Method for producing cultured meat

Abstract

Provided is a method for producing cultured meat. Cultured meat is produced by culturing animal cells in the presence of bovine basic fibroblast growth factor (bbFGF) produced by using coryneform bacteria as expression hosts.

Description

How cultured meat is produced

[0001] The present invention relates to a method for producing cultured meat.

[0002] In recent years, the technology for producing cultured meat has been attracting attention. One of the challenges in producing cultured meat is the high production cost.

[0003] Growth factors such as bovine basic fibroblast growth factor (bbFGF) are typically used in cell culture in regenerative medicine and the like in a highly purified form obtained by chromatography or the like.

[0004] An objective of the present invention is to provide a method for producing cultured meat.

[0005] As a result of intensive research to solve the above problems, the inventors discovered that bovine basic fibroblast growth factor (bbFGF) expressed in coryneform bacteria as a host is effective in producing cultured meat, and thus completed the present invention.

[0006] That is, the present invention can be exemplified as follows: [1] A method for producing bovine basic fibroblast growth factor (bbFGF) for use in producing cultured meat, comprising the step of culturing a coryneform bacterium having a gene construct for secreting and expressing bbFGF to obtain a culture containing bbFGF. [2] The method further comprising the step of removing the cells of the coryneform bacterium from the culture. [3] The method does not comprise the step of purifying bbFGF. [4] The method does not comprise the step of purifying bbFGF by chromatography. [5] The method wherein the gene construct comprises, in the 5' to 3' direction, a promoter sequence functional in coryneform bacteria, a nucleic acid sequence encoding a signal peptide functional in coryneform bacteria, and a nucleic acid sequence encoding bbFGF, and wherein bbFGF is expressed as a fusion protein with the signal peptide. [6] The method wherein the coryneform bacterium is a bacterium of the genus Corynebacterium. [7] The method as described above, wherein the coryneform bacterium is Corynebacterium glutamicum. [8] A method for producing cultured meat, comprising a step of culturing animal cells in the presence of bovine basic fibroblast growth factor (bbFGF), wherein the bbFGF is produced by the method as described above.

[0007] Photograph showing the results of SDS-PAGE analysis of bbFGF secreted and expressed in C. glutamicum as a host. Diagram showing a chromatogram of bbFGF secreted and expressed in C. glutamicum as a host, separated by cation exchange chromatography. Diagram showing the proliferation of bovine muscle stem cells in serum-free medium supplemented with bbFGF secreted and expressed in C. glutamicum as a host. Data (vertical axis) are shown as relative values, with the number of cells at the start of culture set at 1. Diagram showing the proliferation rate of bovine muscle stem cells in serum-free medium supplemented with bbFGF secreted and expressed in C. glutamicum as a host. The horizontal axis indicates group number. Diagram showing the doubling time of bovine muscle stem cells in serum-free medium supplemented with bbFGF secreted and expressed in C. glutamicum as a host. The horizontal axis indicates group number. A diagram showing the proliferation of bovine muscle stem cells in serum-free medium supplemented with bbFGF secreted and expressed in C. glutamicum as a host. Data (vertical axis) are shown as relative values, with the number of cells at the start of culture set at 1. A diagram showing the proliferation rate of bovine muscle stem cells in serum-free medium supplemented with bbFGF secreted and expressed in C. glutamicum as a host. The horizontal axis shows the group number. A diagram showing the doubling time of bovine muscle stem cells in serum-free medium supplemented with bbFGF secreted and expressed in C. glutamicum as a host. The horizontal axis shows the group number.

[0008] The present invention will be described in detail below.

[0009] <1> Active ingredient In the present invention, bovine basic fibroblast growth factor (bbFGF) produced using coryneform bacteria as an expression host is used as an active ingredient. bbFGF produced using coryneform bacteria as an expression host is also referred to as the "active ingredient."

[0010] By utilizing an active ingredient, specifically by culturing animal cells in the presence of the active ingredient, it is possible to promote the proliferation of animal cells, i.e., an effect of promoting the proliferation of animal cells can be obtained. This effect is also referred to as a "proliferation-promoting effect." The proliferation-promoting effect can be confirmed, for example, by culturing animal cells in the presence and absence of the active ingredient and comparing the degree of proliferation of the animal cells. By promoting the proliferation of animal cells, for example, cultured meat can be efficiently produced. In other words, the active ingredient may be for promoting the proliferation of animal cells, specifically, for producing cultured meat.

[0011] The method for producing the active ingredient using a coryneform bacterium as an expression host will be described below.

[0012] The active ingredient can be produced by culturing a coryneform bacterium having a gene construct for secretion and expression of bbFGF. That is, the method for producing the active ingredient may be a method comprising the step of culturing a coryneform bacterium having a gene construct for secretion and expression of bbFGF. This step is also referred to as a "step of culturing a coryneform bacterium." By the step of culturing a coryneform bacterium, bbFGF is secreted and produced, thereby obtaining a culture containing bbFGF (specifically, a culture containing secreted and produced bbFGF). Therefore, the step of culturing a coryneform bacterium may specifically be a step of culturing a coryneform bacterium having a gene construct for secretion and expression of bbFGF to secrete and produce bbFGF. Furthermore, the step of culturing a coryneform bacterium may specifically be a step of culturing a coryneform bacterium having a gene construct for secretion and expression of bbFGF to obtain a culture containing bbFGF.

[0013] The coryneform bacterium (i.e., the coryneform bacterium used to produce an active ingredient) is also referred to as the "bacterium of the present invention." The bacterium of the present invention or a parent strain used to construct the bacterium is also referred to as the "host."

[0014] The above gene construct (i.e., a gene construct for secretory expression of bbFGF) is also referred to as a "bbFGF expression cassette."

[0015] <1-1> Bacteria of the Present Invention The bacterium of the present invention is a coryneform bacterium having a gene construct for secretory expression of bbFGF (ie, a bbFGF expression cassette).

[0016] The bacterium of the present invention has the ability to secrete and produce bbFGF. The bacterium of the present invention has the ability to secrete and produce bbFGF, at least due to the presence of a bbFGF expression cassette.

[0017] "Secretion" of bbFGF refers to the transport of bbFGF outside the bacterial cell (extracellularly). Examples of "extracellular" include the medium and the bacterial cell surface. That is, the secreted bbFGF may be present, for example, in the medium, on the bacterial cell surface, or both in the medium and on the bacterial cell surface. That is, "secretion" of bbFGF is not limited to cases where all bbFGF molecules are ultimately released into the medium, but also includes cases where all bbFGF molecules are present on the bacterial cell surface, or cases where some bbFGF molecules are present in the medium and the remaining molecules are present on the bacterial cell surface.

[0018] Specifically, the "ability to secrete and produce bbFGF" refers to the ability of the bacterium of the present invention, when cultured in a medium, to secrete bbFGF into the medium and / or onto the cell surface and accumulate it to an extent that it can be recovered from the medium and / or the cell surface. The amount accumulated in the medium may be, for example, preferably 10 μg / L or more, more preferably 1 mg / L or more, particularly preferably 100 mg / L or more, and even more preferably 1 g / L or more. Furthermore, the amount accumulated on the cell surface may be, for example, such that when bbFGF from the cell surface is recovered and suspended in an equal volume of liquid as the medium, the bbFGF concentration in the suspension is preferably 10 μg / L or more, more preferably 1 mg / L or more, and particularly preferably 100 mg / L or more.

[0019] Coryneform bacteria are aerobic, gram-positive rod-shaped bacteria. Examples of coryneform bacteria include bacteria of the genera Corynebacterium, Brevibacterium, and Microbacterium. The advantages of using coryneform bacteria include the fact that, compared with filamentous fungi, yeast, Bacillus bacteria, and the like that have traditionally been used for the secretory production of proteins, very little protein is secreted outside the bacterial cells, which is expected to simplify or eliminate the purification process when proteins are secreted and produced. Furthermore, they grow well in simple media containing sugars, ammonia, inorganic salts, and the like, and are therefore excellent in terms of medium cost, culture method, and culture productivity.

[0020] Specific examples of coryneform bacteria include the following species: Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium casei Corynebacterium crenatum Corynebacterium flavescens Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecora melassecola) Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium casei Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium glutamicum) Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum)Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum

[0021] Specific examples of coryneform bacteria include the following strains: Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium alkanolyticum ATCC 21511, Corynebacterium callunae ATCC 15991, Corynebacterium casei JCM 12072, and Corynebacterium crenatum AS1.542 Corynebacterium flavescens ATCC 10340 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium casei ATCC 35513 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020 Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium thiogenitalis ATCC 19240 Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872 Brevibacterium album ATCC 15111 Brevibacterium cerinum ATCC 15112 Microbacterium ammoniaphilum ATCC 15354.

[0022] The genus Corynebacterium also includes bacteria that were previously classified as Brevibacterium but have now been integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1991)). Corynebacterium stationis also includes bacteria that were previously classified as Corynebacterium ammoniagenes but have been reclassified as Corynebacterium stationis based on 16S rRNA sequence analysis and other factors (Int. J. Syst. Evol. Microbiol., 60, 874-879(2010)).

[0023] These strains can be obtained, for example, from the American Type Culture Collection (address: 12301 Parklawn Drive, Rockville, Maryland 20852, PO Box 1549, Manassas, VA 20108, United States of America). Each strain is assigned a corresponding accession number, and can be obtained using this accession number (see http: / / www.atcc.org / ). The accession numbers corresponding to each strain are listed in the catalog of the American Type Culture Collection. These strains can also be obtained, for example, from the depository institution where they were deposited.

[0024] In particular, C. glutamicum AJ12036 (FERM BP-734), a streptomycin (Sm)-resistant mutant isolated from wild-type C. glutamicum ATCC 13869, is predicted to have a mutation in a gene controlling protein secretion functions compared to its parent (wild-type) strain. This mutation is believed to have significantly higher protein secretion production capacity, with the amount of protein accumulated under optimal culture conditions being approximately two to three times higher than that of the wild-type strain, making it a suitable host bacterium (WO2002 / 081694). AJ12036 was originally deposited as an international deposit with the Fermentation Research Institute of the Agency of Industrial Science and Technology (now the National Institute of Technology and Evaluation, Patent Organism Depositary Center (NITE IPOD), 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Postal Code: 292-0818, Address: Room 120) on March 26, 1984, and has been assigned the accession number FERM BP-734.

[0025] Alternatively, a strain with enhanced protein secretory production ability may be selected using mutation or genetic recombination techniques from the above-described coryneform bacteria as a parent strain and used as a host. For example, strains with enhanced protein secretory production ability can be selected after treatment with ultraviolet light or a chemical mutagen such as N-methyl-N'-nitrosoguanidine.

[0026] Furthermore, using such a strain modified so as not to produce cell surface proteins as a host is particularly preferred, as it facilitates the purification of bbFGF secreted into the medium or on the bacterial cell surface. Such modification can be achieved by introducing a mutation into the coding region of the cell surface protein or its expression regulatory region on the chromosome by mutagenesis or genetic recombination. An example of a coryneform bacterium modified so as not to produce cell surface proteins is the C. glutamicum YDK010 strain (WO 2002 / 081694), which is a strain of C. glutamicum AJ12036 (FERM BP-734) that lacks the cell surface protein PS2.

[0027] The bacterium of the present invention can be obtained by appropriately modifying the coryneform bacterium described above (e.g., by introducing a bbFGF expression cassette and, optionally, by introducing other modifications). That is, the bacterium of the present invention may be, for example, a modified strain derived from the coryneform bacterium described above. Specifically, the bacterium of the present invention may be, for example, a modified strain derived from C. glutamicum AJ12036 (FERM BP-734) or a modified strain derived from C. glutamicum ATCC 13869. Note that a modified strain derived from C. glutamicum AJ12036 (FERM BP-734) also corresponds to a modified strain derived from C. glutamicum ATCC 13869. The modifications for constructing the bacterium of the present invention can be performed in any order.

[0028] <1-2> Gene construct for secretory expression of bbFGF and introduction thereof The bacterium of the present invention has a gene construct for secretory expression of bbFGF (ie, a bbFGF expression cassette).

[0029] It is known that secretory proteins are generally translated as preproteins (also referred to as prepeptides) or preproproteins (also referred to as prepropeptides), which are then processed to become mature proteins. Specifically, secretory proteins are generally translated as preproteins or preproproteins, and then the signal peptide, which is the prepart, is cleaved by a protease (commonly referred to as a signal peptidase) to convert them into mature proteins or proproteins. The proprotein is then further cleaved by a protease to become the mature protein. Therefore, in the present invention, signal peptides may be used for the secretory production of bbFGF. Note that preproteins and preproproteins of secretory proteins are sometimes collectively referred to as "secretory protein precursors." A "signal peptide" (also referred to as a "signal sequence") refers to an amino acid sequence present at the N-terminus of a secretory protein precursor and not normally present in the natural mature protein.

[0030] The bbFGF expression cassette may comprise, from 5' to 3', a promoter sequence functional in coryneform bacteria, a nucleic acid sequence encoding a signal peptide functional in coryneform bacteria, and a nucleic acid sequence encoding bbFGF. The nucleic acid sequence encoding the signal peptide may be linked downstream of the promoter sequence so that the signal peptide is expressed under the control of the promoter. The nucleic acid sequence encoding bbFGF may be linked downstream of the nucleic acid sequence encoding the signal peptide so that bbFGF is expressed as a fusion protein with the signal peptide. Such a fusion protein is also referred to as the "fusion protein of the present invention." Note that in the fusion protein of the present invention, the signal peptide and bbFGF may or may not be adjacent to each other. In other words, "bbFGF is expressed as a fusion protein with a signal peptide" does not necessarily mean that bbFGF is expressed adjacent to the signal peptide as a fusion protein with the signal peptide, but also includes cases where bbFGF is expressed as a fusion protein with the signal peptide via another amino acid sequence. For example, as described below, the fusion protein of the present invention may contain an inserted sequence between the signal peptide and bbFGF, such as an amino acid sequence containing Gln-Glu-Thr or an amino acid sequence used for enzymatic cleavage. Furthermore, as described below, the final bbFGF does not need to have a signal peptide. In other words, "bbFGF is expressed as a fusion protein with a signal peptide" means that bbFGF forms a fusion protein with the signal peptide upon expression; the final bbFGF does not necessarily form a fusion protein with the signal peptide. The term "nucleic acid sequence" may also be interpreted as "gene." For example, a nucleic acid sequence encoding bbFGF is also referred to as a "gene encoding bbFGF" or a "bbFGF gene." Examples of nucleic acid sequences include DNA. Furthermore, the bbFGF expression cassette may contain regulatory sequences (e.g., operators, SD sequences, terminators) effective for expressing the fusion protein of the present invention in coryneform bacteria, at appropriate positions to enable their function.

[0031] The promoter used in the present invention is not particularly limited as long as it is a promoter that functions in coryneform bacteria. A "promoter that functions in coryneform bacteria" refers to a promoter that has promoter activity (i.e., gene transcription activity) in coryneform bacteria. Examples of promoters that function in coryneform bacteria include those described below in the section "Methods for increasing protein activity."

[0032] The signal peptide used in the present invention is not particularly limited as long as it is a signal peptide that functions in coryneform bacteria. The signal peptide may be a signal peptide derived from coryneform bacteria (e.g., derived from a host) or may be a signal peptide derived from a heterologous species. A "signal peptide that functions in coryneform bacteria" refers to a peptide that, when linked to the N-terminus of a protein such as bbFGF, enables the coryneform bacteria to secrete the protein. Whether a certain signal peptide functions in coryneform bacteria can be confirmed, for example, by fusing a protein such as bbFGF with the signal peptide, expressing the protein, and confirming whether the protein is secreted.

[0033] Examples of signal peptides include Tat-dependent signal peptides and Sec-dependent signal peptides.

[0034] The term "Tat system-dependent signal peptide" refers to a signal peptide recognized by the Tat system. Specifically, the "Tat system-dependent signal peptide" may be a peptide that, when linked to the N-terminus of a protein such as bbFGF, causes the protein to be secreted by the Tat system secretion apparatus.

[0035] Examples of Tat-dependent signal peptides include the signal peptide of the E. coli TorA protein (trimethylamine-N-oxide reductase), the signal peptide of the E. coli SufI protein (suppressor of ftsI), the signal peptide of the Bacillus subtilis PhoD protein (phosphodiesterase), the signal peptide of the Bacillus subtilis LipA protein (lipoic acid synthase), and the signal peptide of the Arthrobacter globiformis IMD protein (isomaltodextranase). The amino acid sequences of these signal peptides are as follows: TorA signal peptide: MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 18) SufI signal peptide: MSLSRRQFIQASGIALCAGAVPLKASA (SEQ ID NO: 19) PhoD signal peptide: MAYDSRFDEWVQKLKEESFQNNTFDRRKFIQGAGKIAGLSLGLTIAQS (SEQ ID NO: 20) LipA signal peptide: MKFVKRRTTALVTTLMLSVTSLFALQPSAKAAEH (SEQ ID NO: 21) IMD signal peptide: MMNLSRRTLLTTGSAATLAYALGMAGSAQA (SEQ ID NO: 22)

[0036] Tat-dependent signal peptides have a twin-arginine motif, such as S / TRRXFLK (SEQ ID NO: 23) and RRX-#-# (X: naturally occurring amino acid residue, #: hydrophobic amino acid residue).

[0037] The term "Sec system-dependent signal peptide" refers to a signal peptide recognized by the Sec system. Specifically, the term "Sec system-dependent signal peptide" may be a peptide that, when linked to the N-terminus of a protein such as bbFGF, causes the protein to be secreted by the Sec system secretion apparatus.

[0038] Examples of Sec system-dependent signal peptides include signal peptides of cell surface proteins of coryneform bacteria. Cell surface proteins of coryneform bacteria are as described above. Examples of cell surface proteins of coryneform bacteria include PS1 and PS2 (CspB) derived from C. glutamicum (JP Patent Publication No. 6-502548) and SlpA (CspA) derived from C. stationis (JP Patent Publication No. 10-108675). The amino acid sequence of the signal peptide of PS1 from C. glutamicum (PS1 signal peptide) is shown in SEQ ID NO: 25, the amino acid sequence of the signal peptide of PS2 (CspB) from C. glutamicum (PS2 signal peptide) is shown in SEQ ID NO: 26, and the amino acid sequence of the signal peptide of SlpA (CspA) from C. stationis (SlpA signal peptide) is shown in SEQ ID NO: 27.

[0039] The Tat-dependent signal peptide may be a variant of the above-exemplified Tat-dependent signal peptide, so long as it has a twin-arginine motif and maintains its original function. The Sec-dependent signal peptide may be a variant of the above-exemplified Sec-dependent signal peptide, so long as it maintains its original function. The description of conservative variants of bbFGF and the bbFGF gene described below applies mutatis mutandis to variants of signal peptides and the genes encoding them. For example, the signal peptide may be a peptide having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the amino acid sequence of the above-exemplified signal peptide. The term "one or several" in the context of a signal peptide variant specifically refers to preferably 1 to 7, more preferably 1 to 5, even more preferably 1 to 3, and particularly preferably 1 to 2. The terms "TorA signal peptide," "SufI signal peptide," "PhoD signal peptide," "LipA signal peptide," "IMD signal peptide," "PS1 signal peptide," "PS2 signal peptide," and "SlpA signal peptide" are intended to encompass the peptides set forth in SEQ ID NOs: 18 to 22 and 25 to 27, respectively, as well as conservative variants thereof.

[0040] With regard to a Tat system-dependent signal peptide, "maintaining its original function" means that it is recognized by the Tat system, and specifically, it may have the function of secreting the protein by the Tat system secretion apparatus when linked to the N-terminus of a protein such as bbFGF. Whether a peptide functions as a Tat system-dependent signal peptide can be confirmed, for example, by confirming that the secretory production of a protein to which the peptide has been added at its N-terminus is increased by enhancing the Tat system secretion apparatus, or by confirming that the secretory production of a protein to which the peptide has been added at its N-terminus is decreased by deficiency of the Tat system secretion apparatus.

[0041] With regard to a Sec system-dependent signal peptide, "maintaining its original function" means that it is recognized by the Sec system, and specifically, it may have the function of secreting the protein by the Sec system secretion apparatus when linked to the N-terminus of a protein such as bbFGF. Whether a peptide functions as a Sec system-dependent signal peptide can be confirmed, for example, by confirming that the secretory production amount of a protein to which the peptide has been added at its N-terminus is increased by enhancing the Sec system secretion apparatus, or by confirming that the secretory production amount of a protein to which the peptide has been added at its N-terminus is decreased by deficiency of the Sec system secretion apparatus.

[0042] The signal peptide is generally cleaved by a signal peptidase when the translation product is secreted outside the bacterial cell. In other words, the finally obtained bbFGF does not need to have a signal peptide. The gene encoding the signal peptide can be used in its natural form, but it can also be modified to have optimal codons depending on the codon usage frequency of the host used.

[0043] In the bbFGF expression cassette, a nucleic acid sequence encoding an amino acid sequence containing Gln-Glu-Thr may be inserted between the nucleic acid sequence encoding the signal peptide and the nucleic acid sequence encoding bbFGF (WO2013 / 062029). This "amino acid sequence containing Gln-Glu-Thr" is also referred to as the "insertion sequence used in the present invention." Examples of insertion sequences used in the present invention include the amino acid sequences containing Gln-Glu-Thr described in WO2013 / 062029. The insertion sequences used in the present invention are particularly suitable for use in combination with a Sec-dependent signal peptide.

[0044] The insertion sequence used in the present invention is preferably a sequence consisting of three or more amino acid residues from the N-terminus of the mature protein of the cell surface protein CspB of coryneform bacteria (hereinafter also referred to as "mature CspB" or "mature CspB protein"). The "sequence consisting of three or more amino acid residues from the N-terminus" refers to the amino acid sequence from the first amino acid residue at the N-terminus to the third or more amino acid residues.

[0045] The cell surface protein CspB of coryneform bacteria is described below. Specific examples of CspB include CspB from C. glutamicum ATCC13869, CspB from the 28 strains of C. glutamicum described below, and variants thereof. In the amino acid sequence of C. glutamicum ATCC13869 CspB shown in SEQ ID NO: 11, amino acid residues 1 to 30 correspond to the signal peptide, and amino acid residues 31 to 499 correspond to the mature CspB protein. The amino acid sequence of the mature CspB protein from C. glutamicum ATCC13869 excluding the 30 amino acid residues in the signal peptide portion is shown in SEQ ID NO: 28. In the mature CspB from C. glutamicum ATCC13869, amino acid residues 1 to 3 at the N-terminus correspond to Gln-Glu-Thr.

[0046] The insertion sequence used in the present invention is preferably an amino acid sequence extending from the amino acid residue at position 1 to any one of amino acid residues 3 to 50 of mature CspB. The insertion sequence used in the present invention is more preferably an amino acid sequence extending from the amino acid residue at position 1 to any one of amino acid residues 3 to 8, 17, or 50 of mature CspB. The insertion sequence used in the present invention is particularly preferably an amino acid sequence extending from the amino acid residue at position 1 to any one of amino acid residues 4, 6, 17, or 50 of mature CspB.

[0047] The insertion sequence used in the present invention is preferably an amino acid sequence selected from the group consisting of the amino acid sequences A to H below. (A) Gln-Glu-Thr (B) Gln-Glu-Thr-Xaa1 (C) Gln-Glu-Thr-Xaa1-Xaa2 (D) Gln-Glu-Thr-Xaa1-Xaa2-Xaa3 (E) An amino acid sequence in which amino acid residues at positions 4 to 7 of mature CspB are added to Gln-Glu-Thr (F) An amino acid sequence in which amino acid residues at positions 4 to 8 of mature CspB are added to Gln-Glu-Thr (G) An amino acid sequence in which amino acid residues at positions 4 to 17 of mature CspB are added to Gln-Glu-Thr (H) An amino acid sequence in which amino acid residues at positions 4 to 50 of mature CspB are added to Gln-Glu-Thr In the amino acid sequences A to H, Xaa1 is Asn, Gly, Thr, Pro, or Ala, Xaa2 is Pro, Thr, or Val, and Xaa3 is Thr or Tyr. Furthermore, in the amino acid sequences A to H, "amino acid residues at positions 4 to X of mature CspB are added to Gln-Glu-Thr" means that amino acid residues at positions 4 to X of the N-terminus of mature CspB are added to Thr in Gln-Glu-Thr. Typically, the first to third amino acid residues at the N-terminus of mature CspB are Gln-Glu-Thr, and in that case, "an amino acid sequence in which amino acid residues at positions 4 to X of mature CspB are added to Gln-Glu-Thr" is synonymous with the amino acid sequence consisting of amino acid residues at positions 1 to X of mature CspB.

[0048] Specifically, the insertion sequence used in the present invention is preferably an amino acid sequence selected from the group consisting of Gln-Glu-Thr-Asn-Pro-Thr (SEQ ID NO: 32), Gln-Glu-Thr-Gly-Thr-Tyr (SEQ ID NO: 33), Gln-Glu-Thr-Thr-Val-Thr (SEQ ID NO: 34), Gln-Glu-Thr-Pro-Val-Thr (SEQ ID NO: 35), and Gln-Glu-Thr-Ala-Val-Thr (SEQ ID NO: 36).

[0049] The "amino acid residue at position X in mature CspB" refers to the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 28. In the amino acid sequence of any mature CspB, which amino acid residue is "the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 28" can be determined by aligning the amino acid sequence of any mature CspB with the amino acid sequence of SEQ ID NO: 28.

[0050] "Bovine basic fibroblast growth factor (bbFGF)" may refer to bovine-derived basic fibroblast growth factor. The term "bovine" in reference to bbFGF may refer to organisms of the genus Bos. Examples of organisms of the genus Bos include Bos taurus. The nucleotide sequence of the bbFGF gene and the amino acid sequence of bbFGF can be obtained from public databases such as NCBI and technical literature such as patent documents. The nucleotide sequence of the Bos taurus bbFGF gene (modified to take into account the codon usage of C. glutamicum) and the amino acid sequence of bbFGF encoded by the gene are shown in SEQ ID NOs: 45 and 46, respectively.

[0051] The bbFGF gene may be, for example, a gene having the nucleotide sequence of the bbFGF gene exemplified above (e.g., the nucleotide sequence shown in SEQ ID NO: 45). The bbFGF may be, for example, a protein having the amino acid sequence of the bbFGF gene exemplified above (e.g., the amino acid sequence shown in SEQ ID NO: 46). Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or amino acid sequence" may mean that the gene or protein contains the nucleotide sequence or amino acid sequence, and may also include cases where the gene or protein consists of the nucleotide sequence or amino acid sequence.

[0052] The bbFGF gene may be, for example, a variant of a gene having the nucleotide sequence of the bbFGF gene exemplified above. The bbFGF may be, for example, a variant of a protein having the amino acid sequence of the bbFGF exemplified above. The variant is not particularly limited as long as it has the desired function. The variant may be, for example, a conservative variant. A "conservative variant" refers to a variant that maintains the original function. Examples of variants such as conservative variants include homologs and artificially modified versions of genes and proteins having the nucleotide sequence of the bbFGF gene exemplified above and the amino acid sequence of the bbFGF exemplified above. In the case of bbFGF, examples of homologs include those found in bovine (Bos) organisms. The protein identified by the biological species from which it originates is not limited to the protein itself found in that biological species, but also includes proteins having the amino acid sequence of a protein found in that biological species and variants thereof. The variant may or may not be found in that biological species. That is, for example, "bbFGF" is not limited to the basic fibroblast growth factor itself found in bovine (Bos) organisms, but also includes proteins having the amino acid sequence of the basic fibroblast growth factor found in bovine (Bos) organisms and variants thereof.

[0053] "Maintaining the original function" means that the variant of a gene or protein has a function (e.g., activity or property) corresponding to the function (e.g., activity or property) of the original gene or protein. "Maintaining the original function" with respect to a gene means that the variant of a gene encodes a protein whose original function is maintained. "Maintaining the original function" with respect to bbFGF may mean that the variant of bbFGF has the function of promoting the proliferation of animal cells.

[0054] Examples of variants such as conservative variants are given below.

[0055] The bbFGF gene and bbFGF homologs can be easily obtained from public databases by, for example, BLAST or FASTA searches using the nucleotide sequence of the bbFGF gene or the amino acid sequence of the bbFGF gene as a query sequence. Alternatively, bbFGF gene homologs can be obtained by PCR using, for example, the chromosomes of various organisms as templates and oligonucleotides prepared based on the nucleotide sequence of the bbFGF gene as primers.

[0056] The bbFGF gene may encode a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added at one or more positions in the amino acid sequence of the bbFGF exemplified above. For example, the encoded protein may have its N-terminus and / or C-terminus extended or shortened. Note that the term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0057] The substitution, deletion, insertion, or addition of one or several amino acids may be a conservative mutation that maintains the original function of the protein. A typical conservative mutation is a conservative substitution. A conservative substitution is a mutation in which Phe, Trp, and Tyr are substituted for each other when the substitution site is an aromatic amino acid; Leu, Ile, and Val are substituted for each other when the substitution site is a hydrophobic amino acid; Gln and Asn are substituted for each other when the substitution site is a polar amino acid; Lys, Arg, and His are substituted for each other when the substitution site is a basic amino acid; Asp and Glu are substituted for each other when the substitution site is an acidic amino acid; and Ser and Thr are substituted for each other when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered to be conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Il Examples of such substitutions include substitutions of Lys with Leu, Met, Val, or Phe, substitutions of Leu with Ile, Met, Val, or Phe, substitutions of Lys with Asn, Glu, Gln, His, or Arg, substitutions of Met with Ile, Leu, Val, or Phe, substitutions of Phe with Trp, Tyr, Met, Ile, or Leu, substitutions of Ser with Thr or Ala, substitutions of Thr with Ser or Ala, substitutions of Trp with Phe or Tyr, substitutions of Tyr with His, Phe, or Trp, and substitutions of Val with Met, Ile, or Leu. The above-mentioned amino acid substitutions, deletions, insertions, additions, inversions, and the like also include those resulting from naturally occurring mutations (mutants or variants) based on individual differences or differences in species of the organism from which the gene is derived.

[0058] Furthermore, the bbFGF gene may be a gene encoding a protein having an amino acid sequence that is, for example, 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence of the bbFGF exemplified above.

[0059] The bbFGF gene may also be a gene, such as DNA, that hybridizes under stringent conditions with a probe prepared from the nucleotide sequence of the bbFGF gene, such as a sequence complementary to all or part of the nucleotide sequence of the bbFGF gene, as exemplified above. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. One example of such conditions is a condition under which DNAs with high identity, for example, DNAs with an identity of 50% or more, 65% or more, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more, hybridize with each other, while DNAs with lower identity do not hybridize with each other; or a condition in which washing is performed once, preferably two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0060] As mentioned above, the probe used in the hybridization may be a portion of the complementary sequence of the gene. Such a probe can be prepared by PCR using oligonucleotides prepared based on the base sequence of the bbFGF gene as primers and a DNA fragment containing the gene as a template. For example, a DNA fragment of about 300 bp in length can be used as the probe. When a DNA fragment of about 300 bp in length is used as the probe, washing conditions for the hybridization include 50°C, 2×SSC, and 0.1% SDS.

[0061] Furthermore, since codon degeneracy differs depending on the host, the bbFGF gene may be one in which any codon has been replaced with an equivalent codon. That is, the bbFGF gene may be a variant of the bbFGF gene exemplified above due to the degeneracy of the genetic code. For example, the bbFGF gene may be modified to have optimal codons depending on the codon usage frequency of the host used.

[0062] The "identity" between amino acid sequences refers to the identity between amino acid sequences calculated by blastp using default scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The "identity" between nucleotide sequences refers to the identity between nucleotide sequences calculated by blastn using default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0063] The above descriptions regarding variants of bbFGF and the bbFGF gene also apply mutatis mutandis to other proteins and the nucleotide sequences encoding them.

[0064] The bbFGF expression cassette may further contain a nucleic acid sequence encoding an amino acid sequence used for enzymatic cleavage between the nucleic acid sequence encoding the amino acid sequence containing Gln-Glu-Thr and the nucleic acid sequence encoding bbFGF. By inserting the amino acid sequence used for enzymatic cleavage into the fusion protein of the present invention, the expressed fusion protein can be enzymatically cleaved to obtain bbFGF.

[0065] The amino acid sequence used for enzymatic cleavage is not particularly limited as long as it is a sequence that can be recognized and cleaved by an enzyme that hydrolyzes peptide bonds, and a sequence that can be used may be appropriately selected depending on the amino acid sequence of bbFGF. A nucleic acid sequence encoding the amino acid sequence used for enzymatic cleavage can be appropriately designed based on the amino acid sequence. For example, a nucleic acid sequence encoding the amino acid sequence used for enzymatic cleavage can be designed to have optimal codons depending on the codon usage frequency of the host.

[0066] The amino acid sequence used for enzymatic cleavage is preferably a recognition sequence for a protease with high substrate specificity. Specific examples of such amino acid sequences include the recognition sequences for Factor Xa protease and proTEV protease. Factor Xa protease recognizes the amino acid sequence Ile-Glu-Gly-Arg (IEGR) (SEQ ID NO: 37) in proteins, and proTEV protease recognizes the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln (ENLYFQ) (SEQ ID NO: 38) in proteins, and specifically cleaves the C-terminus of each sequence.

[0067] The N-terminal region of the finally obtained bbFGF may or may not be identical to the naturally occurring protein. For example, the N-terminal region of the finally obtained bbFGF may have one or several additional amino acids added or deleted compared to the naturally occurring protein. Note that the above-mentioned "one or several" varies depending on the full length and structure of bbFGF, but specifically preferably means 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and particularly preferably 1 to 3.

[0068] The method for introducing a bbFGF expression cassette into a coryneform bacterium is not particularly limited. "Introduction of a bbFGF expression cassette" refers to having the gene construct maintained in the host. "Introduction of a bbFGF expression cassette" is not limited to the case where a pre-constructed gene construct is introduced into the host all at once, but also includes the case where at least the bbFGF gene is introduced into the host and the gene construct is constructed within the host. In the bacterium of the present invention, the bbFGF expression cassette may be present on a vector that replicates autonomously outside the chromosome, such as a plasmid, or may be integrated into the chromosome. Introduction of a bbFGF expression cassette can be carried out, for example, in the same manner as the introduction of a gene in the "method for increasing protein activity" described below.

[0069] The bbFGF expression cassette can be introduced into a host using, for example, a vector containing the gene construct. For example, the bbFGF expression cassette can be ligated to a vector to construct an expression vector for the gene construct, and the gene construct can be introduced into the host by transforming the host with the expression vector. Furthermore, for example, when a vector has a promoter that functions in coryneform bacteria, an expression vector for the bbFGF expression cassette can also be constructed by ligating a nucleotide sequence encoding the fusion protein of the present invention downstream of the promoter. There are no particular limitations on the vector, as long as it is capable of autonomous replication in coryneform bacteria. Vectors that can be used in coryneform bacteria are as described above.

[0070] Alternatively, the bbFGF expression cassette can be introduced into the host chromosome using a transposon, such as an artificial transposon. When a transposon is used, the bbFGF expression cassette is introduced into the chromosome by homologous recombination or its own transposition ability. Alternatively, the bbFGF expression cassette can be introduced into the host chromosome by other methods utilizing homologous recombination. Examples of methods utilizing homologous recombination include linear DNA, a plasmid containing a temperature-sensitive replication origin, a conjugatively transferable plasmid, or a suicide vector lacking a replication origin functional in the host. Alternatively, at least the bbFGF gene may be introduced into the chromosome to construct the bbFGF expression cassette on the chromosome. In this case, some or all of the components of the bbFGF expression cassette, other than the bbFGF gene, may be originally present on the host chromosome. Specifically, for example, a bbFGF expression cassette can be constructed on a chromosome of a host cell by simply using a promoter sequence originally present on the chromosome of the host cell and a nucleic acid sequence encoding a signal peptide connected downstream of the promoter sequence, and replacing only the gene connected downstream of the nucleic acid sequence encoding the signal peptide with the bbFGF gene. Introduction of a portion of the bbFGF expression cassette, such as the bbFGF gene, into a chromosome can be carried out in the same manner as introduction of the bbFGF expression cassette into a chromosome.

[0071] A bbFGF expression cassette and its components (e.g., promoter sequence, nucleic acid sequence encoding a signal peptide, nucleic acid sequence encoding bbFGF) can be obtained, for example, by cloning. Specifically, for example, a bbFGF gene can be obtained by cloning from an organism that has bbFGF, and then modified, for example, by introducing a nucleotide sequence encoding a signal peptide or a promoter sequence, to obtain a bbFGF expression cassette. Alternatively, a bbFGF expression cassette and its components can be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)). The obtained gene construct and its components can be used as is or with appropriate modifications.

[0072] The method for introducing the bbFGF expression cassette into the coryneform bacterium is not particularly limited, and commonly used methods such as the protoplast method (Gene, 39, 281-286 (1985)), electroporation method (Bio / Technology, 7, 1067-1070 (1989)), and electric pulse method (Japanese Patent Laid-Open Publication No. 2-207791) can be used.

[0073] <1-3> Other Properties The bacterium of the present invention may have any desired properties as long as it is capable of secreting and producing bbFGF. For example, the bacterium of the present invention may have reduced activity of a cell surface protein (WO2013 / 065869, WO2013 / 065772, WO2013 / 118544, WO2013 / 062029). The bacterium of the present invention may also be modified to reduce the activity of a penicillin-binding protein (WO2013 / 065869). The bacterium of the present invention may also be modified to increase the expression of a gene encoding a metallopeptidase (WO2013 / 065772). The bacterium of the present invention may also be modified to have a mutant ribosomal protein S1 gene (mutant rpsA gene) (WO2013 / 118544). The bacterium of the present invention may also be modified to have a mutant phoS gene (WO2016 / 171224). The bacterium of the present invention may also be modified to reduce the activity of the RegX3 protein (WO2018 / 074578). The bacterium of the present invention may also be modified to reduce the activity of the HrrSA system (WO2018 / 074579). The bacterium of the present invention may also be modified to increase the activity of the Tat system secretion apparatus. The bacterium of the present invention may also be modified to reduce the activity of protein O-mannosyltransferase (PMT) (Martina Mahne et al., The Corynebacterium glutamicum gene pmt encodes a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins. FEMS Microbiol Lett. 2006 Jun;259(2):226-33.). These properties or modifications can be used alone or in appropriate combination.

[0074] <1-3-1> Introduction of a mutant phoS gene The bacterium of the present invention may be modified to harbor a mutant phoS gene. "Harboring a mutant phoS gene" is also referred to as "having a mutant phoS gene" or "having a mutation in the phoS gene." Furthermore, "harboring a mutant phoS gene" is also referred to as "having a mutant PhoS protein" or "having a mutation in the PhoS protein."

[0075] The phoS gene and the PhoS protein are described below. The phoS gene encodes the PhoS protein, a sensor kinase in the PhoRS system. The PhoRS system is a two-component regulatory system that initiates a response to environmental phosphate deficiency. The PhoRS system consists of the sensor kinase PhoS, encoded by the phoS gene, and the response regulator PhoR, encoded by the phoR gene.

[0076] A PhoS protein having a "specific mutation" is also called a "mutant PhoS protein," and the gene encoding it is also called a "mutant phoS gene." In other words, a "mutant phoS gene" is a phoS gene having a "specific mutation." A PhoS protein that does not have a "specific mutation" is also called a "wild-type PhoS protein," and the gene encoding it is also called a "wild-type phoS gene." In other words, a "wild-type phoS gene" is a phoS gene that does not have a "specific mutation." Note that the term "wild-type" used here is a convenient description to distinguish it from a "mutant," and is not limited to naturally occurring ones, as long as they do not have a "specific mutation." A "specific mutation" will be described later.

[0077] Examples of wild-type phoS genes include the phoS genes of coryneform bacteria. Specific examples of phoS genes of coryneform bacteria include the phoS genes of C. glutamicum YDK010 strain, C. glutamicum ATCC13032 strain, C. glutamicum ATCC14067 strain, C. callunae, C. crenatum, and C. efficiens. The nucleotide sequence of the phoS gene of the C. glutamicum YDK010 strain is shown in SEQ ID NO: 1. The amino acid sequences of the wild-type PhoS proteins encoded by these phoS genes are shown in SEQ ID NOs: 2 to 7, respectively.

[0078] The wild-type phoS gene may be a variant of the wild-type phoS gene exemplified above, so long as it does not have the "specific mutation" and maintains its original function. Similarly, the wild-type PhoS protein may be a variant of the wild-type PhoS protein exemplified above, so long as it does not have the "specific mutation" and maintains its original function. In other words, the term "wild-type phoS gene" is not limited to the wild-type phoS gene exemplified above, but also includes its conservative variants that do not have the "specific mutation." Similarly, the term "wild-type PhoS protein" is not limited to the wild-type PhoS protein exemplified above, but also includes its conservative variants that do not have the "specific mutation." The above descriptions regarding the bbFGF gene and conservative variants of bbFGF apply mutatis mutandis to the wild-type PhoS protein and variants of the wild-type phoS gene. For example, the wild-type phoS gene may be a gene that encodes a protein having an amino acid sequence in which one or more amino acids at one or more positions in the above amino acid sequence have been substituted, deleted, inserted, and / or added, so long as it does not have a "specific mutation" and the original function is maintained. Furthermore, for example, the wild-type phoS gene may be a gene that encodes a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire above amino acid sequence, so long as it does not have a "specific mutation" and the original function is maintained.

[0079] Note that "maintaining the original function" may mean that, in the case of a wild-type PhoS protein, a protein variant retains the function of a PhoS protein (e.g., the function of a protein consisting of the amino acid sequences set forth in SEQ ID NOS: 2 to 7). Furthermore, "maintaining the original function" may mean that, in the case of a wild-type PhoS protein, a protein variant retains the function of a sensor kinase in the PhoRS system. That is, "the function of a PhoS protein" may specifically mean the function of a sensor kinase in the PhoRS system. "The function of a sensor kinase in the PhoRS system" may specifically mean the function of conjugating with the PhoR protein, which is a response regulator, to elicit a response to environmental phosphate deficiency. More specifically, "the function of a sensor kinase in the PhoRS system" may mean the function of sensing environmental phosphate deficiency, undergoing autophosphorylation, and activating the PhoR protein by phosphoryl transfer.

[0080] Whether a PhoS protein variant functions as a sensor kinase of the PhoRS system can be confirmed, for example, by introducing a gene encoding the variant into a phoS gene-deficient strain of a coryneform bacterium and determining whether the responsiveness to phosphate deficiency is complemented. Complementation of the responsiveness to phosphate deficiency can be detected, for example, by improved growth under phosphate-deficient conditions or by induction of expression of a gene known to be induced under phosphate-deficient conditions (J. Bacteriol., 188, 724-732 (2006)). Examples of phoS gene-deficient strains of coryneform bacteria that can be used include the phoS gene-deficient strain of C. glutamicum YDK010 and the phoS gene-deficient strain of C. glutamicum ATCC13032.

[0081] In the wild-type PhoS protein, the histidine residue that undergoes autophosphorylation is preferably conserved. That is, the conservative mutation preferably occurs at an amino acid residue other than the histidine residue that undergoes autophosphorylation. The "histidine residue that undergoes autophosphorylation" refers to the histidine residue at position 276 in the wild-type PhoS protein. Furthermore, the wild-type PhoS protein preferably has a conserved sequence of, for example, the wild-type PhoS protein exemplified above. That is, the conservative mutation preferably occurs at an amino acid residue that is not conserved in, for example, the wild-type PhoS protein exemplified above.

[0082] The mutant PhoS protein has a "specific mutation" in the amino acid sequence of the wild-type PhoS protein as described above.

[0083] In other words, the mutant PhoS protein may be identical to the wild-type PhoS protein or a conservative variant thereof exemplified above, except for the "specific mutation." Specifically, for example, the mutant PhoS protein may be a protein having the amino acid sequence set forth in SEQ ID NOs: 2 to 7, except for the "specific mutation." Furthermore, specifically, for example, the mutant PhoS protein may be a protein having an amino acid sequence containing one or more amino acid substitutions, deletions, insertions, and / or additions in the amino acid sequence set forth in SEQ ID NOs: 2 to 7, except for the "specific mutation." Furthermore, specifically, for example, the mutant PhoS protein may be a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more identical to the amino acid sequence set forth in SEQ ID NOs: 2 to 7, except for the "specific mutation."

[0084] In other words, the mutant PhoS protein may be a variant of the wild-type PhoS protein exemplified above that has a "specific mutation" and further contains conservative mutations at positions other than the "specific mutation." Specifically, for example, the mutant PhoS protein may be a protein having an amino acid sequence shown in SEQ ID NOs: 2 to 7 that has a "specific mutation" and further contains one or more amino acid substitutions, deletions, insertions, and / or additions at positions other than the "specific mutation."

[0085] The mutant phoS gene is not particularly limited as long as it encodes the above-mentioned mutant PhoS protein.

[0086] The "specific mutation" contained in the mutant PhoS protein will be explained below.

[0087] The "specific mutation" is not particularly limited as long as it changes the amino acid sequence of the wild-type PhoS protein as described above and is effective for the secretory production of heterologous proteins such as bbFGF.

[0088] The "specific mutation" is preferably a mutation that improves the secretory production of a heterologous protein such as bbFGF. "Improving the secretory production of a heterologous protein" means that a coryneform bacterium (modified strain) modified to have a mutant phoS gene can secrete and produce a greater amount of heterologous protein than an unmodified strain. The "unmodified strain" refers to a control strain that does not have a mutation in the phoS gene, i.e., a control strain that does not have a mutant phoS gene, and may be, for example, a wild-type strain or a parent strain. The term "secreting and producing a greater amount of heterologous protein than an unmodified strain" is not particularly limited as long as the amount of secreted heterologous protein is increased compared to an unmodified strain. For example, it may mean that the amount of heterologous protein accumulated in the medium and / or on the bacterial cell surface is preferably 1.1-fold or more, more preferably 1.2-fold or more, even more preferably 1.3-fold or more, even more preferably 2-fold or more, and particularly preferably 5-fold or more, of the unmodified strain. Furthermore, "secreting and producing a greater amount of heterologous protein than a non-modified strain" may mean that when an unconcentrated culture supernatant of a non-modified strain is subjected to SDS-PAGE and stained with CBB, the heterologous protein cannot be detected, but when an unconcentrated culture supernatant of a modified strain is subjected to SDS-PAGE and stained with CBB, the heterologous protein can be detected. Note that "improving the secretory production amount of a heterologous protein" does not necessarily mean that the secretory production amount of all heterologous proteins needs to be improved; it is sufficient if the secretory production amount of the heterologous protein set as the target for secretory production is improved. "Improving the secretory production amount of a heterologous protein" may specifically mean, for example, improving the secretory production amount of a heterologous protein described in the Examples.

[0089] Whether a certain mutation improves the secretory production of a heterologous protein such as bbFGF can be confirmed, for example, by creating a strain based on a strain belonging to the coryneform bacteria that has been modified to have a gene encoding a PhoS protein with the mutation, quantifying the amount of heterologous protein secreted when the modified strain is cultured in a medium, and comparing this with the amount of heterologous protein secreted when the unmodified strain (unmodified strain) is cultured in a medium.

[0090] The amino acid sequence change is preferably an amino acid residue substitution. That is, the "specific mutation" is preferably a substitution of any amino acid residue in the wild-type PhoS protein with another amino acid residue. The amino acid residue substituted by the "specific mutation" may be a single residue, or a combination of two or more residues. The amino acid residue substituted by the "specific mutation" may preferably be an amino acid residue other than an autophosphorylated histidine residue. The amino acid residue substituted by the "specific mutation" may more preferably be an amino acid residue in the HisKA domain other than an autophosphorylated histidine residue. The "autophosphorylated histidine residue" refers to the histidine residue at position 276 of the wild-type PhoS protein. The "HisKA domain" refers to the region consisting of amino acid residues 266 to 330 of the wild-type PhoS protein. The amino acid residue substituted by the "specific mutation" may particularly preferably be the tryptophan residue at position 302 (W302) of the wild-type PhoS protein.

[0091] In the above mutations, the substituted amino acid residues include those other than the original amino acid residues among K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), and Q (Gln). The substituted amino acid residue can be selected, for example, from those that improve the secretory production of heterologous proteins such as bbFGF.

[0092] When W302 is substituted, the amino acid residue after substitution can be an amino acid residue other than aromatic amino acids and histidine. Specific examples of "amino acid residues other than aromatic amino acids and histidine" include K (Lys), R (Arg), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), and Q (Gln). More specific examples of "amino acid residues other than aromatic amino acids and histidine" include K (Lys), A (Ala), V (Val), S (Ser), C (Cys), M (Met), D (Asp), and N (Asn).

[0093] The "specific mutation" in the phoS gene refers to a mutation in the nucleotide sequence that causes the above-mentioned "specific mutation" in the encoded PhoS protein.

[0094] The "amino acid residue at position X of the wild-type PhoS protein" refers to the amino acid residue corresponding to the amino acid residue at position X in SEQ ID NO: 2. For example, "W302" refers to the amino acid residue corresponding to the tryptophan residue at position 302 in SEQ ID NO: 2. The above amino acid residue positions indicate relative positions, and their absolute positions may change due to amino acid deletion, insertion, addition, etc. For example, if a wild-type PhoS protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 is deleted or inserted at a position N-terminal to position X, the original amino acid residue at position X becomes the X-1 or X+1 amino acid residue, respectively, counting from the N-terminus, and is still considered to be the "amino acid residue at position X of the wild-type PhoS protein." Specifically, in the amino acid sequences of the wild-type PhoS proteins set forth in SEQ ID NOs: 2 to 7, "W302" refers to the tryptophan residues at positions 302, 302, 302, 321, 275, and 286, respectively. In the amino acid sequences of the wild-type PhoS proteins shown in SEQ ID NOs: 2 to 7, the "histidine residue at position 276 of the wild-type PhoS protein (the histidine residue that is autophosphorylated)" refers to the histidine residues at positions 276, 276, 276, 295, 249, and 260, respectively. In the amino acid sequences of the wild-type PhoS proteins shown in SEQ ID NOs: 2 to 7, the "region consisting of amino acid residues at positions 266 to 330 of the wild-type PhoS protein (HisKA domain)" refers to the regions consisting of amino acid residues at positions 266 to 330, 266 to 330, 266 to 330, 285 to 349, 239 to 303, and 250 to 314, respectively.

[0095] Note that "W302" as used herein is typically a tryptophan residue, but does not necessarily have to be a tryptophan residue. That is, when the wild-type PhoS protein has an amino acid sequence other than those set forth in SEQ ID NOS: 2 to 7, "W302" may not be a tryptophan residue. Therefore, for example, "a mutation in which W302 is substituted with a cysteine ​​residue" is not limited to a mutation in which "W302" is a tryptophan residue and the tryptophan residue is substituted with a cysteine ​​residue. It also encompasses a mutation in which "W302" is K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), Y (Tyr), M (Met), D (Asp), E (Glu), N (Asn), or Q (Gln) and the amino acid residue is substituted with a cysteine ​​residue. The same applies to other mutations.

[0096] In the amino acid sequence of any PhoS protein, which amino acid residue corresponds to the amino acid residue at position X in SEQ ID NO: 2 can be determined by aligning the amino acid sequence of the PhoS protein with the amino acid sequence of SEQ ID NO: 2. Alignment can be performed using, for example, known genetic analysis software. Specific examples of such software include DNASIS manufactured by Hitachi Solutions and GENETYX manufactured by Genetyx (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1), 72-96, 1991; Barton GJ et al., Journal of molecular biology, 198(2), 327-37, 1987).

[0097] A mutant phoS gene can be obtained, for example, by modifying a wild-type phoS gene so that the encoded PhoS protein has the above-mentioned "specific mutation." The wild-type phoS gene that is the source of the modification can be obtained, for example, by cloning from an organism having a wild-type phoS gene or by chemical synthesis. A mutant phoS gene can also be obtained without using a wild-type phoS gene. For example, a mutant phoS gene can be obtained directly by chemical synthesis. The obtained mutant phoS gene can be further modified and used.

[0098] Genetic modification can be performed using known techniques. For example, a desired mutation can be introduced into a desired site in DNA by site-directed mutagenesis. Examples of site-directed mutagenesis include PCR-based methods (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)).

[0099] Hereinafter, a method for modifying a coryneform bacterium so that it has a mutant phoS gene will be described.

[0100] Modification of a coryneform bacterium to have a mutant phoS gene can be achieved by introducing the mutant phoS gene into the coryneform bacterium. Modification of a coryneform bacterium to have a mutant phoS gene can also be achieved by introducing the above-mentioned "specific mutation" into the phoS gene on the chromosome of the coryneform bacterium. Introduction of a mutation into a gene on the chromosome can be achieved by natural mutation, mutagen treatment, or genetic engineering techniques.

[0101] The method for introducing a mutant phoS gene into a coryneform bacterium is not particularly limited. In the bacterium of the present invention, the mutant phoS gene may be maintained in an expressible state under the control of a promoter that functions in the coryneform bacterium. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be a promoter native to the phoS gene or a promoter of another gene. In the bacterium of the present invention, the mutant phoS gene may be present on an extrachromosomally autonomously replicating vector such as a plasmid, or may be integrated into the chromosome. The bacterium of the present invention may have only one copy of the mutant phoS gene, or may have two or more copies. The bacterium of the present invention may have only one type of mutant phoS gene, or may have two or more types of mutant phoS genes. Introduction of the mutant phoS gene can be performed, for example, in the same manner as the introduction of genes in the methods for increasing gene expression or the introduction of a bbFGF expression cassette, as described below.

[0102] The bacterium of the present invention may or may not have a wild-type phoS gene, but preferably does not have one.

[0103] A coryneform bacterium lacking the wild-type phoS gene can be obtained by disrupting the wild-type phoS gene on its chromosome. Disruption of the wild-type phoS gene can be carried out by known techniques. Specifically, for example, the wild-type phoS gene can be disrupted by deleting part or all of the promoter region and / or coding region of the wild-type phoS gene.

[0104] Furthermore, by replacing the wild-type phoS gene on the chromosome with a mutant phoS gene, it is possible to obtain a coryneform bacterium that has been modified to have the mutant phoS gene but not the wild-type phoS gene. Examples of methods for performing such gene replacement include methods using linear DNA, such as "Red-driven integration" (Datsenko, K. A., and Wanner, BL Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), a method combining Red-driven integration with an excision system derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184:5200-5203 (2002)) (see WO2005 / 010175), methods using a plasmid containing a temperature-sensitive replication origin, methods using a conjugatively transferable plasmid, and methods using a suicide vector that does not have a replication origin that functions in the host (U.S. Pat. No. 6,303,383, JP 05-007491 A).

[0105] The PhoS protein functions in conjunction with the PhoR protein, a response regulator, i.e., it triggers a response to environmental phosphate deficiency. Therefore, the bacterium of the present invention has a phoR gene so that the mutant PhoS protein can function. The phoR gene encodes the PhoR protein, which is a response regulator of the PhoRS system. "Having the phoR gene" is also referred to as "having the PhoR protein." Generally, it is sufficient for the PhoR protein inherent in the bacterium of the present invention to function in conjunction with the mutant PhoS protein. Alternatively, an appropriate phoR gene may be introduced into the bacterium of the present invention in addition to or instead of the phoR gene inherent in the bacterium of the present invention. The phoR gene to be introduced is not particularly limited, as long as it encodes a PhoR protein that functions in conjunction with the mutant PhoS protein.

[0106] Examples of the phoR gene include the phoR gene of coryneform bacteria. Specific examples of the phoR gene of coryneform bacteria include the phoR genes of C. glutamicum YDK010 strain, C. glutamicum ATCC13032 strain, C. glutamicum ATCC14067 strain, C. callunae, C. crenatum, and C. efficiens. The nucleotide sequence of the phoR gene and the amino acid sequence of the PhoR protein of the C. glutamicum ATCC13032 strain are shown in SEQ ID NOs: 8 and 9, respectively.

[0107] The phoR gene may be a variant of the exemplified phoR gene, as long as the original function is maintained. Similarly, the PhoR protein may be a variant of the exemplified PhoR protein, as long as the original function is maintained. In other words, the term "phoR gene" encompasses not only the exemplified phoR genes but also their conservative variants. Similarly, the term "PhoR protein" encompasses not only the exemplified PhoR proteins but also their conservative variants. The above descriptions regarding the bbFGF gene and conservative variants of bbFGF can be applied mutatis mutandis to variants of the PhoR protein and phoR gene. For example, the phoR gene may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions in the above amino acid sequence have been substituted, deleted, inserted, and / or added, as long as the original function is maintained. For example, the phoR gene may be a gene encoding a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence, so long as the original function is maintained. Note that, in the case of PhoR protein, "maintaining the original function" may mean that the protein variant retains the function of the PhoR protein (e.g., the function of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 9). Also, in the case of PhoR protein, "maintaining the original function" may mean that the protein variant retains the function as a response regulator of the PhoRS system. Specifically, "the function as a PhoR protein" may mean the function as a response regulator of the PhoRS system. Specifically, "the function as a response regulator of the PhoRS system" may mean the function of conjugating with the sensor kinase PhoS protein to elicit a response to environmental phosphate deficiency.More specifically, the "function of the PhoRS system as a response regulator" may be a function that is activated by phosphoryl transfer from the autophosphorylated PhoS protein upon sensing phosphate deficiency in the environment, thereby controlling the expression of genes that respond to phosphate deficiency in the environment.

[0108] Whether a PhoR protein variant functions as a response regulator of the PhoRS system can be confirmed, for example, by introducing a gene encoding the variant into a phoR gene-deficient strain of a coryneform bacterium and determining whether the responsiveness to phosphate deficiency is complemented. Complementation of the responsiveness to phosphate deficiency can be detected, for example, by improved growth under phosphate deficiency conditions or by induction of expression of a gene known to be induced under phosphate deficiency conditions (J. Bacteriol., 188, 724-732 (2006)). Examples of phoR gene-deficient strains of coryneform bacteria that can be used include the phoR gene-deficient strain of C. glutamicum YDK010 and the phoR gene-deficient strain of C. glutamicum ATCC13032.

[0109] <1-3-2> Decreased Activity of Cell Surface Protein The bacterium of the present invention may have reduced activity of a cell surface protein. Specifically, the bacterium of the present invention may have reduced activity of a cell surface protein compared to a non-modified strain. "Decreased activity of a cell surface protein" may particularly mean a reduction in the number of molecules of the cell surface protein per cell. Cell surface proteins and the genes encoding them are described below.

[0110] Cell surface proteins are proteins that make up the cell surface (S-layer) of bacteria and archaea. Examples of cell surface proteins of coryneform bacteria include PS1, PS2 (CspB), and SlpA (CspA). Specific examples of cell surface proteins of coryneform bacteria include PS1 and PS2 (CspB) of C. glutamicum (JP Patent Publication No. 6-502548) and SlpA (CspA) of C. stationis (JP Patent Publication No. 10-108675). Among these, reducing the activity of the PS2 protein is preferred.

[0111] The nucleotide sequence of the cspB gene of C. glutamicum ATCC13869 and the amino acid sequence of the PS2 protein (CspB protein) encoded by the gene are shown in SEQ ID NOs: 10 and 11, respectively.

[0112] For example, the amino acid sequences of CspB homologs from 28 strains of C. glutamicum have been reported (J. Biotechnol., 112, 177-193 (2004)). The GenBank accession numbers of these 28 C. glutamicum strains and their cspB gene homologs in the NCBI database are shown below (the numbers in parentheses indicate the GenBank accession numbers).C. glutamicum ATCC13058(AY524990) C. glutamicum ATCC13744(AY524991) C. glutamicum ATCC13745(AY524992) C. glutamicum ATCC14017(AY524993) C. glutamicum ATCC14020(AY525009) C. glutamicum ATCC14067(AY524994) C. glutamicum ATCC14068(AY525010) C. glutamicum ATCC14747(AY525011) C. glutamicum ATCC14751(AY524995) C. glutamicum ATCC14752(AY524996) C. glutamicum ATCC14915(AY524997) C. glutamicum ATCC15243(AY524998) C. glutamicum ATCC15354(AY524999) C. glutamicum ATCC17965(AY525000) C. glutamicum ATCC17966(AY525001) C. glutamicum ATCC19223(AY525002) C. glutamicum ATCC19240(AY525012) C. glutamicum ATCC21341(AY525003) C. glutamicum ATCC21645(AY525004) C. glutamicum ATCC31808(AY525013) C. glutamicum ATCC31830(AY525007) C. glutamicum ATCC31832(AY525008) C. glutamicum LP-6(AY525014) C. glutamicum DSM20137(AY525015) C. glutamicum DSM20598(AY525016) C. glutamicum DSM46307(AY525017) C. glutamicum 22220(AY525005) C. glutamicum 22243(AY525006)。

[0113] Because the nucleotide sequence of a gene encoding a cell surface protein may differ depending on the species or strain of the coryneform bacterium, the gene encoding the cell surface protein may be a variant of the gene encoding the above-exemplified cell surface protein, as long as the original function is maintained. Similarly, the cell surface protein may be a variant of the above-exemplified cell surface protein, as long as the original function is maintained. That is, for example, the term "cspB gene" encompasses not only the above-exemplified cspB genes but also their conservative variants. Similarly, the term "CspB protein" encompasses not only the above-exemplified CspB proteins but also their conservative variants. The above descriptions regarding the bbFGF gene and conservative variants of bbFGF can be applied mutatis mutandis to variants of cell surface proteins and genes encoding them. For example, the gene encoding the cell surface protein may be a gene encoding a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the above-exemplified amino acid sequence, as long as the original function is maintained. Furthermore, for example, the gene encoding the cell surface protein may be a gene encoding a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence, so long as the original function is maintained. Note that, in the case of a cell surface protein, "maintaining the original function" may mean, for example, that the cell surface protein has the property of increasing the secretory production of a heterologous protein such as bbFGF when its activity is reduced in a coryneform bacterium compared to a non-modified strain.

[0114] The term "the property of increasing the amount of secreted production of a heterologous protein when the activity is reduced in a coryneform bacterium compared to a non-modified strain" refers to the property of conferring on the coryneform bacterium the ability to secrete and produce a greater amount of heterologous protein than a non-modified strain when the activity is reduced in the coryneform bacterium. A "non-modified strain" refers to a control strain in which the activity of the cell surface protein is not reduced, and may be, for example, a wild-type strain or a parent strain. The term "secreting and producing a greater amount of heterologous protein than a non-modified strain" is not particularly limited as long as the amount of secreted production of the heterologous protein is increased compared to a non-modified strain, and may mean, for example, that the amount of heterologous protein accumulated in the medium and / or on the cell surface is preferably 1.1-fold or more, more preferably 1.2-fold or more, even more preferably 1.3-fold or more, and particularly preferably 2-fold or more of the non-modified strain. Furthermore, "secreting and producing a greater amount of a heterologous protein than a non-modified strain" may also mean that when an unconcentrated culture supernatant of a non-modified strain is subjected to SDS-PAGE and stained with CBB, the heterologous protein cannot be detected, but when an unconcentrated culture supernatant of a modified strain is subjected to SDS-PAGE and stained with CBB, the heterologous protein can be detected.

[0115] Whether a protein has the property of increasing the secretory production of a heterologous protein such as bbFGF when its activity is reduced in a coryneform bacterium compared to an unmodified strain can be confirmed by creating a strain based on a strain belonging to coryneform bacteria that has been modified to reduce the activity of the protein, quantifying the amount of the heterologous protein secreted and produced when the modified strain is cultured in a medium, and comparing this with the amount of the heterologous protein secreted and produced when the unmodified strain (unmodified strain) is cultured in a medium.

[0116] "Decreased cell surface protein activity" includes cases where a coryneform bacterium has been modified to reduce the activity of the cell surface protein, as well as cases where the activity of the cell surface protein is originally reduced in the coryneform bacterium. "Decreased cell surface protein activity in the coryneform bacterium" also includes cases where the coryneform bacterium does not originally have a cell surface protein. That is, an example of a coryneform bacterium with reduced cell surface protein activity is a coryneform bacterium that does not originally have a cell surface protein. An example of a coryneform bacterium that does not originally have a cell surface protein is a coryneform bacterium that does not originally have a gene encoding a cell surface protein. Note that "coryneform bacterium does not originally have a cell surface protein" may mean that the coryneform bacterium does not originally have one or more proteins selected from cell surface proteins found in other strains of the species to which the coryneform bacterium belongs. For example, "C. glutamicum does not originally have cell surface proteins" may mean that the C. glutamicum strain does not originally have one or more proteins selected from cell surface proteins found in other C. glutamicum strains, i.e., PS1 and / or PS2 (CspB). An example of a coryneform bacterium that does not originally have cell surface proteins is C. glutamicum ATCC 13032, which does not originally have the cspB gene.

[0117] <1-3-3> Protein Secretion System The bacterium of the present invention has a protein secretion system. The bacterium of the present invention may inherently have a protein secretion system. The protein secretion system is not particularly limited, as long as it is capable of secreting proteins such as bbFGF. Examples of protein secretion systems include the Sec system (Sec system secretion apparatus) and the Tat system (Tat system secretion apparatus). The bacterium of the present invention may be modified to increase the activity of the protein secretion system (e.g., the Tat system secretion apparatus). Specifically, the bacterium of the present invention may be modified to increase the activity of the protein secretion system (e.g., the Tat system secretion apparatus) compared to an unmodified strain. The activity of the Tat system secretion apparatus can be increased, for example, by increasing the expression of one or more genes selected from genes encoding the Tat system secretion apparatus. More specifically, the bacterium of the present invention may be modified to increase the expression of one or more genes selected from genes encoding the Tat system secretion apparatus. Increased activity of the Tat system secretion apparatus is particularly advantageous when secreting and producing bbFGF using a Tat system-dependent signal peptide. A method for increasing the expression of genes encoding the Tat-based secretion system is described in Japanese Patent No. 4730302.

[0118] Genes encoding the Tat system secretion apparatus include the tatA gene, the tatB gene, the tatC gene, and the tatE gene.

[0119] Specific examples of genes encoding the Tat system secretion apparatus include the tatA gene, tatB gene, and tatC gene of C. glutamicum. The tatA gene, tatB gene, and tatC gene of C. glutamicum ATCC 13032 correspond to the complementary sequence of the sequence from positions 1571065 to 1571382, the sequence from positions 1167110 to 1167580, and the sequence from positions 1569929 to 1570873, respectively, in the genome sequence registered in the NCBI database as GenBank accession NC_003450 (VERSION NC_003450.3 GI:58036263). The TatA, TatB, and TatC proteins of C. glutamicum ATCC 13032 have been registered as GenBank accession numbers NP_600707 (version NP_600707.1 GI:19552705, locus_tag="NCgl1434"), NP_600350 (version NP_600350.1 GI:19552348, locus_tag="NCgl1077"), and NP_600706 (version NP_600706.1 GI:19552704, locus_tag="NCgl1433"), respectively. The nucleotide sequences of the tatA gene, tatB gene, and tatC gene of C. glutamicum ATCC 13032 and the amino acid sequences of the TatA protein, TatB protein, and TatC protein are shown in SEQ ID NOs: 12 to 17.

[0120] Specific examples of genes encoding the Tat system secretion apparatus include the tatA, tatB, tatC, and tatE genes of E. coli. The tatA, tatB, tatC, and tatE genes of E. coli K-12 MG1655 correspond to the sequences of positions 4019968 to 4020237, 4020241 to 4020756, 4020759 to 4021535, and 658170 to 658373, respectively, in the genome sequence registered in the NCBI database as GenBank accession NC_000913 (VERSION NC_000913.2 GI:49175990). The TatA, TatB, TatC, and TatE proteins of E. coli K-12 MG1655 were identified in GenBank accession numbers NP_418280 (version NP_418280.4 GI:90111653, locus_tag="b3836"), YP_026270 (version YP_026270.1 GI:49176428, locus_tag="b3838"), NP_418282 (version NP_418282.1 GI:16131687, locus_tag="b3839"), and NP_415160 (version NP_415160.1 GI:16131687), respectively. It is registered as GI:16128610, locus_tag="b0627").

[0121] The gene encoding the Tat system secretion apparatus may be a variant of the gene encoding the Tat system secretion apparatus exemplified above, so long as the original function is maintained. Similarly, the Tat system secretion apparatus may be a variant of the Tat system secretion apparatus exemplified above, so long as the original function is maintained. That is, for example, the terms "tatA gene," "tatB gene," "tatC gene," and "tatE gene" encompass the tatA gene, tatB gene, tatC gene, and tatE gene exemplified above, respectively, as well as conservative variants thereof. Similarly, the terms "TatA protein," "TatB protein," "TatC protein," and "TatE protein" encompass the TatA protein, TatB protein, TatC protein, and TatE protein exemplified above, as well as conservative variants thereof, respectively. The above descriptions regarding the bbFGF gene and conservative variants of bbFGF apply mutatis mutandis to variants of the Tat system secretion apparatus and the gene encoding it. For example, a gene encoding a Tat system secretion system may be a gene encoding a protein having an amino acid sequence in which one or more amino acids at one or more positions have been substituted, deleted, inserted, and / or added, as long as the original function is maintained. Furthermore, for example, a gene encoding a Tat system secretion system may be a gene encoding a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence, as long as the original function is maintained. Note that, in the case of a Tat system secretion system, "maintaining the original function" may mean that the protein having a Tat system-dependent signal peptide added to its N-terminus is capable of being secreted extracellularly.

[0122] Increased activity of the Tat secretion apparatus can be confirmed, for example, by confirming an increase in the secretory production of a protein having a Tat-dependent signal peptide added to its N-terminus. The secretory production of a protein having a Tat-dependent signal peptide added to its N-terminus may be increased, for example, by 1.5-fold or more, 2-fold or more, or 3-fold or more compared to that of an unmodified strain.

[0123] <1-3-4> Reduced PMT Activity The bacterium of the present invention may have reduced activity of protein O-mannosyltransferase (PMT). Specifically, the bacterium of the present invention may have reduced PMT activity compared to a non-modified strain. "Reduced PMT activity" may particularly mean a reduction in the number of PMT molecules per cell. PMT and the gene encoding it are described below.

[0124] "Protein O-mannosyltransferase (PMT)" may refer to a protein that has the activity of catalyzing the O-maltosylation of a secreted protein. This activity is also referred to as "PMT activity." A gene encoding PMT is also referred to as "PMT gene."

[0125] The nucleotide sequence of the PMT gene and the amino acid sequence of PMT of coryneform bacteria can be obtained, for example, from public databases such as NCBI, or from technical literature such as patent documents. The nucleotide sequence of the PMT gene (also referred to as the "pmt1 gene") of the C. glutamicum ATCC 13869 strain and the amino acid sequence of the PMT (also referred to as "PMT1") encoded by this gene are shown in SEQ ID NOs: 39 and 40, respectively.

[0126] Because the nucleotide sequence of the PMT gene may differ depending on the species or strain to which the coryneform bacterium belongs, the PMT gene may be a variant of the PMT gene exemplified above, so long as the original function is maintained. Similarly, the PMT may be a variant of the PMT gene exemplified above, so long as the original function is maintained. In other words, the term "PMT gene" encompasses not only the PMT genes exemplified above, but also their conservative variants. Similarly, the term "PMT" encompasses not only the PMT genes exemplified above, but also their conservative variants. The above descriptions regarding the bbFGF gene and conservative variants of bbFGF can be applied mutatis mutandis to variants of PMTs and genes encoding them. For example, the PMT gene may be a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions in the above amino acid sequence have been substituted, deleted, inserted, and / or added, so long as the original function is maintained. Furthermore, for example, the PMT gene may be a gene encoding a protein having an amino acid sequence that is 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identical to the entire amino acid sequence described above, so long as the original function is maintained. In the case of PMT, "maintaining the original function" may mean, for example, having PMT activity.

[0127] Whether a protein has PMT activity can be confirmed, for example, by creating a strain based on a strain belonging to the coryneform bacteria that has been modified to reduce the activity of the protein, and confirming that the degree of O-glycosylation of the protein secreted and produced when the modified strain is cultured in a medium is reduced compared to the degree of O-glycosylation of the protein secreted and produced when the unmodified strain (unmodified strain) is cultured in a medium.

[0128] <1-4> Methods for Increasing Protein Activity Methods for increasing protein activity (including methods for increasing gene expression) are described below.

[0129] "Increased protein activity" means that the activity of the protein is increased compared to that of an unmodified strain. Specifically, "increased protein activity" means that the activity of the protein per cell is increased compared to that of an unmodified strain. Here, "unmodified strain" refers to a control strain that has not been modified to reduce the activity of the target protein. Examples of unmodified strains include wild-type strains and parent strains. Specific examples of unmodified strains include the type strains of each bacterial species. Specific examples of unmodified strains also include the strains exemplified in the description of coryneform bacteria. That is, in one embodiment, the activity of the protein may be increased compared to that of the type strain (i.e., the type strain of the species to which the bacterium of the present invention belongs). In another embodiment, the activity of the protein may be increased compared to that of the C. glutamicum ATCC 13869 strain. In another embodiment, the activity of the protein may be increased compared to that of the C. glutamicum ATCC 13032 strain. In another embodiment, the activity of the protein may be increased compared to that of C. glutamicum AJ12036 (FERM BP-734). In another embodiment, the activity of the protein may be increased compared to that of the C. glutamicum YDK010 strain. "Increased protein activity" is also referred to as "enhanced protein activity." More specifically, "increased protein activity" may mean an increase in the number of molecules of the protein per cell and / or an increase in the function of the protein per molecule compared to a non-modified strain. That is, the "activity" in "increased protein activity" is not limited to the catalytic activity of the protein, but may also refer to the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. The "number of protein molecules per cell" may mean the average number of molecules of the protein per cell. Furthermore, "increasing the activity of a protein" includes not only increasing the activity of a target protein in a strain that originally has the activity of that protein, but also imparting the activity of that protein to a strain that does not originally have the activity of that protein.Furthermore, as long as the resulting protein activity is increased, the activity of a suitable target protein may be imparted after reducing or eliminating the activity of a target protein that the host naturally possesses.

[0130] The degree of increase in protein activity is not particularly limited as long as the protein activity is increased compared to that of an unmodified strain. The protein activity may be increased, for example, by 1.5 times or more, 2 times or more, or 3 times or more compared to that of an unmodified strain. Furthermore, if the unmodified strain does not have the activity of the target protein, the protein may be produced by introducing a gene encoding the protein, and for example, the protein may be produced to an extent that its activity can be measured.

[0131] Modifications that increase the activity of a protein can be achieved, for example, by increasing the expression of the gene encoding the protein. "Increased gene expression" means that the expression of the gene is increased compared to an unmodified strain such as a wild-type strain or a parent strain. "Increased gene expression" specifically means that the expression level of the gene per cell is increased compared to an unmodified strain. "Expression level of the gene per cell" may refer to the average expression level of the gene per cell. "Increased gene expression" may more specifically mean that the transcription level (mRNA level) of the gene is increased and / or the translation level (protein level) of the gene is increased. "Increased gene expression" is also referred to as "enhanced gene expression." Gene expression may be increased, for example, by 1.5-fold or more, 2-fold or more, or 3-fold or more compared to an unmodified strain. "Increased gene expression" includes not only increasing the expression level of the target gene in a strain in which the target gene is originally expressed, but also expressing the gene in a strain in which the target gene is not originally expressed. That is, "gene expression is increased" may mean, for example, introducing a target gene into a strain that does not harbor the gene, thereby expressing the gene.

[0132] Increased gene expression can be achieved, for example, by increasing the copy number of the gene.

[0133] Increasing the copy number of a gene can be achieved by introducing the gene into a host chromosome. Introduction of a gene into a chromosome can be achieved, for example, by homologous recombination (Miller, JH, Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Gene introduction methods that utilize homologous recombination include, for example, methods using linear DNA such as Red-driven integration (Datsenko, K. A., and Wanner, BL, Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), methods using plasmids containing a temperature-sensitive replication origin, methods using conjugatively transferable plasmids, methods using suicide vectors lacking a replication origin that functions in the host, and transduction methods using phages. Specifically, a host can be transformed with recombinant DNA containing a target gene, and the gene can be introduced into the host chromosome by homologous recombination with the target site on the host chromosome. The structure of the recombinant DNA used for homologous recombination is not particularly limited as long as it allows homologous recombination to occur in the desired manner. For example, a host can be transformed with linear DNA containing a target gene, with base sequences at both ends of the gene that are homologous to the target site on the chromosome, respectively, and homologous recombination can occur upstream and downstream of the target site, thereby replacing the target site with the gene. The recombinant DNA used for homologous recombination may contain a marker gene for selecting transformants. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination targeting a base sequence that exists in multiple copies on a chromosome. Examples of sequences that exist in multiple copies on a chromosome include repetitive DNA sequences and inverted repeats at both ends of transposons. Homologous recombination may also be performed targeting an appropriate sequence on a chromosome, such as a gene unnecessary for the production of a target substance.Genes can also be randomly introduced into chromosomes using transposons or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1). Such chromosome modification techniques using homologous recombination are not limited to the introduction of target genes, but can also be used for any modification of chromosomes, such as modification of expression regulatory sequences.

[0134] The introduction of the target gene into the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by PCR using primers prepared based on the sequence of the gene.

[0135] The copy number of a gene can also be increased by introducing a vector containing the gene into a host. For example, a DNA fragment containing a target gene can be ligated to a vector that functions in the host to construct an expression vector for the gene, and the host can be transformed with the expression vector to increase the copy number of the gene. A DNA fragment containing a target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism containing the target gene as a template. A vector capable of autonomous replication within host cells can be used. The vector is preferably a multicopy vector. Furthermore, the vector preferably contains a marker such as an antibiotic resistance gene for the selection of transformants. The vector may also contain a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric. Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901-2903 (1984)); plasmids having drug resistance genes improved from these; pCRY30 (JP-A-3-210184); pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX (JP-A-2-72876, U.S. Pat. No. 5,185,262); pCRY2 and pCRY3 (JP-A-1-191686); pAJ655, pAJ611, and pAJ1844 (JP-A-1983) -192900); pCG1 (JP 57-134500); pCG2 (JP 58-35197); pCG4 and pCG11 (JP 57-183799); pVK7 (JP 10-215883); pVK9 (US2006-0141588); pVC7 (JP 9-070291); pVS7 (WO2013 / 069634); pPK4 (JP 9-322774); pPK5 (WO2018 / 074579).Specific examples of vectors capable of autonomous replication in coryneform bacteria include pVC7N, a variant of pVC7 (Shuhei Hashiro et al., High copy number mutants derived from Corynebacterium glutamicum cryptic plasmid pAM330 and copy number control, J Biosci Bioeng, 2019 May;127(5):529-538.), and pVC7H1, pVC7H2, pVC7H3, pVC7H4, pVC7H5, pVC7H6, and pVC7H7 (WO2018 / 179834). Specific examples of vectors capable of autonomous replication in coryneform bacteria include pPK4H1, pPK4H2, pPK4H3, pPK4H4, pPK4H5, and pPK4H6 (WO2018 / 179834). Examples of vectors include pVC-based vectors and pPK-based vectors. pVC-based vectors include pVC7 and its variants, vectors in which the antibiotic resistance gene has been replaced with another antibiotic resistance gene, and vectors with 90% or more, 95% or more, 97% or more, or 99% or more nucleotide sequence identity therewith. pPK-based vectors include pPK4, pPK5, and their variants, vectors in which the antibiotic resistance gene has been replaced with another antibiotic resistance gene, and vectors with 90% or more, 95% or more, 97% or more, or 99% or more nucleotide sequence identity therewith. More particularly, examples of vectors include pVC7, pVC7N, pPK4, and pPK5.

[0136] When a gene is introduced, it is sufficient that the gene is retained in the host in an expressible manner. Specifically, it is sufficient that the gene is retained so that it is expressed under the control of a promoter that functions in the host. The promoter is not particularly limited as long as it functions in the host. A "promoter that functions in the host" refers to a promoter that has promoter activity in the host. The promoter may be a promoter derived from the host or a heterologous promoter. The promoter may be a promoter native to the gene to be introduced or a promoter of another gene. Furthermore, the promoter may be inducible or constitutive for gene expression.

[0137] Examples of promoters that can be used in coryneform bacteria include promoters of genes involved in the glycolysis pathway, the pentose phosphate pathway, the TCA cycle, the amino acid biosynthesis pathway, and cell surface proteins. Specific examples of promoters for amino acid biosynthesis genes include the glutamate dehydrogenase gene for glutamate biosynthesis, the glutamine synthetase gene for glutamine synthesis, the aspartokinase gene for lysine biosynthesis, the homoserine dehydrogenase gene for threonine biosynthesis, the acetohydroxyacid synthase gene for isoleucine and valine biosynthesis, the 2-isopropylmalate synthase gene for leucine biosynthesis, the glutamate kinase gene for proline and arginine biosynthesis, the phosphoribosyl-ATP pyrophosphorylase gene for histidine biosynthesis, the deoxyarabinoheptulosonate phosphate (DAHP) synthase gene for aromatic amino acid biosynthesis such as tryptophan, tyrosine, and phenylalanine, and the phosphoribosylpyrophosphate (PRPP) amidotransferase gene, inosinate dehydrogenase gene, and guanylate synthase gene for nucleic acid biosynthesis such as inosinate and guanylate. Furthermore, promoters that can be used in coryneform bacteria also include the stronger promoters described below.

[0138] A terminator for terminating transcription can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a terminator derived from the host or a heterologous terminator. The terminator may be a terminator inherent to the gene to be introduced or a terminator of another gene. Specific examples of terminators include the terminator of bacteriophage BFK20.

[0139] Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, "Basic Microbiology Lectures 8: Genetic Engineering, Kyoritsu Shuppan, 1987," and they can be used.

[0140] Furthermore, when two or more genes are introduced, it is sufficient that each gene is retained in the host in an expressible manner. For example, two or more genes may all be retained on a single expression vector, or all may be retained on a chromosome. Alternatively, two or more genes may be retained separately on multiple expression vectors, or may be retained separately on a single or multiple expression vectors and on a chromosome. Alternatively, two or more genes may be introduced as an operon.

[0141] The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The gene to be introduced may be a gene derived from the host or a gene derived from a heterologous species. The gene to be introduced can be obtained, for example, by PCR using primers designed based on the nucleotide sequence of the gene and the genomic DNA of an organism carrying the gene or a plasmid carrying the gene as a template. Alternatively, the gene to be introduced may be totally synthesized based on the nucleotide sequence of the gene (Gene, 60(1), 115-127 (1987)). The obtained gene can be used as is or after appropriate modification. In other words, by modifying the gene, its variants can be obtained. Gene modification can be performed using known techniques. For example, site-directed mutagenesis can be used to introduce a desired mutation into a target site in DNA. For example, site-directed mutagenesis can be used to modify the coding region of a gene so that the encoded protein contains substitutions, deletions, insertions, and / or additions of amino acid residues at specific sites. Site-directed mutagenesis methods include PCR-based methods (Higuchi, R., 61, in PCR Technology, Erlich, HA Eds., Stockton Press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and phage-based methods (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). Alternatively, gene variants may be totally synthesized.

[0142] When a protein functions as a complex consisting of multiple subunits, all or only a portion of the multiple subunits may be modified, as long as the resulting protein activity is increased. That is, for example, when increasing protein activity by increasing gene expression, the expression of all or only a portion of the multiple genes encoding the subunits may be enhanced. It is usually preferable to enhance the expression of all of the multiple genes encoding the subunits. Furthermore, each subunit constituting the complex may be derived from a single organism, or from two or more different organisms, as long as the complex has the function of the target protein. That is, for example, genes encoding multiple subunits derived from the same organism may be introduced into a host, or genes derived from different organisms may be introduced into a host.

[0143] Increased gene expression can also be achieved by improving gene transcription efficiency. Increased gene expression can also be achieved by improving gene translation efficiency. Gene transcription efficiency and translation efficiency can be improved, for example, by modifying expression regulatory sequences. "Expression regulatory sequence" is a general term for sites that affect gene expression. Examples of expression regulatory sequences include promoters, Shine-Dalgarno (SD) sequences (also known as ribosome binding sites (RBS)), and spacer regions between the RBS and the start codon. Expression regulatory sequences can be determined using promoter search vectors or genetic analysis software such as GENETYX. These expression regulatory sequences can be modified, for example, by a method using a temperature-sensitive vector or the Red-driven integration method (WO2005 / 010175).

[0144] The efficiency of gene transcription can be improved, for example, by replacing the promoter of a gene on a chromosome with a stronger promoter. A "stronger promoter" refers to a promoter that enhances gene transcription compared to the wild-type promoter that is originally present. Examples of stronger promoters that can be used in coryneform bacteria include the artificially engineered P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679(2000)), pta, aceA, aceB, adh, and amyE promoters that can be induced by acetate, ethanol, pyruvate, etc., and other strong promoters such as cspB, SOD, and tuf (EF-Tu) promoters (Journal of Biotechnology 104 (2003) 311-323, Appl. Environ Microbiol. 2005 Dec;71(12):8587-96), lac promoter, tac promoter, trc promoter, F1 promoter, T7 promoter, T5 promoter, T3 promoter, and SP6 promoter. Furthermore, stronger promoters can be obtained by using various reporter genes to obtain highly active versions of existing promoters. For example, promoter activity can be increased by adjusting the -35 and -10 regions of the promoter region to resemble consensus sequences (WO 00 / 18935). Examples of highly active promoters include various tac-like promoters (Katashkina JI et al., Russian Federation Patent Application 2006134574). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

[0145] The translation efficiency of a gene can be improved, for example, by replacing the Shine-Dalgarno (SD) sequence (also known as the ribosome binding site (RBS)) of a gene on a chromosome with a stronger SD sequence. A "stronger SD sequence" refers to an SD sequence that improves mRNA translation compared to the native wild-type SD sequence. An example of a stronger SD sequence is the RBS of gene 10 from phage T7 (Olins PO et al., Gene, 1988, 73, 227-235). Furthermore, it is known that substitution, insertion, or deletion of several nucleotides in the spacer region between the RBS and the start codon, particularly in the sequence immediately upstream of the start codon (5'-UTR), significantly affects mRNA stability and translation efficiency. Gene translation efficiency can also be improved by modifying these sequences.

[0146] The translation efficiency of a gene can also be improved by, for example, codon modification. For example, the translation efficiency of a gene can be improved by replacing rare codons present in the gene with synonymous codons that are used more frequently. That is, the gene to be introduced may be modified to have optimal codons depending on the codon usage frequency of the host used. Codon substitution can be performed, for example, by site-directed mutagenesis. Alternatively, a gene fragment with substituted codons may be totally synthesized. The codon usage frequencies in various organisms are disclosed in the "Codon Usage Database" (http: / / www.kazusa.or.jp / codon; Nakamura, Y. et al., Nucl. Acids Res., 28, 292 (2000)).

[0147] Furthermore, increasing gene expression can also be achieved by amplifying regulators that increase gene expression, or by deleting or weakening regulators that decrease gene expression.

[0148] The above-mentioned methods for increasing gene expression may be used alone or in any combination.

[0149] Modifications that increase protein activity can also be achieved by, for example, enhancing the specific activity of the protein. Proteins with enhanced specific activity can be obtained, for example, by searching for and obtaining proteins from various organisms. Highly active proteins can also be obtained by introducing mutations into existing proteins. The mutations introduced may be, for example, substitutions, deletions, insertions, and / or additions of one or several amino acids at one or several positions in the protein. Mutations can be introduced, for example, by site-directed mutagenesis, as described above. Mutations can also be introduced by, for example, mutagenesis. Examples of mutagenesis include X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS). Alternatively, random mutations can be induced by directly treating DNA with hydroxylamine in vitro. Specific activity enhancement can be used alone or in any combination with the above-mentioned methods for enhancing gene expression.

[0150] The transformation method is not particularly limited, and conventionally known methods can be used, such as a method reported for Escherichia coli K-12 in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), or a method reported for Bacillus subtilis in which DNA is introduced into competent cells prepared from cells in the growth stage (Duncan, C.H., Wilson, G.A. and Young, F.E., 1977, Gene 1: 153-167). Alternatively, a method known for Bacillus subtilis, actinomycetes, and yeasts can be used in which the recipient cells are transformed into protoplasts or spheroplasts, which readily incorporate recombinant DNA, and then the recombinant DNA is introduced into the recipient cells (Chang, S. and Choen, SN, 1979, Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM, and Hopwood, OA, 1978, Nature 274: 398-400; Hinnen, A., Hicks, JB, and Fink, GR, 1978, Proc. Natl. Acad. Sci. USA 75: 1929-1933). Alternatively, an electric pulse method, as reported for coryneform bacteria (JP 2-207791), can be used.

[0151] The increase in protein activity can be confirmed by measuring the activity of the protein.

[0152] Increased protein activity can also be confirmed by confirming increased expression of the gene encoding the protein, which can be confirmed by confirming increased transcription of the gene or increased amount of protein expressed from the gene.

[0153] Increased gene transcription levels can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a wild-type strain or a non-modified strain such as the parent strain. Methods for assessing mRNA levels include Northern hybridization, RT-PCR, microarrays, RNA-seq, etc. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA (e.g., number of molecules per cell) may be increased, for example, by 1.5-fold or more, 2-fold or more, or 3-fold or more compared to that of a non-modified strain.

[0154] The increase in the amount of the protein can be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of the protein (e.g., the number of molecules per cell) may be increased by, for example, 1.5-fold or more, 2-fold or more, or 3-fold or more compared to that of an unmodified strain.

[0155] The above-mentioned methods for increasing protein activity can be used to enhance the activity of any protein or enhance the expression of any gene.

[0156] <1-5> Methods for reducing protein activity Methods for reducing protein activity are described below. Note that the methods for reducing protein activity described below can also be used to disrupt wild-type PhoS protein.

[0157] "Decreased protein activity" means that the activity of the protein is reduced compared to that of a non-modified strain. Specifically, "decreased protein activity" means that the activity of the protein per cell is reduced compared to that of a non-modified strain. Here, "non-modified strain" refers to a control strain that has not been modified to reduce the activity of the target protein. Examples of non-modified strains include wild-type strains and parent strains. Specific examples of non-modified strains include type strains of each bacterial species. Specific examples of non-modified strains also include the strains exemplified in the description of coryneform bacteria. That is, in one embodiment, the activity of the protein may be reduced compared to that of a type strain (i.e., the type strain of the species to which the bacterium of the present invention belongs). In another embodiment, the activity of the protein may be reduced compared to that of C. glutamicum ATCC 13032. In another embodiment, the activity of the protein may be reduced compared to that of C. glutamicum ATCC 13869. In another embodiment, the activity of the protein may be reduced compared to that of C. glutamicum AJ12036 (FERM BP-734). In another embodiment, the activity of the protein may be reduced compared to that of the C. glutamicum YDK010 strain. Note that "reduced protein activity" also encompasses cases where the activity of the protein is completely lost. More specifically, "reduced protein activity" may mean a reduction in the number of molecules of the protein per cell and / or a reduction in the function of the protein per molecule compared to a non-modified strain. That is, the "activity" in "reduced protein activity" is not limited to the catalytic activity of the protein, but may also refer to the transcription amount (mRNA amount) or translation amount (protein amount) of the gene encoding the protein. The "number of molecules of protein per cell" may refer to the average number of molecules of the protein per cell. Note that "reduced number of molecules of protein per cell" also encompasses cases where the protein is completely absent. Furthermore, "the function per molecule of a protein is reduced" also includes a case where the function per molecule of the protein is completely lost.The degree of reduction in protein activity is not particularly limited, as long as the protein activity is reduced compared to that of an unmodified strain. For example, the protein activity may be reduced to 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0158] Modifications that reduce the activity of a protein can be achieved, for example, by reducing the expression of the gene encoding the protein. "Reduced gene expression" means that the expression of the gene is reduced compared to an unmodified strain. "Reduced gene expression" specifically means that the expression level of the gene per cell is reduced compared to an unmodified strain. "Expression level of the gene per cell" may refer to the average expression level of the gene per cell. "Reduced gene expression" may more specifically mean a reduction in the transcription level (mRNA level) of the gene and / or a reduction in the translation level (protein level) of the gene. "Reduced gene expression" also includes cases where the gene is not expressed at all. "Reduced gene expression" is also referred to as "attenuated gene expression." Gene expression may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0159] Decreased gene expression may be due to, for example, decreased transcription efficiency, decreased translation efficiency, or a combination thereof. Decreased gene expression can be achieved, for example, by modifying the expression regulatory sequence of the gene. "Expression regulatory sequence" is a general term for sites that affect gene expression, such as promoters, Shine-Dalgarno (SD) sequences (also known as ribosome binding sites (RBS)), and spacer regions between the RBS and the start codon. Expression regulatory sequences can be determined, for example, using promoter search vectors or gene analysis software such as GENETYX. When modifying an expression regulatory sequence, preferably one or more bases, more preferably two or more bases, and particularly preferably three or more bases are modified in the expression regulatory sequence. Decreased gene transcription efficiency can be achieved, for example, by replacing the promoter of a gene on a chromosome with a weaker promoter. A "weaker promoter" refers to a promoter that weakens gene transcription compared to the native wild-type promoter. An example of a weaker promoter is an inducible promoter. That is, an inducible promoter can function as a weaker promoter under non-inducing conditions (e.g., in the absence of an inducer). Part or all of the expression regulatory sequence may also be deleted (deleted). Reduced gene expression can also be achieved, for example, by manipulating factors involved in expression control. Factors involved in expression control include small molecules (inducers, inhibitors, etc.), proteins (transcription factors, etc.), and nucleic acids (siRNA, etc.) involved in transcription and translation control. Reduced gene expression can also be achieved, for example, by introducing a mutation into the coding region of the gene that reduces gene expression. For example, gene expression can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host. Furthermore, gene expression itself can be reduced, for example, by gene disruption as described below.

[0160] Furthermore, a modification that reduces the activity of a protein can be achieved, for example, by disrupting the gene encoding the protein. "Disrupting a gene" means that the gene is modified so that it does not produce a protein that functions normally. "Not producing a protein that functions normally" includes cases where no protein is produced from the gene at all, and cases where the gene produces a protein with reduced or lost function per molecule (e.g., activity or properties).

[0161] Gene disruption can be achieved, for example, by deleting (deleting) the gene on a chromosome. "Gene deletion" refers to the deletion of part or all of the coding region of a gene. Furthermore, the entire gene may be deleted, including the sequences before and after the coding region of the gene on the chromosome. The sequences before and after the coding region of the gene may include, for example, a gene expression regulatory sequence. As long as a reduction in protein activity can be achieved, the region to be deleted may be any region, such as the N-terminal region (the region encoding the N-terminal side of the protein), an internal region, or a C-terminal region (the region encoding the C-terminal side of the protein). Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. The region to be deleted may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the entire length of the coding region of the gene. Furthermore, it is preferable that the reading frames of the sequences before and after the region to be deleted do not match. Reading frame mismatches can result in frameshifts downstream of the region to be deleted.

[0162] Gene disruption can also be achieved by, for example, introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting one or two bases (frameshift mutation) (Journal of Biological Chemistry 272:8611-8617(1997), Proceedings of the National Academy of Sciences, USA 95 5511-5515(1998), Journal of Biological Chemistry 26 116, 20833-20839(1991)).

[0163] Gene disruption can also be achieved, for example, by inserting another base sequence into the coding region of the gene on the chromosome. The insertion site may be anywhere in the gene, but the longer the inserted base sequence, the more reliably the gene can be inactivated. Furthermore, it is preferable that the reading frames of the sequences before and after the insertion site do not match. A mismatch in the reading frame can cause a frameshift downstream of the insertion site. The other base sequence is not particularly limited as long as it reduces or eliminates the activity of the encoded protein, and examples include marker genes such as antibiotic resistance genes and genes useful for producing target substances.

[0164] Gene disruption may be carried out, particularly to delete (delete) the amino acid sequence of the encoded protein. In other words, modification that reduces the activity of a protein can be achieved, for example, by deleting the amino acid sequence of the protein (partial or entire region of the amino acid sequence), specifically by modifying the gene to encode a protein from which the amino acid sequence (partial or entire region of the amino acid sequence) has been deleted. The term "deletion of the amino acid sequence of a protein" refers to the deletion of part or entire region of the amino acid sequence of a protein. The term "deletion of the amino acid sequence of a protein" refers to the absence of the original amino acid sequence in the protein, and also encompasses cases in which the original amino acid sequence is changed to a different amino acid sequence. For example, a region that has been changed to a different amino acid sequence due to frameshifting may be considered a deleted region. While deletion of the amino acid sequence of a protein typically shortens the overall length of the protein, it may also be possible for the overall length of the protein to remain unchanged or to be extended. For example, deletion of part or entire region of the coding region of a gene can delete the region encoded by the deleted region in the amino acid sequence of the encoded protein. For example, by introducing a stop codon into the coding region of a gene, the region coded for by the region downstream of the introduction site in the amino acid sequence of the encoded protein can be deleted. For example, by causing a frameshift in the coding region of a gene, the region coded for by the frameshift site can be deleted. The position and length of the region to be deleted in the deletion of an amino acid sequence can be determined mutatis mutandis from the explanation of the position and length of the region to be deleted in the deletion of a gene.

[0165] The above-described modification of a gene on a chromosome can be achieved, for example, by creating a disrupted gene modified so that it does not produce a normally functioning protein, transforming a host with recombinant DNA containing the disrupted gene, and inducing homologous recombination between the disrupted gene and the wild-type gene on the chromosome, thereby replacing the wild-type gene on the chromosome with the disrupted gene. In this case, incorporating a marker gene into the recombinant DNA according to the host's traits, such as its nutritional requirements, facilitates manipulation. Examples of disrupted genes include genes lacking part or all of the coding region of a gene, genes with missense mutations, genes with nonsense mutations, genes with frameshift mutations, and genes with insertion sequences such as transposons or marker genes. Even if a protein encoded by a disrupted gene is produced, it will have a different three-dimensional structure from the wild-type protein, resulting in reduced or lost function. The structure of the recombinant DNA used for homologous recombination is not particularly limited, as long as it allows homologous recombination to occur in the desired manner. For example, a host can be transformed with linear DNA containing a disrupted gene, the linear DNA having upstream and downstream sequences of a wild-type gene on a chromosome at both ends, and homologous recombination can occur upstream and downstream of the wild-type gene, thereby replacing the wild-type gene with the disrupted gene.Such gene disruption by gene replacement using homologous recombination has already been established, and includes methods using linear DNA, such as a method called "Red-driven integration" (Datsenko, K. A., and Wanner, BL Proc. Natl. Acad. Sci. USA 97:6640-6645 (2000)), a method combining the Red-driven integration method with an excision system derived from λ phage (Cho, EH, Gumport, RI, Gardner, JFJ Bacteriol. 184: 5200-5203 (2002)) (see WO2005 / 010175), methods using a plasmid containing a temperature-sensitive replication origin, methods using a conjugatively transferable plasmid, and methods using a suicide vector that does not have a replication origin that functions in the host (U.S. Pat. No. 6,303,383, JP 05-007491 A). Such a method of modifying a chromosome using homologous recombination is not limited to disrupting a target gene, but can be used for any modification of a chromosome, such as modification of an expression regulatory sequence.

[0166] Modifications that reduce the activity of a protein may also be performed by, for example, mutation treatments, such as X-ray irradiation, ultraviolet irradiation, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0167] The above-mentioned methods for reducing protein activity may be used alone or in any combination.

[0168] The decrease in the activity of the protein can be confirmed by measuring the activity of the protein.

[0169] A decrease in protein activity can also be confirmed by confirming a decrease in expression of the gene encoding the protein. A decrease in gene expression can be confirmed by confirming a decrease in the transcription level of the gene or a decrease in the amount of protein expressed from the gene.

[0170] The reduction in the transcription level of a gene can be confirmed by comparing the amount of mRNA transcribed from the gene with that of a non-modified strain. Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, microarray, RNA-Seq, etc. (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The amount of mRNA may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of a non-modified strain.

[0171] The reduction in protein amount can be confirmed by performing SDS-PAGE and checking the intensity of the separated protein bands. The reduction in protein amount can also be confirmed by Western blotting using an antibody (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual / Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001). The protein amount (e.g., number of molecules per cell) may be reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% of that of an unmodified strain.

[0172] Gene disruption can be confirmed by determining the nucleotide sequence, restriction enzyme map, or full length of a part or all of the gene, depending on the means used for disruption.

[0173] The above-mentioned methods for reducing protein activity can be used to reduce the activity of any protein or the expression of any gene.

[0174] <1-6> Production of bbFGF By culturing the bacterium of the present invention, bbFGF is secreted and produced, thereby obtaining a culture containing bbFGF.

[0175] The medium used for culturing is not particularly limited, as long as the bacterium of the present invention can grow and bbFGF can be produced. Examples of the medium include conventional media used for culturing bacteria such as coryneform bacteria. Specific examples of the medium include conventional media containing a carbon source, a nitrogen source, inorganic ions, etc. Organic trace nutrients such as vitamins and amino acids can also be added to the medium as needed. The culture conditions are not particularly limited, as long as the bacterium of the present invention can grow and bbFGF can be produced. Culture can be performed under, for example, conventional conditions used for culturing bacteria such as coryneform bacteria. The types and concentrations of medium components and the culture conditions can be appropriately determined depending on various conditions, such as the type of coryneform bacteria.

[0176] Examples of carbon sources that can be used include carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, or other suitable carbon sources. Examples of nitrogen sources that can be used include ammonia gas, aqueous ammonia, ammonium salts, or other suitable nitrogen sources. Examples of inorganic ions that can be used include calcium ions, magnesium ions, phosphate ions, potassium ions, and iron ions, as needed. Culture can be carried out for approximately 1 to 7 days under aerobic conditions, for example, at a pH of 5.0 to 8.5 and a temperature range of 15 to 37°C. Culture conditions for L-amino acid production by coryneform bacteria and conditions described in methods for producing proteins using Sec-dependent or Tat-dependent signal peptides can also be used (see WO 01 / 23591 and WO 2005 / 103278). When an inducible promoter is used to express a gene such as the bbFGF gene, gene expression can be induced as appropriate. By culturing the bacterium of the present invention under these conditions, bbFGF is secreted and produced, thereby obtaining a culture containing bbFGF.

[0177] The secretion and production of bbFGF can be confirmed by performing SDS-PAGE using a culture supernatant and / or a fraction containing the bacterial cell surface as a sample and determining the molecular weight of the separated protein band. The secretion and production of bbFGF can also be confirmed by Western blotting using an antibody, using the culture supernatant and / or a fraction containing the bacterial cell surface as a sample (Molecular cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001)). The secretion and production of bbFGF can also be confirmed by detecting the N-terminal amino acid sequence of the secreted protein using a protein sequencer. The secretion and production of bbFGF can also be confirmed by determining the mass of the secreted protein using a mass spectrometer. If bbFGF is an enzyme or has some measurable physiological activity, the secretion and production of bbFGF can be confirmed by measuring the physiological activity of bbFGF using the culture supernatant and / or a fraction containing the bacterial cell surface as a sample.

[0178] Cultures containing bbFGF (specifically, cultures containing secreted and produced bbFGF) may be used as an active ingredient in the production of cultured meat either directly or after appropriate treatments such as sterilization, reduction, purification, solvent substitution, and drying. These treatments may be performed alone or in appropriate combinations. For example, at least sterilization may be performed. "Sterilization" may refer to the removal of cells of the bacterium of the present invention from a culture containing bbFGF. Sterilization may be performed by solid-liquid separation methods such as natural sedimentation, centrifugation, and filtration. Filtration may be performed using a filter such as a PVDF membrane. "Reduction" may refer to the reduction of bbFGF, specifically, to placing a fraction containing bbFGF (e.g., a culture, culture supernatant, or purified bbFGF solution) in a reducing state. Reduction may be performed, for example, by adding a reducing agent such as DTT. "Purification" may refer to the purification of bbFGF, specifically, the purification of bbFGF from a fraction containing bbFGF (e.g., a culture or culture supernatant). "Purification of bbFGF" may refer to the separation of bbFGF from other components. Components other than bbFGF are also referred to as "impurities." Examples of impurities include those contained in a fraction containing bbFGF (e.g., a culture supernatant). Note that sterilization does not fall under "purification of bbFGF." In other words, bacterial cells of the present invention do not fall under "impurities" in the purification of bbFGF. Purification can be performed by protein separation and purification methods such as salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange chromatography, reverse-phase chromatography, and affinity chromatography. In one embodiment, purification of bbFGF is not necessary. The term "no purification of bbFGF" may be used interchangeably with the term "bbFGF is not purified.""No purification of bbFGF" may mean, for example, that bbFGF is used as an active ingredient in a state where 70% (w / w) or more, 80% (w / w) or more, 90% (w / w) or more, 95% (w / w) or more, 97% (w / w) or more, or 99% (w / w) or more of the contaminants remain (i.e., coexist with bbFGF). In one embodiment, purification of bbFGF by chromatography may not be necessary. By not purifying bbFGF (particularly by chromatography), for example, the production cost of bbFGF can be reduced, thereby reducing the production cost of cultured meat. bbFGF produced using coryneform bacteria as an expression host can be suitably used in the culture of animal cells (specifically, the production of cultured meat) without high-purity purification by chromatography or other methods. "Solvent replacement" may mean replacing the liquid portion of a bbFGF-containing fraction (e.g., a culture, culture supernatant, or purified bbFGF solution). A buffer solution such as PBS may be used for solvent substitution. The buffer solution may contain a reducing agent such as DTT. "Drying" may refer to drying of bbFGF, specifically, drying a fraction containing bbFGF (e.g., a culture, culture supernatant, or purified bbFGF solution). Drying can be carried out by drying methods such as freeze-drying or spray-drying. When bbFGF is secreted onto the bacterial cell surface, bbFGF can be solubilized by solubilization methods such as increasing the salt concentration or using a surfactant. The solubilized bbFGF-containing fraction, like the bbFGF-containing culture, may be used as an active ingredient in the production of cultured meat either directly or after appropriate treatments such as sterilization, reduction, purification, solvent substitution, and drying.

[0179] The form of the active ingredient is not particularly limited. The active ingredient may be in any form, such as powder, flakes, paste, liquid, etc. For example, a fraction containing bbFGF may be dried and powdered and used as the active ingredient.

[0180] <2> Method of the Present Invention The method of the present invention is a method for producing cultured meat, which includes a step of culturing animal cells in the presence of an active ingredient (i.e., bbFGF produced using a coryneform bacterium as an expression host). This step is also referred to as the "animal cell culturing step." Specifically, the animal cell culturing step may be a step of culturing animal cells in the presence of the active ingredient to form cultured meat.

[0181] The animal cells are not particularly limited as long as they can be cultured to form cultured meat. Cultured meat may be composed primarily of muscle tissue. Therefore, "animal cells can be cultured to form cultured meat" may mean, for example, that the animal cells can differentiate to form muscle tissue through culture, and specifically, that the animal cells can differentiate into myotube cells through culture to form muscle tissue. Examples of animal cells include stem cells such as mesenchymal stem cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells), as well as myoblasts. Examples of animals include animals used for meat production. Examples of animals used for meat production include cattle, sheep, goats, pigs, and rabbits. Examples of animals used for meat production include cattle and pigs in particular.

[0182] The animal cells are cultured in the presence of an active ingredient. "The animal cells are cultured in the presence of an active ingredient" may mean, for example, that the medium used for culturing the animal cells contains the active ingredient. "The animal cells are cultured in the presence of an active ingredient" may mean, for example, that the active ingredient is supplied to the culture system during the culturing of the animal cells.

[0183] The medium composition and culture conditions are not particularly limited, as long as culture is performed in the presence of an active ingredient, and cultured meat is formed from the cultured cells. The medium composition and culture conditions can be appropriately set depending on various conditions, such as the type of animal cell. Other than the presence of an active ingredient, culture can be performed using, for example, a conventional medium and conventional conditions used for the formation of muscle tissue by differentiation of animal cells (e.g., the production of cultured meat), either as is or with appropriate modifications.

[0184] Culture can be carried out using, for example, a liquid medium. Culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Continuous culture includes perfusion culture and chemostat culture. The medium at the start of culture is also called the "initial medium" or "basal medium." The medium supplied to a culture system (e.g., initial medium) in fed-batch culture is also called the "feed medium." The medium supplied to a culture system (e.g., initial medium) in continuous culture (not limited to perfusion culture) is also called the "perfusion medium." Supplying a feed medium or perfusion medium to a culture system in fed-batch or continuous culture is also simply called "medium supply." Medium supply may be carried out throughout the entire culture period or only for a portion of the culture period. Medium supply may be carried out continuously or intermittently. During culture (particularly continuous culture such as perfusion culture), the culture medium may be withdrawn. The culture medium may be withdrawn throughout the entire culture period, or only during a portion of the culture period. The culture medium may be withdrawn continuously or intermittently. The culture medium may be withdrawn and the medium may be supplied simultaneously, or not. Culturing may be carried out three-dimensionally, for example, using a scaffold that matches the shape of the cultivated meat to be formed.

[0185] The media used for culture, for example, the basal medium, the feed medium, and the perfusion medium, can be selected independently.

[0186] The medium used for the culture may be a commercially available medium or an appropriately prepared medium.

[0187] The medium used for the culture includes a medium containing components essential for culturing animal cells (for example, a carbon source, a nitrogen source, inorganic salts, etc.).

[0188] Specific examples of media used for culture include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM / F12, McCoy's 5A medium, Minimum Essential Medium (MEM), Eagle's Minimum Essential Medium (EMEM), alpha Modified Eagle's Minimum Essential Medium (αMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Iscove's Modified Dulbecco's Medium (IMDM), MCDB131 medium, William's Medium E, and Fischer's Medium.

[0189] Furthermore, specific examples of media used for culture (for example, media particularly used for culturing stem cells (particularly pluripotent stem cells)) include STEMPRO (registered trademark) hESC SFM medium (Life Technologies), mTeSR1 medium (STEMCELL Technologies), TeSR2 medium (STEMCELL Technologies), TeSR-E8 medium (STEMCELL Technologies), Essential 8 medium (Life Technologies), HEScGRO (trademark) Serum-Free Medium for hES cells (Millipore), PluriSTEM (trademark) Human ES / iPS Medium (EMD Millipore), NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Beit-Haemek), NutriStem (trademark) XF / FF Culture Medium (Stemgent), AF NutriStem (registered trademark) hESC XF medium (Biological Industries Israel Examples of suitable medium include hESF9 medium, hESF-FX medium, CDM medium, DEF-CS 500 Xeno-Free 3D Spheroid Culture Medium (Cellartis), and StemFlex medium (Thermo Fisher Scientific).

[0190] Other commercially available media include animal cell culture media such as CELLiST Basal Media BASAL3, BASAL4P, and BASAL10 (Ajinomoto Co., Inc.), Opti-MEM (Thermo Fisher Scientific), RPMI 1640 (Thermo Fisher Scientific), CD293 (Thermo Fisher Scientific), CHO-S-SFMII (Thermo Fisher Scientific), CHO-SF (Sigma-Aldrich), EX-CELL CD CHO (Sigma-Aldrich), EX-CELL™302 (Sigma-Aldrich), IS CHO-CD (Irvine Scientific), and IS CHO-CDXP (Irvine Scientific).

[0191] The media exemplified above may be used for culture by adding, for example, an active ingredient.

[0192] The medium may contain various medium components. Examples of medium components include carbon sources, amino acid sources, peptides, proteins, vitamins, fatty acids, lipids, inorganic components, pH buffers, growth factors, cytokines, hormones, cell adhesion factors, extracellular matrix components, serum, antibiotics, and gene expression inducers. Any of these medium components may be essential or effective for, for example, the survival or proliferation of animal cells. Any of these medium components may be pre-contained in the medium exemplified above, or may be added to the medium exemplified above.

[0193] Examples of carbon sources include sugars such as glucose, fructose, sucrose, and maltose.

[0194] The amino acid source may include amino acids. In addition, both peptides and proteins may be examples of amino acid sources. The amino acids may include glycine, alanine, valine, leucine, isoleucine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, proline, and ornithine. The amino acids may be, for example, L-form.

[0195] Examples of peptides include dipeptides and tripeptides. Specific examples of peptides include glycylglycylglycine and soybean peptide. The descriptions of amino acids also apply mutatis mutandis to the amino acids that make up peptides.

[0196] Examples of proteins include albumin and transferrin.

[0197] Vitamins include vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E, and vitamin K, and precursors thereof.

[0198] Fatty acids include oleic acid, arachidonic acid, and linoleic acid.

[0199] Lipids include cholesterol.

[0200] Examples of inorganic components include sodium, potassium, calcium, magnesium, phosphorus, and various trace elements (e.g., Co, Cu, F, Fe, Mn, Mo, Ni, Se, Si, Ni, Bi, V, and Zn).Specific examples of inorganic components include inorganic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and sodium dihydrogen phosphate.

[0201] Examples of pH buffers include sodium bicarbonate, phosphate, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and N-[tris(hydroxymethyl)methyl]glycine (Tricine).

[0202] Growth factors include fibroblast growth factor (FGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-α, transforming growth factor (TGF)-β, vascular endothelial growth factor (VEGF), activin A, and insulin-like growth factor-1 (IGF-1).

[0203] Cytokines include interleukins.

[0204] Hormones include dexamethasone, hydrocortisone, estradiol, progesterone, glucagon, and insulin.

[0205] Examples of cell adhesion factors or extracellular matrix components include Type I collagen, Type II collagen, fibronectin, laminin, poly-L-lysine, and poly-D-lysine.

[0206] Antibiotics include amphotericin B, kanamycin, gentamicin, streptomycin, and penicillin.

[0207] In one embodiment, the medium may be substantially free of serum. For example, the medium may be substantially free of serum throughout the entire culture period. "The medium is substantially free of serum" may mean that the serum content in the medium is 1% (w / w) or less, 0.1% (w / w) or less, 0.01% (w / w) or less, or 0.001% (w / w) or less, and may also include a case where the serum content in the medium is 0 (zero) (i.e., the medium does not contain serum).

[0208] Various components, such as active ingredients, may be contained in the initial medium, feed medium, perfusion medium, or a combination thereof. That is, various components, such as active ingredients, may be supplied to the medium alone or in any combination during the culture process. These components may be supplied once, multiple times, or continuously. The compositions (e.g., the types and / or concentrations of the components) of the initial medium, feed medium, and perfusion medium may or may not be the same. That is, the types of components contained in the initial medium may or may not be the same as the types of components contained in the feed medium or perfusion medium. Furthermore, the concentrations of the components contained in the initial medium may or may not be the same as the concentrations of the components contained in the feed medium or perfusion medium. For example, when a feed medium is used for perfusion culture, the compositions of the initial medium and the feed medium may be the same. Furthermore, two or more feed media or perfusion media with different compositions (e.g., the types and / or concentrations of the components contained) may be used. For example, when the feed medium or perfusion medium is supplied intermittently multiple times, the composition of the feed medium or perfusion medium may or may not be the same for each supply. In addition, various components such as active ingredients may be supplied to the medium in a form not contained in the feed medium or perfusion medium, such as a powder.

[0209] The seeding amount of animal cells at the start of culture is, for example, 1 × 10 in terms of viable cell number. 3 cells / mL or more, 1×10 4 cells / mL or more, 1×10 5 cells / mL or more, 1×10 6cells / mL or more, or 1×10 7 cells / mL or more, but not more than 1 x 10 8 cells / mL or less, 1×10 7 cells / mL or less, 1×10 6 cells / mL or less, 1×10 5 cells / mL or less, or 1 x 10 4 The seeding amount of animal cells at the start of culture may be, for example, 1 × 10 cells / mL or less in terms of the number of viable cells. 3 ~1×10 4 cells / mL, 1 × 10 4 ~1×10 5 cells / mL, 1 × 10 5 ~1×10 6 cells / mL, 1 × 10 6 ~1×10 7 cells / mL, or 1 x 10 7 ~1×10 8 The seeding amount of animal cells at the start of culture may be, for example, 1 × 10 cells / mL in terms of the number of viable cells. 3 ~1×10 8 cells / mL, 1 × 10 4 ~1×10 7 cells / mL, or 1 x 10 5 ~1×10 6 The number of viable cells may be measured using, for example, a viable cell autoanalyzer, Vi-CELL TM Measurement can be performed using XR (Beckman Coulter).

[0210] The culture may be carried out under, for example, 5 to 15% CO ２ CO etc. ２The cultivation may be carried out under a pH-containing atmosphere. The pH of the medium may be, for example, near neutral. "Near neutral" may mean, for example, pH 6 to 8, pH 6.5 to 7.5, or pH 6.8 to 7.2. The pH of the medium can be adjusted as needed during cultivation. The pH of the medium can be adjusted using various alkaline or acidic substances such as ammonia gas, ammonia water, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, and magnesium hydroxide. The cultivation temperature may be, for example, 30 to 38°C. The culture period may be, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 12 days or more, 15 days or more, or 20 days or more, or 60 days or less, 50 days or less, 40 days or less, 30 days or less, 25 days or less, 20 days or less, 15 days or less, 12 days or less, 10 days or less, 9 days or less, 8 days or less, or 7 days or less, or any compatible combination thereof. Specific examples of the culture period may be, for example, 1 to 60 days, 3 to 25 days, or 5 to 20 days. Culture may continue, for example, until the cultured meat has been formed to a desired extent.

[0211] The concentration of the active ingredient in the medium may be, for example, 0.01 ng / mL or more, 0.02 ng / mL or more, 0.05 ng / mL or more, 0.1 ng / mL or more, 0.2 ng / mL or more, 0.5 ng / mL or more, 1 ng / mL or more, 2 ng / mL or more, 5 ng / mL or more, 10 ng / mL or more, 15 ng / mL or more, 20 ng / mL or more, or 25 ng / mL or more, or 30 ng / mL or less, 25 ng / mL or less, 20 ng / mL or less, 15 ng / mL or less, 10 ng / mL or less, 5 ng / mL or less, 2 ng / mL or less, 1 ng / mL or less, 0.5 ng / mL or less, 0.2 ng / mL or less, 0.1 ng / mL or less, 0.05 ng / mL or less, or 0.02 ng / mL or less, or any compatible combination thereof. The concentration of the active ingredient in the medium may be, for example, 0.01 to 0.02 ng / mL, 0.02 to 0.05 ng / mL, 0.05 to 0.1 ng / mL, 0.1 to 0.2 ng / mL, 0.2 to 0.5 ng / mL, 0.5 to 1 ng / mL, 1 to 2 ng / mL, 2 to 5 ng / mL, 5 to 10 ng / mL, 10 to 15 ng / mL, 15 to 20 ng / mL, 20 to 25 ng / mL, or 25 to 30 ng / mL. The concentration of the active ingredient in the medium may be, for example, 0.01 to 30 ng / mL, 0.02 to 20 ng / mL, or 0.05 to 15 ng / mL.

[0212] Each of the various components, such as the active ingredient, may be contained in the medium throughout the entire culture period, or only during a portion of the culture period. In other words, "culture is carried out in a medium containing a certain component" or "a certain component is contained in the medium during culture" means that the component is contained in the medium during at least a portion of the culture period, and does not necessarily have to be contained in the medium throughout the entire culture period. Each of the various components, such as the active ingredient, may be contained in the medium at the start of culture, or may be supplied to the medium after the start of culture. Furthermore, each of the various components, such as the active ingredient, may be contained in the medium at the start of culture, and further supplied to the medium after the start of culture (e.g., after the active ingredient has been consumed).

[0213] Each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above throughout the entire culture period, or only during a portion of the culture period. That is, "culture is carried out in a medium containing a certain component at a certain concentration," "a certain component is contained in the medium at a certain concentration during culture," or "the concentration of a certain component in the medium during culture is a certain concentration" means that the concentration of the component in the medium is within the range for at least a portion of the culture period, but does not necessarily need to be within the range for the entire culture period. Each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above, for example, at the start of culture, or may be supplied to the medium after the start of culture to achieve the concentrations exemplified above. Furthermore, each of the various components, such as the active ingredient, may be contained in the medium at the concentrations exemplified above at the start of culture, and then further supplied to the medium after the start of culture (e.g., after the component has been consumed) to achieve the concentrations exemplified above.

[0214] The length of the "partial culture period" is not particularly limited as long as a proliferation-promoting effect is obtained. The length of the "partial culture period" can be appropriately set depending on various conditions, such as the type of animal cells and the length of the culture period. The "partial culture period" may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the total culture period. Furthermore, the "partial culture period" may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the total culture period after the animal cells have differentiated into myotubes. Furthermore, a "partial period" may be, for example, a period of 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 12 days or more, or 15 days or more.

[0215] Furthermore, the concentrations of various components, such as active ingredients, in the culture medium may be set to the concentrations exemplified above, for example, as average values ​​over a specific period during culture. That is, "culture is performed in a medium containing a certain component at a certain concentration," "a certain component is contained in the medium at a certain concentration during culture," or "the concentration of a certain component in the medium during culture is a certain concentration" may mean that the average concentration of the component in the medium over a specific period during culture falls within the range of concentrations. The "average concentration of a certain component in the medium over a specific period during culture" is not particularly limited as long as it allows for understanding fluctuations in the concentration of the component over a specific period during culture. For example, it may mean the average concentration of the component in the medium measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes over a specific period during culture. The "specific period during culture" may refer to the entire culture period or a portion of the culture period. The "partial culture period" is as described above.

[0216] Each of the various components, such as the active ingredient, may be supplied to the medium throughout the entire culture period, or may be supplied to the medium only during a portion of the culture period. The "part of the culture period" is as described above. Each of the various components, such as the active ingredient, may be supplied to the medium continuously or intermittently, for example. Each of the various components, such as the active ingredient, may be supplied to the medium every day, or every few days, for example.

[0217] The concentrations of various components, such as active ingredients, in the feed medium or perfusion medium may be within the range of the concentrations of the components in the media exemplified above, or may be, for example, 1-fold or more, 1.1-fold or more, 1.3-fold or more, 1.5-fold or more, 2-fold or more, 3-fold or more, 5-fold or more, 7-fold or more, 10-fold or more, 15-fold or more, or 20-fold or more, or 100-fold or less, 70-fold or less, 50-fold or less, 30-fold or less, 20-fold or less, 15-fold or less, 10-fold or less, 7-fold or less, 5-fold or less, 3-fold or less, 2-fold or less, or any combination thereof that is consistent therewith. The concentrations of various components such as active ingredients in the feed medium or perfusion medium may be, for example, 1 to 2 times, 1.1 to 2 times, 1.3 to 2 times, 1.5 to 2 times, 2 to 3 times, 3 to 5 times, 5 to 7 times, 7 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 30 times, 20 to 50 times, 20 to 70 times, or 20 to 100 times the concentrations of the components in the above-exemplified media. The concentrations of various components such as active ingredients in the feed medium or perfusion medium may be, for example, 1 to 100 times, 2 to 50 times, or 5 to 20 times the concentrations of the components in the above-exemplified media.

[0218] The concentrations of any of the components, including the active ingredient, can be measured by known methods used to detect or identify compounds, such as HPLC, UPLC, LC / MS, GC / MS, and NMR.

[0219] Cultured meat can be obtained by culturing animal cells as described above.

[0220] The present invention will now be described in more detail with reference to the following non-limiting examples.

[0221] (Reference Example 1) Construction of Corynebacterium glutamicum lacking the O-mannosyltransferase gene pmt1 (1) Construction of vector pBS5TΔpmt1 for deleting the pmt1 gene It is known that O-glycosylation occurs in secreted proteins expressed in C. glutamicum as a host, and that deletion of protein O-mannosyltransferase PMT1 can eliminate the ability to modify O-glycosylation of secreted proteins (Martina Mahne et al., The Corynebacterium glutamicum gene pmt encoding a glycosyltransferase related to eukaryotic protein-O-mannosyltransferases is essential for glycosylation of the resuscitation promoting factor (Rpf2) and other secreted proteins. FEMS Microbiol Lett. 2006 Jun;259(2):226-33).

[0222] The genome sequence of the C. glutamicum ATCC13869 strain and the nucleotide sequence of the pmt1 gene encoding protein O-mannosyltransferase PMT1 have already been determined (GenBank Accession No. AP017557, NCBI locus_tag CGBL_0109730). The nucleotide sequence of the pmt1 gene of the C. glutamicum ATCC 13869 strain and the amino acid sequence of PMT1 encoded by the gene are shown in SEQ ID NOs: 39 and 40, respectively.

[0223] PurElute TMUsing chromosomal DNA from C. glutamicum ATCC13869 strain prepared using the Genomic DNA Kit (EdgeBio) as a template, approximately 1 kbp upstream of the 5' end of the pmt1 gene was amplified by PCR using primers set forth in SEQ ID NOs: 41 and 42, and approximately 1 kbp downstream of the 3' end of the pmt1 gene was amplified by PCR using primers set forth in SEQ ID NOs: 43 and 44. Next, using both amplified DNA fragments as templates, PCR was performed using the DNA set forth in SEQ ID NOs: 41 and 44 as primers to obtain a DNA fragment of approximately 2 kbp in which both DNA fragments were fused. PCR was performed using Pyrobest® DNA polymerase (Takara Bio) under the conditions recommended by the manufacturer. This DNA fragment was inserted into the SmaI site of pBS5T described in WO2006 / 057450 to obtain the pmt1 gene deletion vector pBS5TΔpmt1. Ligation reactions were performed using the DNA Ligation Kit (Takara Bio).<Mighty Mix> (Takara Bio) was used, and the reaction conditions followed the protocol recommended by the manufacturer.

[0224] (2) Construction of pmt1 gene-deficient C. glutamicum YDK0107 strain. The C. glutamicum YDK0107 strain described in WO2016 / 171224 was transformed with pBS5TΔpmt1 constructed in (1). The C. glutamicum YDK0107 strain is a spontaneous mutant derived from the C. glutamicum YDK010 parent strain, carrying the W302C mutation in the phoS gene (WO2016 / 171224). The C. glutamicum YDK010 strain is a C. glutamicum AJ12036 (FERM BP-734) strain lacking the cell surface protein CspB (WO2002 / 081694). From the resulting transformants, strains were selected according to the method described in WO2006 / 057450, and the YDK0107Δpmt1 strain lacking the pmt1 gene was obtained.

[0225] (Example 1) Secretory expression of bovine basic fibroblast growth factor (bbFGF) by Corynebacterium glutamicum (1-1) Construction of a bbFGF secretory expression plasmid containing a TorA signal sequence The amino acid sequence of bovine fibroblast growth factor-2 (bovine basic fibroblast growth factor; hereinafter referred to as bbFGF) has already been determined (UniProt Accession No. P03969). This amino acid sequence is shown in SEQ ID NO:46.

[0226] A nucleotide sequence encoding bbFGF was designed taking into consideration the codon usage frequency of C. glutamicum. The designed nucleotide sequence is shown in SEQ ID NO:45.

[0227] Next, we designed a fusion protein (TorAss-bbFGF) of bbFGF with the 39-amino acid signal peptide of the E. coli TorA protein (UniProt Accession No. P33225) to enable secretory expression of bbFGF in C. glutamicum. The nucleotide sequence and amino acid sequence encoding the designed TorAss-bbFGF are shown in SEQ ID NO: 47 and SEQ ID NO: 48, respectively. The cspB gene promoter from C. glutamicum ATCC13869 was ligated upstream of the nucleotide sequence encoding TorAss-bbFGF, and a KpnI site was added to the 5'-end and an ApaI site to the 3'-end. An expression cassette for TorAss-bbFGF was designed and synthesized.

[0228] A bbFGF secretion expression plasmid, pPK10_TorAss-bbFGF, utilizing the TorA signal sequence was constructed by inserting the TorAss-bbFGF expression cassette into the KpnI-ApaI site of pPK10, described in WO2021 / 112249. Nucleotide sequencing of the inserted fragment confirmed that the TorAss-bbFGF expression cassette was constructed as designed. Nucleotide sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and a 3500xL Genetic Analyzer (Applied Biosystems).

[0229] (1-2) Secretory Expression of bbFGF in C. glutamicum The C. glutamicum YDK0107Δpmt1 strain described in Reference Example 1 was transformed with pPK10_TorAss-bbFGF to obtain the YDK0107Δpmt1 / pPK10_TorAss-bbFGF strain. The resulting transformant was cultured at 30°C for 72 hours in MMTG liquid medium (120 g glucose, 3 g magnesium sulfate heptahydrate, 30 g ammonium sulfate, 1.5 g potassium dihydrogen phosphate, 0.03 g ferrous sulfate heptahydrate, 0.03 g manganese sulfate pentahydrate, 0.45 mg thiamine hydrochloride, 0.45 mg biotin, 0.15 g DL-methionine, soybean hydrochloric acid hydrolyzate (total nitrogen: 0.2 g), 50 g calcium carbonate, and adjusted to 1 L with water and pH 7.0) containing 25 mg / L kanamycin. After the incubation, each culture was centrifuged, and 6.5 μL of the resulting culture supernatant was subjected to reducing SDS-PAGE using NuPAGE® 12% Bis-Tris Gel (Thermo Fisher Scientific) and then stained with Quick-CBB (Wako). As a result, a protein band presumed to be bbFGF was detected in the culture supernatant of the YDK0107Δpmt1 / pPK10_TorAss-bbFGF strain (Figure 1, Lanes 2-5).

[0230] Example 2: Evaluation of the Biological Activity of Bovine Basic Fibroblast Growth Factor (bbFGF) Secreted by Corynebacterium glutamicum - 1 (2-1) Preparation of Crudely Purified bbFGF Fraction A The C. glutamicum YDK0107Δpmt1 / pPK10_TorAss-bbFGF strain was cultured in a fermenter according to a known method (Hiroshi Itaya and Yoshimi Kikuchi. Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria. Appl Microbiol Biotechnol. 2008; 78(4): 621-625.). The supernatant of the culture was diluted 6.6-fold with ultrapure water and sterile-filtered using a 0.2 μm PVDF membrane filter. This fraction was designated as Crudely Purified Fraction A and was stored frozen at -80°C until its biological activity was evaluated.

[0231] (2-2) Activity Measurement of Crudely Purified Fraction A of bbFGF The activity of the crudely purified fraction A obtained above was measured by cell proliferation activity using BALB / c 3T3 cells. The activity standard was human-derived bFGF International Standard (WHO International Standard FGF-2, Human, rDNA derived, NIBSC code: 90 / 712, Specific Activity; 8.0 x 10 5 The bbFGF concentration in crude fraction A was determined by reverse-phase HPLC using bFGF (basic FGF) Solution, Human, Recombinant (154 aa), Animal-Free (Nacalai Tesque, Cat. #19155-07) as a standard. The cell proliferation activity (specific activity) of crude fraction A was 3.6 x 10 6 U / mg.

[0232] Example 3: Evaluation of the Biological Activity of Bovine Basic Fibroblast Growth Factor (bbFGF) Secreted by Corynebacterium glutamicum - 2 (3-1) Preparation of Crudely Purified bbFGF Fraction B The C. glutamicum YDK0107Δpmt1 / pPK10_TorAss-bbFGF strain was cultured in a fermenter according to a known method (Hiroshi Itaya and Yoshimi Kikuchi. Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria. Appl Microbiol Biotechnol. 2008; 78(4): 621-625.). Dithiothreitol was added to the centrifuged culture supernatant to a concentration of 1.03 mg / mL. After standing for 15 minutes, the mixture was diluted 6.6-fold with ultrapure water and sterile filtered using a 0.2 μm PVDF membrane filter. This was designated as crude fraction B and was stored frozen at -80°C until evaluation of its biological activity.

[0233] (3-2) Activity Measurement of Crudely Purified bbFGF Fraction B The activity of the crudely purified fraction B obtained above was measured by cell proliferation activity using BALB / c 3T3 cells. The activity standard was human-derived bFGF International Standard (WHO International Standard FGF-2, Human, rDNA derived, NIBSC code: 90 / 712, Specific Activity; 8.0 x 10 5 The bbFGF concentration in crude fraction B was determined by reverse-phase HPLC using bFGF (basic FGF) Solution, Human, Recombinant (154 aa), Animal-Free (Nacalai Tesque, Cat. #19155-07) as a standard. The cell proliferation activity (specific activity) of crude fraction B was 2.5 x 10 6 U / mg.

[0234] Example 4: Evaluation of the biological activity of bovine basic fibroblast growth factor (bbFGF) secreted by Corynebacterium glutamicum - 3 (4-1) Preparation of crude bbFGF fraction C: The C. glutamicum YDK0107Δpmt1 / pPK10_TorAss-bbFGF strain was cultured in a fermenter according to a known method (Hiroshi Itaya and Yoshimi Kikuchi. Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria. Appl Microbiol Biotechnol. 2008; 78(4): 621-625.). Dithiothreitol was added to the centrifuged culture supernatant to a concentration of 1.03 mg / mL. After standing for 15 minutes, the solvent was exchanged with a solution of 154 mg / L dithiothreitol in PBS (Thermo Fisher Scientific Cat#: 14190) using a 3000 molecular weight ultrafiltration membrane. Sterile filtration was performed using a 0.22 μm PVDF membrane filter. This fraction was designated as crude fraction C and was stored frozen at -80°C until biological activity evaluation.

[0235] (4-2) Activity Measurement of Crudely Purified bbFGF Fraction C The activity of the crudely purified fraction C obtained above was measured by cell proliferation activity using BALB / c 3T3 cells. The activity standard was human-derived bFGF International Standard (WHO International Standard FGF-2, Human, rDNA derived, NIBSC code: 90 / 712, Specific Activity; 8.0 x 10 5 The bbFGF concentration in crude fraction C was determined by reverse-phase HPLC using bFGF (basic FGF) Solution, Human, Recombinant (154 aa), Animal-Free (Nacalai Tesque, Cat. #19155-07) as a standard. The cell proliferation activity (specific activity) of crude fraction C was 3.1 x 10 6U / mg.

[0236] Example 5: Evaluation of the Biological Activity of Bovine Basic Fibroblast Growth Factor (bbFGF) Secreted by Corynebacterium glutamicum - 4 (5-1) Preparation of Crudely Purified bbFGF Fraction D The C. glutamicum YDK0107Δpmt1 / pPK10_TorAss-bbFGF strain was cultured in a fermenter according to a known method (Hiroshi Itaya and Yoshimi Kikuchi. Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria. Appl Microbiol Biotechnol. 2008; 78(4): 621-625.). The pH of the culture supernatant was adjusted to 6.5 with 50% acetic acid, and then dithiothreitol (DTT) was added to a concentration of 27 mg / mL for dissolution. After standing for 15 minutes, water was added to adjust the conductivity to 9.5 mS / cm (25°C). The sample was filtered through a 0.22 μm PVDF membrane filter and used as the load solution for cation exchange chromatography. The column was equilibrated with A-buffer (5.7 g disodium hydrogen phosphate, 11.7 g sodium dihydrogen phosphate, 0.15 g DTT, 5.4 g sodium chloride, and 908 mL of water) using a HiTrap SP FF (Cytiva, Cat#: 17504501) and then loaded with the aforementioned load solution. After re-equilibration with A-buffer, the column was eluted with a gradient (0-100%, 20 CV) of B-buffer (3.6 g disodium hydrogen phosphate, 0.15 g DTT, 6.0 g sodium chloride, and 1000 mL of water, adjusted to pH 8.0 with A-buffer, to which sodium chloride was added to a concentration of 1 M) (Figure 2). The peak fraction was collected and designated crude fraction D, which was stored frozen at -80°C until bioactivity evaluation.

[0237] (5-2) Activity Measurement of Crudely Purified bbFGF Fraction D The activity of the crudely purified fraction D obtained above was measured by cell proliferation activity using BALB / c 3T3 cells. The activity standard was human-derived bFGF International Standard (WHO International Standard FGF-2, Human, rDNA derived, NIBSC code: 90 / 712, Specific Activity; 8.0 x 10 5 The bbFGF concentration in crude fraction D was determined by reverse-phase HPLC using bFGF (basic FGF) Solution, Human, Recombinant (154 aa), Animal-Free (Nacalai Tesque, Cat. #19155-07) as a standard. The cell proliferation activity (specific activity) of crude fraction D was 2.3 x 10 6 U / mg.

[0238] Example 6: Evaluation of the Biological Activity of Bovine Basic Fibroblast Growth Factor (bbFGF) Secreted by Corynebacterium glutamicum - 5 (6-1) Preparation of Crudely Purified bbFGF Fraction E The C. glutamicum YDK0107Δpmt1 / pPK10_TorAss-bbFGF strain was cultured in a fermenter according to a known method (Hiroshi Itaya and Yoshimi Kikuchi. Secretion of Streptomyces mobaraensis pro-transglutaminase by coryneform bacteria. Appl Microbiol Biotechnol. 2008; 78(4): 621-625.). The pH of the culture supernatant was adjusted to 6.5 with 50% acetic acid, and then dithiothreitol was added to dissolve the culture at a concentration of 27 mg / mL. After allowing to stand for 15 minutes, water was added to adjust the conductivity to 9.5 mS / cm (25°C). The solution was filtered through a 0.22 μm PVDF membrane filter and used as the load solution for cation exchange chromatography. The column was equilibrated with A-buffer (5.7 g disodium hydrogen phosphate, 11.7 g sodium dihydrogen phosphate, 1.7 g EDTA, 5.4 g sodium chloride, and 908 mL of water) using a HiTrap SP FF (Cytiva, Cat#: 17504501) and then loaded with the aforementioned load solution. After re-equilibration with A-buffer, the column was eluted with a gradient (0-100%: 20 CV) of B-buffer (3.6 g disodium hydrogen phosphate, 1.9 g EDTA, 6.0 g sodium chloride, and 1000 mL of water, with sodium chloride added to a concentration of 1 M) (Figure 3). The peak fractions were pooled (Figure 3, pool) and subjected to solvent exchange using a 3000 molecular weight ultrafiltration membrane with a solution of PBS (Thermo Fisher Scientific, Cat#: 14190) supplemented with dithiothreitol at a concentration of 154 mg / L. Sterile filtration was then performed using a 0.22 μm PVDF membrane filter. This fraction was designated crude fraction E and was stored frozen at -80°C until bioactivity evaluation.

[0239] (6-2) Activity Measurement of Crudely Purified bbFGF Fraction E The activity of the crudely purified fraction E obtained above was measured by cell proliferation activity using BALB / c 3T3 cells. The activity standard was human-derived bFGF International Standard (WHO International Standard FGF-2, Human, rDNA derived, NIBSC code: 90 / 712, Specific Activity; 8.0 x 10 5 The bbFGF concentration in crude fraction E was determined by reverse-phase HPLC using bFGF (basic FGF) Solution, Human, Recombinant (154 aa), Animal-Free (Nacalai Tesque, Cat. #19155-07) as a standard. The cell proliferation activity (specific activity) of crude fraction E was 2.6 x 10 6 U / mg.

[0240] As shown in Examples 2 to 6, all fractions exhibited high cell proliferation activity. This indicates that bbFGF produced using coryneform bacteria as an expression host can be suitably used for culturing animal cells (specifically, for producing cultured meat). In particular, it was found that bbFGF produced using coryneform bacteria as an expression host can be suitably used for culturing animal cells (specifically, for producing cultured meat) without the need for highly purified bbFGF by chromatography or other methods.

[0241] (Example 7) Evaluation of the biological activity of bovine basic fibroblast growth factor (bbFGF) secreted by Corynebacterium glutamicum - 6 (7-1) Preparation of bovine muscle stem cells Muscle tissue was collected from edible beef shank, and CD56-positive cells were isolated using a magnetically activated cell sorting system (MACS) to prepare bovine muscle stem cells.

[0242] (7-2) Evaluation of bbFGF activity using bovine muscle stem cells in serum-free medium The bovine muscle stem cells obtained above were suspended in serum-free medium, which consisted of Advanced DMEM (Thermo Fisher Scientific, Cat No. 12491015) supplemented with 20% FBS (Thermo Fisher Scientific, Cat No. 10270106), 10% horse serum (Thermo Fisher Scientific, Cat No. 16050-130), and 1% GlutaMAX. TM Supplement (Thermo Fisher Scientific, Cat No. 35050061) was added to the cell suspension at 1 μg / cm 2 5300 cells / 2 mL / cm were cultured in a 6-well plate (BD FALCON Cat. #353046) coated with fibronectin (Corning Cat. #F2006). 2 On the fourth day of culture, the cells were subcultured in serum-free medium without bbFGF or serum-free medium with bbFGF, and the medium was changed every other day. R Basic03 (Ajinomoto) supplemented with 5 ng / mL HGF (Peprotech Cat. #294HGN005) and 0.5 mg / mL soy protein acid hydrolysate (Sigma Cat. #S1674) was used. For bbFGF-containing serum-free medium, bbFGF-nonserum-free medium was supplemented with bbFGF crude fraction A, bbFGF crude fraction E, or Animal-Free Recombinant Bovine FGF-basic (Peprotech Cat. #AF-450-62) at 0.1, 1, or 10 ng / mL. Table 1 summarizes the group numbers and media. Cell counts and passages were performed every 4 days, and the growth rate and doubling time were calculated for two consecutive passages from day 4 to day 12.

[0243]

[0244] The results are shown in Figures 4 to 6. bbFGF crude fraction A, bbFGF crude fraction E, and animal-free recombinant bovine FGF-basic all promoted the proliferation of bovine muscle stem cells in serum-free medium. This demonstrates that bbFGF produced using coryneform bacteria as an expression host can be suitably used in the culture of animal cells (specifically, the production of cultured meat). In particular, it demonstrates that bbFGF produced using coryneform bacteria as an expression host can be suitably used in the culture of animal cells (specifically, the production of cultured meat) without the need for highly purified bbFGF by chromatography or other methods.

[0245] (Example 8) Evaluation of the biological activity of bovine basic fibroblast growth factor (bbFGF) secreted by Corynebacterium glutamicum - 7 (8-1) Preparation of bovine muscle stem cells Muscle tissue was collected from edible beef shank, and CD56-positive cells were isolated using a magnetically activated cell sorting system (MACS) to prepare bovine muscle stem cells.

[0246] (8-2) Evaluation of bbFGF activity using bovine muscle stem cells The bovine muscle stem cells obtained above were suspended in serum-containing medium. The serum-containing medium was Advanced DMEM (Thermo Fisher Scientific, Cat No. 12491015) supplemented with 20% FBS (Thermo Fisher Scientific, Cat No. 10270106), 10% horse serum (Thermo Fisher Scientific, Cat No. 16050-130), and 1% GlutaMAX. TMThe cells were cultured in a 6-well plate (BD FALCON Cat. #353046) coated with 1 μg / cm² fibronectin (Corning Cat. #F2006) at a cell density of 5300 cells / 2 mL / cm², and the medium was changed every other day. On the fourth day of culture, the cells were suspended and passaged in serum-free medium without bbFGF or serum-free medium with bbFGF, and the medium was changed every other day. StemFit was used as the serum-free medium without bbFGF. R Basic03 (Ajinomoto) supplemented with 5 ng / mL HGF (Peprotech Cat. #294HGN005) and 0.5 mg / mL soy protein acid hydrolysate (Sigma Cat. #S1674) was used. For bbFGF-containing serum-free medium, bbFGF-non-serum-free medium supplemented with bbFGF crude fraction B, bbFGF crude fraction C, bbFGF crude fraction D, or Animal-Free Recombinant Bovine FGF-basic (Peprotech Cat. #AF-450-62) at 0.1, 1, or 10 ng / mL was used. Table 2 summarizes the group numbers and media. The cell suspension was cultured at 1 μg / cm. 2 5300 cells / 2 mL / cm were cultured in a 6-well plate (BD FALCON Cat. #353046) coated with fibronectin (Corning Cat. #F2006). 2 The cells were seeded at a cell density of 1000 x g / ml, and the medium was changed every other day. Cell counts and subcultures were performed every 4 days, and the growth rate and doubling time of two consecutive passages from day 4 to day 12 were calculated.

[0247]

[0248] The results are shown in Figures 7 to 9. bbFGF crudely purified fraction B, bbFGF crudely purified fraction C, bbFGF crudely purified fraction D, and animal-free recombinant bovine FGF-basic all promoted the proliferation of bovine muscle stem cells in serum-free medium. This demonstrates that bbFGF produced using coryneform bacteria as an expression host can be suitably used in the culture of animal cells (specifically, the production of cultured meat). In particular, it demonstrates that bbFGF produced using coryneform bacteria as an expression host can be suitably used in the culture of animal cells (specifically, the production of cultured meat) without the need for highly purified bbFGF by chromatography or other methods.

[0249] The present invention makes it possible to efficiently produce cultured meat.

[0250] [Explanation of the sequence listing] SEQ ID NO: 1: Nucleotide sequence of the phoS gene of C. glutamicum YDK010 SEQ ID NO: 2: Amino acid sequence of the PhoS protein of C. glutamicum YDK010 SEQ ID NO: 3: Amino acid sequence of the PhoS protein of C. glutamicum ATCC 13032 SEQ ID NO: 4: Amino acid sequence of the PhoS protein of C. glutamicum ATCC 14067 SEQ ID NO: 5: Amino acid sequence of the PhoS protein of C. callunae SEQ ID NO: 6: Amino acid sequence of the PhoS protein of C. crenatum SEQ ID NO: 7: Amino acid sequence of the PhoS protein of C. efficiens SEQ ID NO: 8: Nucleotide sequence of the phoR gene of C. glutamicum ATCC 13032 SEQ ID NO: 9: Amino acid sequence of the PhoR protein of C. glutamicum ATCC 13032 SEQ ID NO: 10: Nucleotide sequence of the cspB gene of C. glutamicum ATCC 13869 SEQ ID NO: 11: Amino acid sequence of the PhoS protein of C. glutamicum ATCC SEQ ID NO: 12: Nucleotide sequence of the tatA gene of C. glutamicum ATCC 13032 SEQ ID NO: 13: Amino acid sequence of the TatA protein of C. glutamicum ATCC 13032 SEQ ID NO: 14: Nucleotide sequence of the tatB gene of C. glutamicum ATCC 13032 SEQ ID NO: 15: Amino acid sequence of the TatB protein of C. glutamicum ATCC 13032 SEQ ID NO: 16: Nucleotide sequence of the tatC gene of C. glutamicum ATCC 13032 SEQ ID NO: 17: C.SEQ ID NO: 18: Amino acid sequence of the TorA signal peptide SEQ ID NO: 19: Amino acid sequence of the SufI signal peptide SEQ ID NO: 20: Amino acid sequence of the PhoD signal peptide SEQ ID NO: 21: Amino acid sequence of the LipA signal peptide SEQ ID NO: 22: Amino acid sequence of the IMD signal peptide SEQ ID NO: 23: Amino acid sequence of the twin-arginine motif SEQ ID NO: 24: Skipped sequence SEQ ID NO: 25: Amino acid sequence of the PS1 signal peptide SEQ ID NO: 26: Amino acid sequence of the PS2 signal peptide SEQ ID NO: 27: Amino acid sequence of the SlpA signal peptide SEQ ID NO: 28: Amino acid sequence of the CspB mature protein of C. glutamicum ATCC 13869 SEQ ID NOs: 29-31: Skipped sequences SEQ ID NOs: 32-36: Amino acid sequences of one embodiment of the insertion sequence used in the present invention SEQ ID NO: 37: Recognition sequence for Factor Xa protease SEQ ID NO: 38: Recognition sequence for ProTEV protease SEQ ID NO: 39: Nucleotide sequence of the PMT gene of C. glutamicum ATCC 13869. SEQ ID NO: 40: Amino acid sequence of PMT of C. glutamicum ATCC 13869. SEQ ID NOs: 41-44: Primers. SEQ ID NO: 45: Nucleotide sequence of the bbFGF gene. SEQ ID NO: 46: Amino acid sequence of bbFGF. SEQ ID NO: 47: Nucleotide sequence of TorAss-bbFGF. SEQ ID NO: 48: Amino acid sequence of TorAss-bbFGF.

Claims

1. A method for producing bovine basic fibroblast growth factor (bbFGF) for the production of cultured meat, A method comprising the step of culturing a Corynebacterium having a gene construct for the secretion and expression of bbFGF to obtain a culture containing bbFGF.

2. The method according to claim 1, further comprising the step of removing the cells of the Corynebacterium from the culture.

3. The method according to claim 1 or 2, wherein the step of purifying bbFGF is not included.

4. The claim 1 or 2, which does not include the step of purifying bbFGF by chromatography. The method.

5. The aforementioned gene construct comprises, in the 5' to 3' direction, a promoter sequence that functions in Corynebacteria, a nucleic acid sequence that encodes a signal peptide that functions in Corynebacteria, and bbFGF. It contains nucleic acid sequences that Claim 1 or otherwise, bbFGF is expressed as a fusion protein with the signal peptide. The method described in 2.

6. The claim states that the Corynebacterium bacterium is a bacterium of the genus Corynebacterium. The method described in 1 or 2.

7. The method according to claim 1 or 2, wherein the Corynebacterium bacterium is Corynebacterium glutamicum.

8. A method for producing cultured meat, The process includes culturing animal cells in the presence of bovine basic fibroblast growth factor (bbFGF), A method wherein bbFGF is produced by the method described in claim 1 or 2.