') A polynucleotide containing a sequence encoding the polypeptide of (4), (5'') A polynucleotide comprising a sequence encoding the polypeptide of (5), and (6'') A polynucleotide containing a sequence encoding the polypeptide of (6) above A polynucleotide according to claim 5, selected from the following.

7. A sequence having 90% or more sequence identity with any of sequence numbers 8 to 13, or Sequences in which 1 to 30 bases are deleted, substituted, inserted, or added in any of the sequences of sequence numbers 8 to 13. The polynucleotide according to claim 5, comprising:

8. The polynucleotide according to claim 5, comprising any sequence of sequence numbers 8 to 13.

9. A vector comprising the polynucleotide described in claim 5.

10. A succinate-producing microorganism comprising one or more antibody-mimicking molecules according to claim 1, one or more polynucleotides according to claim 5, or one or more vectors according to claim 9.

11. The aforementioned one or more antibody-mimicking molecules The polypeptides described in (2) above, and Any polypeptide from (3) to (6) above The succinic acid-producing microorganism according to claim 10.

12. The succinic acid-producing microorganism according to claim 10, wherein the one or more antibody-mimicking molecules are a polypeptide containing the sequence of SEQ ID NO: 3 and a polypeptide containing any of the sequences of SEQ ID NOs: 4 to 7.

13. The one or more of the aforementioned polynucleotides The polynucleotides of (2'') and Any of the polynucleotides (3'') to (6'') above The succinic acid-producing microorganism according to claim 10.

14. The succinic acid-producing microorganism according to claim 10, wherein the one or more polynucleotides are a polynucleotide containing the sequence of SEQ ID NO: 9 and a polynucleotide containing any of the sequences of SEQ ID NOs: 10 to 13.

15. The aforementioned one or more types of vectors A vector containing the polynucleotide (2'') described above, and A vector containing any of the polynucleotides (3'') to (6'') above. The succinic acid-producing microorganism according to claim 10.

16. The succinic acid-producing microorganism according to claim 10, wherein the one or more vectors are a vector containing a polynucleotide including the sequence of SEQ ID NO: 9, and a vector containing a polynucleotide including any of the sequences of SEQ ID NOs: 10 to 13.

17. The succinic acid-producing microorganism according to claim 10, which is Escherichia coli.

18. A method for producing succinic acid, comprising culturing the succinic acid-producing microorganism described in claim 10.

19. A method for activating phosphoenolpyruvate carboxylase, comprising treating phosphoenolpyruvate carboxylase in the presence of one or more antibody-mimetic molecules according to claim 1, one or more polynucleotides according to claim 5, or one or more vectors according to claim 9.

20. The aforementioned one or more antibody mimic molecules The polypeptides described in (2) above, and Any polypeptide from (3) to (6) above The method according to claim 19.

21. The one or more antibody mimic molecules mentioned above The method according to claim 19, wherein the polypeptide comprises the sequence of SEQ ID NO: 3 and the polypeptide comprises any sequence of SEQ ID NOs: 4 to 7.

22. Use of one or more antibody-mimetic molecules according to claim 1, one or more polynucleotides according to claim 5, or one or more vectors according to claim 9 for activating phosphoenolpyruvate carboxylase.

23. The one or more antibody mimic molecules mentioned above The polypeptides described in (2) above, and Any polypeptide from (3) to (6) above The use described in claim 22.

24. The one or more antibody mimic molecules mentioned above The use according to claim 22, wherein the polypeptide is a polypeptide containing the sequence of SEQ ID NO: 3, and a polypeptide containing any sequence of SEQ ID NOs: 4 to 7.

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