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WO2013118544A1 - Procédé de sécrétion/production de protéine - Google Patents

Procédé de sécrétion/production de protéine Download PDF

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Publication number
WO2013118544A1
WO2013118544A1 PCT/JP2013/050681 JP2013050681W WO2013118544A1 WO 2013118544 A1 WO2013118544 A1 WO 2013118544A1 JP 2013050681 W JP2013050681 W JP 2013050681W WO 2013118544 A1 WO2013118544 A1 WO 2013118544A1
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protein
gene
mutation
amino acid
strain
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PCT/JP2013/050681
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Japanese (ja)
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吉彦 松田
寛 板屋
慶実 菊池
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味の素株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a method for secretory production of a heterologous protein and a coryneform bacterium having a specific mutation.
  • Non-patent Document 1 Bacillus bacteria
  • Non-patent Document 2 methanol-assimilating yeast Pichia pastoris
  • Aspergillus filamentous fungi Non-Patent Documents 3 and 4
  • Patent Document 1 Non-patent document 5
  • secretion of proteases such as subtilisin
  • non-patent document 6 protein secretion using signal peptides of cell surface proteins PS1 and PS2 (also referred to as CspB) of coryneform bacteria
  • Patent document 2 PS2 Secretion of fibronectin binding protein using (CspB) signal peptide
  • Non-patent Document 7 PS2 Secretion of protransglutaminase using PS2 (CspB) and SlpA (also referred to as CspA) signal peptides
  • Patent Document 3 mutation Protein secretion using a type secretory device
  • Patent Document 5 Protransgle by mutant strain Secretion of Minaze
  • Non-Patent Document 8 The general protein secretion pathway is the Sec system, which is widely present from prokaryotes to eukaryotes. Recently, a protein secretion pathway that is completely different from the Sec system has been found in the thylakoid membrane of plant cell chloroplasts.
  • This new secretory pathway is named Tat system (Twin-Arginine Translocation system) because the arginine-arginine sequence is commonly present in the signal sequence of the secreted protein (Non-patent Document 8). It was.
  • Tat system Twin-Arginine Translocation system
  • proteins are secreted in a state prior to the formation of higher-order structures
  • proteins are known to be secreted through the cell membrane after forming higher-order structures in the cell.
  • Non-Patent Document 9 In coryneform bacteria, there is a report of protein secretory production using a Tat-dependent signal peptide (Patent Document 6).
  • Ribosomal protein S1 (also referred to as RpsA protein) is the largest protein constituting the 30S subunit of ribosome, and controls the binding of 16S rRNA in the 30S subunit and the SD sequence of mRNA (Non-patent Document 10). .
  • RpsA protein is an essential protein for growth (Non-patent Document 11).
  • mutations in RpsA protein are effective for L-amino acid production in intestinal bacteria (Patent Document 7).
  • ribosomal protein S12 also referred to as RpsL protein
  • RpsL protein mutant ribosomal protein S12
  • cell-free extracts of Escherichia coli with mutant ribosomal protein S12 can be used for protein synthesis by cell-free protein synthesis systems. It is known to be useful (Patent Document 8).
  • coryneform bacteria it is not known that mutation of ribosomal protein S1 is effective for secretory production of heterologous proteins, particularly for heterologous protein production using the Tat system.
  • a specific mutation in ribosomal protein S1 is effective for secretory production of a heterologous protein.
  • a coryneform bacterium having a mutation at a specific site of ribosomal protein S1 is not known.
  • An object of the present invention is to develop a novel technique for improving the secretory production of a heterologous protein by a coryneform bacterium and to provide a secretory production method for the heterologous protein by a coryneform bacterium.
  • the present inventors have mutated ribosomal protein S1 of coryneform bacteria in a method for producing a heterologous protein using coryneform bacteria as an expression host. As a result of the introduction, it was found that the ability to secrete and produce heterologous proteins by the Tat secretion apparatus was improved, and the present invention was completed.
  • a method for producing a heterologous protein comprising culturing a coryneform bacterium having a gene construct for secretory expression of a heterologous protein and recovering the secreted heterologous protein,
  • the coryneform bacterium is modified so as to have a mutation in the ribosomal protein S1 gene that improves the secretory production of a heterologous protein;
  • the method wherein the gene construct comprises, in 5 ′ to 3 ′ direction, a promoter sequence that functions in coryneform bacteria, a nucleic acid sequence that encodes a signal peptide that functions in coryneform bacteria, and a nucleic acid sequence that encodes a heterologous protein.
  • the mutation is a mutation that improves the secretory production of a heterologous protein more than twice as compared with a control strain having no mutation in the ribosomal protein S1 gene.
  • the mutation is a mutation that substitutes the amino acid residue at position 173 of the wild-type ribosomal protein S1 with another amino acid residue.
  • the substitution of the amino acid residue is substitution of a glutamic acid residue with a glycine residue.
  • the signal peptide is a Tat-dependent signal peptide.
  • the Tat-dependent signal peptide is selected from the group consisting of a TorA signal peptide, a SufI signal peptide, a PhoD signal peptide, a LipA signal peptide, and an IMD signal peptide.
  • the coryneform bacterium is further modified so that expression of one or more genes selected from a gene encoding a Tat secretion apparatus is increased.
  • the gene encoding the Tat secretion apparatus is selected from the group consisting of a tatA gene, a tatB gene, a tatC gene, and a tatE gene.
  • coryneform bacterium is a genus Corynebacterium.
  • coryneform bacterium is Corynebacterium glutamicum.
  • coryneform bacterium is a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734).
  • the coryneform bacterium is a coryneform bacterium in which the activity of a cell surface protein is reduced.
  • a coryneform bacterium modified to have a mutation in the ribosomal protein S1 gene A coryneform bacterium, wherein the mutation is a mutation that replaces the amino acid residue at position 173 of the wild-type ribosomal protein S1 with another amino acid residue.
  • the coryneform bacterium, wherein the substitution of the amino acid residue is a substitution of a glutamic acid residue with a glycine residue.
  • the coryneform bacterium which is Corynebacterium glutamicum.
  • coryneform bacterium described above, which is a modified strain derived from Corynebacterium glutamicum AJ12036 (FERM BP-734). [18] The coryneform bacterium, wherein the activity of cell surface protein is reduced.
  • FIG. 1 is a photograph showing the results of SDS-PAGE when protransglutaminase fused with the TorA signal sequence of E. coli was expressed in the C. glutamicum YDK010 strain and its RpsA (E173G) mutant.
  • FIG. 2 is a diagram showing alignment of amino acid sequences of RpsA proteins derived from various coryneform bacteria. The “*” at the bottom indicates a completely matched amino acid.
  • the amino acid sequences of RpsA proteins of C. glutamicum THM1, C. glutamicum YDK010, C. glutamicum ATCC13032, C. efficiens YS-314, and C. stationis ATCC6872 are shown in SEQ ID NOs: 2, 4, 8, respectively.
  • FIG. 3 is a photograph showing the results of SDS-PAGE when C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain are expressed with protein glutaminase with a pro-structure part fused with TorA signal sequence of E. coli. is there.
  • FIG. 4 is a photograph showing the results of SDS-PAGE when Arthrobacter globiformis isomaltdextranase (including signal sequence) was expressed in C. glutamicum YDK010 strain and its RpsA (E173G) mutant.
  • FIG. 5 shows SDS-expressing pro-transglutaminase fused with the TorA signal sequence of E. coli in C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain and their Tat secretion apparatus-enhanced strains. It is a photograph which shows the result of PAGE.
  • Fig. 6 shows the expression of protein glutaminase with a pro-structure part fused with the TorA signal sequence of E. coli in C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain and their Tat secretion apparatus-enhanced strains. It is a photograph which shows the result of SDS-PAGE.
  • FIG. 6 shows the expression of protein glutaminase with a pro-structure part fused with the TorA signal sequence of E. coli in C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain and their Tat secretion apparatus-enhance
  • FIG. 7 shows the SDS when expressing the isomaltdextranase (including signal sequence) of Arthrobacter globiformis in C. glutamicum YDK010 strain and its RpsA (E173G) mutant strain and their Tat secretion apparatus-enhanced strains. It is a photograph showing the result of -PAGE.
  • FIG. 8 shows the results of SDS-PAGE when protransglutaminase was expressed in the C. glutamicum YDK010 strain and its RpsA (E173G) mutant and fused with the signal sequence of ClpAstationis ATCC6872 SlpA (CspA). It is a photograph.
  • the present invention comprises culturing a coryneform bacterium having a gene construct for secretory expression of a heterologous protein, and recovering the secreted heterologous protein.
  • a method wherein the coryneform bacterium is modified so as to have a mutation in the ribosomal protein S1 gene hereinafter also referred to as “the method of the present invention” or “the method for producing a heterologous protein of the present invention”. provide.
  • Coryneform bacterium used in the method of the present invention ⁇ 1-1-1> Coryneform bacterium having the ability to secrete and produce a heterologous protein
  • the coryneform bacterium used in the method of the present invention is capable of secreting a heterologous protein. By having a genetic construct for expression, it has the ability to secrete and produce heterologous proteins.
  • coryneform bacterium used in the method of the present invention is also referred to as “the bacterium of the present invention” or “the coryneform bacterium of the present invention”.
  • the gene construct for secretory expression of the heterologous protein possessed by the bacterium of the present invention is also referred to as “gene construct used in the present invention”.
  • secreting means that the protein is transferred outside the bacterial cell (outside the cell).
  • a protein is “secreted” when all the molecules of the protein are finally completely free in the medium, as well as when all the molecules of the protein are present on the surface of the cell. This includes the case where some molecules of the protein are present in the medium and the remaining molecules are present on the surface of the cells.
  • the ability to secrete and produce a heterologous protein means that when the bacterium of the present invention is cultured in a medium, the heterologous protein is secreted in the medium or on the surface of the cell, and in the medium or on the surface of the cell.
  • the accumulation amount is, for example, preferably 10 ⁇ g / L or more, more preferably 1 ⁇ mg / L or more, particularly preferably 100 ⁇ mg / L or more, and still more preferably 1 ⁇ g / L or more as the accumulation amount in the medium. It's okay.
  • the accumulated amount is preferably the concentration of the heterologous protein in the suspension when the heterogeneous protein on the cell surface is collected and suspended in the same amount of liquid as the medium, for example, as the amount accumulated on the cell surface.
  • the amount may be 10 ⁇ g / L or more, more preferably 1 ⁇ mg / L or more, and particularly preferably 100 ⁇ mg / L or more.
  • the “protein” secreted and produced in the present invention is a concept including an embodiment called a peptide or polypeptide.
  • heterologous protein refers to a protein that is exogenous to coryneform bacteria that express and secrete the protein.
  • the heterologous protein may be, for example, a protein derived from a microorganism, a protein derived from a plant, a protein derived from an animal, a protein derived from a virus, or an artificial protein.
  • the protein may be a protein whose amino acid sequence is designed.
  • the heterologous protein may be a monomeric protein or a multimer protein.
  • a multimeric protein refers to a protein that can exist as a multimer composed of two or more subunits.
  • each subunit may be linked by a covalent bond such as a disulfide bond, or may be linked by a non-covalent bond such as a hydrogen bond or a hydrophobic interaction, or by a combination thereof. May be.
  • Multimers preferably contain one or more intermolecular disulfide bonds.
  • the multimer may be a homomultimer composed of a single type of subunit or a heteromultimer composed of two or more types of subunits.
  • the multimeric protein is a heteromultimer, at least one subunit among the subunits constituting the multimer may be a heterologous protein. That is, all the subunits may be derived from different species, or only some of the subunits may be derived from different species.
  • the heterologous protein may be a protein that is naturally secreted or may be a protein that is non-secreted in nature, but is preferably a protein that is naturally secreted. In addition, the heterologous protein may or may not be a secreted protein that is naturally dependent on the Tat system. Specific examples of the “heterologous protein” will be described later.
  • the heterogeneous protein to be produced may be only one type, or two or more types.
  • the heterologous protein is a heteromultimer, only one type of subunit may be produced, or two or more types of subunits may be produced.
  • “secreting production of a heterologous protein” means that not only the subunits constituting the target heterologous protein but all the subunits are secreted and produced, and only some subunits are secreted and produced. Is also included.
  • the coryneform bacterium is an aerobic Gram-positive bacillus, and examples of the coryneform bacterium include Corynebacterium, Brevibacterium, and Microbacterium. And genus bacteria. Coryneform bacteria were previously classified as genus Brevibacterium, but now also include bacteria integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). . Corynebacterium bacteria were previously classified as Corynebacterium ammoniagenes, but some have been reclassified as Corynebacterium stationis by 16S rRNA sequence analysis. Included (Int. J. Syst. Evol.
  • coryneform bacteria is that, compared to filamentous fungi, yeast, Bacillus bacteria, etc., which are conventionally used for secretory production of heterologous proteins, there are very few proteins that are secreted outside the cells. Simplification and omission of the purification process can be expected, and it grows well on simple media containing sugar, ammonia, inorganic salts, etc., and excels in medium cost, culture method, and culture productivity And so on.
  • coryneform bacteria include the following species. Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium callunae Corynebacterium glutamicum Corynebacterium lilium Corynebacterium melassecola Corynebacterium thermoaminogenes (Corynebacterium efficiens) Corynebacterium herculis Brevibacterium divaricatum Brevibacterium flavum Brevibacterium immariophilum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium ammoniagenes (Corynebacterium stationis) Brevibacterium album Brevibacterium cerinum Microbacterium ammoniaphilum
  • coryneform bacteria include the following strains. Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium alkanolyticum ATCC 21511 Corynebacterium callunae ATCC 15991 Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734 Corynebacterium lilium ATCC 15990 Corynebacterium melassecola ATCC 17965 Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539) Corynebacterium herculis ATCC 13868 Brevibacterium divaricatum ATCC 14020 Brevibacterium flavum ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205) Brevibacterium immariophilum ATCC 14068 Brevibacterium lactofermentum ATCC 13869 Brevibacter
  • strains can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.
  • Corynebacterium glutamicum AJ12036 isolated as a streptomycin (Sm) -resistant mutant from wild-type Corynebacterium glutamicum ATCC 13869 is more protein-rich than its parent strain (wild-type). It is predicted that there is a mutation in the functional gene related to secretion of the protein, and the secretory production ability of the heterologous protein is extremely high, about 2 to 3 times as the accumulated amount under the optimum culture condition, and is suitable as a host bacterium.
  • AJ12036 was established on March 26, 1984, by the National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation, Patent Biological Deposit Center, East 1-1-1 Tsukuba, Japan, Central No. 6, postal code 305-8566) was originally deposited as an international deposit and was given the accession number FERM BP-734.
  • Corynebacterium ynethermoaminogenes AJ12340 (FERM BP-1539) was founded on March 13, 1987 at the National Institute of Advanced Industrial Science and Technology (currently the National Institute for Product Evaluation Technology, Patent Biological Depositary Center, East 1 Tsukuba, Japan). -1-1 The original deposit was made as an international deposit in the 6th post, ZIP code 305-8566), and the deposit number FERM BP-1539 was assigned. Also, Brevibacterium flavum AJ12418 (FERM-2BP-2205) was founded on December 24, 1988, by the National Institute of Advanced Industrial Science and Technology (currently an independent administrative agency, Product Evaluation Technology Foundation, Patent Biological Depositary Center, East 1 of Tsukuba City, Japan. 1-1 Deposited as an international deposit at the 6th post, zip code 305-8566), and assigned the deposit number FERM BP-2205.
  • the above-described coryneform bacterium as a parent strain may be selected as a host by selecting a strain having enhanced protein secretory production ability using a mutation method or a gene recombination method. For example, after treatment with a chemical mutagen such as ultraviolet irradiation or N-methyl-N′-nitrosoguanidine, a strain with enhanced protein secretory production ability can be selected.
  • a chemical mutagen such as ultraviolet irradiation or N-methyl-N′-nitrosoguanidine
  • a strain modified so as not to produce cell surface protein from such a strain is used as a host, purification of a heterologous protein secreted in the medium or on the surface of the cell body is facilitated, which is particularly preferable.
  • modification can be carried out by introducing mutation into the coding region of cell surface protein on the chromosome or its expression regulatory region by mutation or gene recombination.
  • coryneform bacteria modified so as not to produce cell surface proteins include C. glutamicum YDK010 strain (WO2004 / 029254), which is a cell line protein PS2 deficient strain of C. glutamicum AJ12036 (FERM BP-734). .
  • the bacterium of the present invention may have a reduced cell surface protein activity.
  • cell surface layer protein and the gene which codes it are demonstrated.
  • the cell surface protein is a protein constituting the cell surface layer (S layer) of bacteria and archaea.
  • Examples of cell surface proteins of coryneform bacteria include PS1 and PS2 (also referred to as CspB) of C. glutamicum, and SlpA (also referred to as CspA) of C. stationis. In these, it is preferable to reduce the activity of PS2 protein.
  • the base sequence of the cspB gene of C. glutamicum ATCC13869 and the amino acid sequence of the PS2 protein encoded by the gene are shown in SEQ ID NOs: 26 and 27, respectively.
  • 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)
  • the gene encoding the cell surface protein encodes a protein whose original function is maintained.
  • “Maintaining the original function” may mean, for example, having the property of increasing the secretory production amount of a heterologous protein as compared to an unmodified strain when the activity is reduced in a coryneform bacterium.
  • a gene encoding a cell surface protein has the property of increasing the secreted production amount of a heterologous protein compared to an unmodified strain when the activity is reduced in a protein having the original function maintained, for example, a coryneform bacterium.
  • the description regarding the ribosomal protein S1 and the variant of the gene encoding the same can be applied mutatis mutandis.
  • the property of increasing the secretory production of heterologous proteins when the activity is reduced in coryneform bacteria compared to non-modified strain means that a non-modified strain such as a wild strain is reduced in activity in coryneform bacteria. Alternatively, it refers to the property of giving coryneform bacteria the ability to secrete and produce a larger amount of heterologous protein than the parent strain.
  • the secretory production of a larger amount of a heterologous protein than that of an unmodified strain is not particularly limited as long as the amount of secretory production of the heterologous protein is increased as compared with that of the unmodified strain, but for example, in the medium and / or on the cell surface
  • the amount of accumulation is preferably 10% or more, more preferably 20% or more, particularly preferably 30% or more, and even more preferably 100% or more of the heterologous protein, as compared with the unmodified strain. Good.
  • secretory production of a larger amount of heterologous protein than the unmodified strain means that the heterogeneous protein cannot be detected when the culture supernatant of the unmodified strain that has not been concentrated is subjected to SDS-PAGE and stained with CBB. It may be possible to detect a heterologous protein when the culture supernatant of a modified strain that has not been subjected to SDS-PAGE and stained with CBB.
  • a modified strain is prepared, and the amount of a heterologous protein secreted and produced when the modified strain is cultured in a medium is quantified, and secreted and produced when the unmodified strain (non-modified strain) is cultured in a medium. This can be confirmed by comparing with the amount of the heterologous protein.
  • the activity of the cell surface protein is reduced means that the coryneform bacterium has been modified so that the activity of the cell surface protein is reduced, and that This includes cases where the activity is reduced.
  • “When the activity of the cell surface protein is originally reduced in the coryneform bacterium” includes the case where the coryneform bacterium originally does not have the cell surface protein. That is, examples of coryneform bacteria in which the activity of cell surface proteins is reduced include coryneform bacteria that originally have no cell surface proteins. Examples of “when the coryneform bacterium originally has no cell surface protein” include a case where the coryneform bacterium originally does not have a gene encoding the cell surface protein.
  • the coryneform bacterium originally has no cell surface protein means that the coryneform bacterium is selected from one or more cell surface proteins found in other strains of the species to which the coryneform bacterium belongs. It may be that there is no protein originally.
  • C. glutamicum originally has no cell surface protein means that the C. glutamicum strain is one or more proteins selected from cell surface proteins found in other C. glutamicum strains, For example, it may be inherently free of PS1 and / or PS2 (CspB).
  • Examples of coryneform bacteria that originally have no cell surface protein include C. glutamicum ATCC 13032 that originally does not have a cspB gene.
  • Protein activity decreases means that the activity of the target protein is reduced compared to a non-modified strain such as a wild strain or a parent strain, and includes cases where the activity is completely lost. .
  • the activity of the protein is decreased means that the number of molecules per cell of the protein is decreased and / or the function per molecule of the protein compared to the unmodified strain. Means that it is decreasing. That is, “activity” in the case of “decrease in protein activity” is not limited to the catalytic activity of the protein, and may mean the transcription amount (mRNA amount) of the gene encoding the protein or the amount of the protein. Note that “the number of molecules per cell of the protein is decreased” includes a case where the protein does not exist at all. Moreover, “the function per molecule of the protein is reduced” includes the case where the function per molecule of the protein is completely lost.
  • the modification that reduces the activity of the protein is achieved, for example, by reducing the expression of a gene encoding the protein.
  • “the expression of the gene is reduced” is also referred to as “the expression of the gene is weakened”.
  • the decrease in gene expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof.
  • Reduction of gene expression can be achieved, for example, by modifying an expression regulatory sequence such as a gene promoter or Shine-Dalgarno (SD) sequence.
  • SD Shine-Dalgarno
  • the expression control sequence is preferably modified by 1 base or more, more preferably 2 bases or more, particularly preferably 3 bases or more. Further, part or all of the expression regulatory sequence may be deleted.
  • Factors involved in expression control include small molecules (such as inducers and inhibitors) involved in transcription and translation control, proteins (such as transcription factors), nucleic acids (such as siRNA), and the like.
  • the modification that decreases the activity of the protein can be achieved, for example, by destroying a gene encoding the protein.
  • Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome.
  • the entire gene including the sequences before and after the gene on the chromosome may be deleted.
  • the region to be deleted may be any region such as an N-terminal region, an internal region, or a C-terminal region as long as a decrease in protein activity can be achieved.
  • the longer region to be deleted can surely inactivate the gene.
  • it is preferable that the reading frames of the sequences before and after the region to be deleted do not match.
  • gene disruption is, for example, introducing an amino acid substitution (missense mutation) into a coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or adding or deleting 1 to 2 bases. It can also be achieved by introducing a 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)).
  • gene disruption can be achieved, for example, by inserting another sequence into the coding region of the gene on the chromosome.
  • the insertion site may be any region of the gene, but the longer the inserted sequence, the more reliably the gene can be inactivated.
  • Other sequences are not particularly limited as long as they reduce or eliminate the activity of the encoded protein, and examples include marker genes such as antibiotic resistance genes and genes useful for heterologous protein production.
  • Modifying a gene on a chromosome as described above includes, for example, deleting a partial sequence of the gene and preparing a deleted gene modified so as not to produce a normally functioning protein. This can be achieved by replacing the gene on the chromosome with the deleted gene by transforming the bacterium with the recombinant DNA containing, and causing homologous recombination between the deleted gene and the gene on the chromosome. At this time, the recombinant DNA can be easily manipulated by including a marker gene in accordance with a trait such as auxotrophy of the host. Even if the protein encoded by the deletion-type gene is produced, it has a three-dimensional structure different from that of the wild-type protein, and its function is reduced or lost.
  • the modification that reduces the activity of the protein may be performed by, for example, a mutation treatment.
  • Mutation treatment includes X-ray irradiation or ultraviolet irradiation, or N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), methylmethanesulfonate (MMS), etc.
  • MNNG N-methyl-N′-nitro-N-nitrosoguanidine
  • EMS ethyl methanesulfonate
  • MMS methylmethanesulfonate
  • the decrease in the activity of the target protein can be confirmed by measuring the activity of the protein.
  • the activity of the protein is, for example, 50% or less, preferably 20% or less, more preferably 10% or less, still more preferably 5% or less, and particularly preferably 0% compared to the unmodified strain. descend.
  • the decrease in the expression of the target gene can be confirmed by confirming that the transcription amount of the gene has decreased, or by confirming that the amount of the target protein expressed from the gene has decreased.
  • the amount of mRNA is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the unmodified strain.
  • the amount of protein is preferably reduced to, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0% as compared to the unmodified strain.
  • the destruction of the target gene can be confirmed by determining a part or all of the base sequence, restriction enzyme map, full length, etc. of the gene according to the means used for the destruction.
  • Coryneform bacteria having the ability to secrete and produce heterologous proteins can be obtained by introducing and retaining the gene construct used in the present invention in the coryneform bacteria as described above.
  • the gene construct used in the present invention and its introduction method will be described later.
  • mutant ribosomal protein S1 gene The bacterium of the present invention has been modified to have a mutation in the ribosomal protein S1 gene.
  • “having a mutation in the ribosomal protein S1 gene” means having a mutant ribosomal protein S1 gene.
  • the bacterium of the present invention can be obtained by modifying a coryneform bacterium having the ability to secrete and produce a heterologous protein so as to have a mutant ribosomal protein S1 gene.
  • the bacterium of the present invention can also be obtained by imparting the ability to secrete and produce a heterologous protein after modifying the coryneform bacterium so as to have a mutant ribosomal protein S1 gene.
  • the modification for constructing the bacterium of the present invention can be performed in any order.
  • the bacterium of the present invention may be obtained from a strain that has been capable of secreting and producing a heterologous protein before being modified so as to have a mutant ribosomal protein S1 gene. Further, the bacterium of the present invention could not secrete and produce a heterologous protein even if it had a gene construct for secretory expression of the heterologous protein before being modified to have the mutant ribosomal protein S1 gene. It may be obtained from a strain and modified to have a mutated ribosomal protein S1 gene so that a heterologous protein can be secreted and produced.
  • ribosomal protein S1 and the gene encoding it will be described.
  • a ribosomal protein S1 having “mutation” described later is also referred to as a mutant ribosomal protein S1
  • a gene encoding it is also referred to as a mutant ribosomal protein S1 gene.
  • ribosomal protein S1 having no “mutation” described later is also referred to as wild-type ribosomal protein S1
  • a gene encoding it is also referred to as wild-type ribosomal protein S1 gene.
  • Examples of the wild-type ribosomal protein S1 gene include the rpsA gene of C. glutamicum YDK010 strain, C. glutamicum ATCC13032 strain, C. efficiens YS-314 strain, and C. stationis ATCC6872 strain.
  • the nucleotide sequences of these rpsA genes are shown in SEQ ID NOs: 3, 7, 9, and 11, respectively.
  • the amino acid sequences of RpsA proteins encoded by these rpsA genes are shown in SEQ ID NOs: 4, 8, 10, and 12, respectively.
  • the wild-type ribosomal protein S1 may be a variant of the RpsA protein as long as it does not have a “mutation” described later and has a function as the ribosomal protein S1. Such variants may be referred to as “conservative variants”.
  • the conservative variant may be, for example, a homologue or artificial variant of the above RpsA protein.
  • the “function as ribosomal protein S1” may be, for example, a function of controlling the binding between 16S rRNA in the 30S subunit and the SD sequence of mRNA.
  • the wild-type ribosomal protein S1 preferably has, for example, a conserved sequence in the RpsA protein.
  • FIG. 2 shows the result of alignment between RpsA proteins including the above RpsA proteins.
  • the wild-type ribosomal protein S1 gene may be a variant of the rpsA gene as long as it encodes the RpsA protein or a conservative variant thereof.
  • the gene encoding the homologue of the RpsA protein can be easily obtained from a public database by BLAST search or FASTA search using the above-mentioned coryneform rpsA gene (SEQ ID NO: 3, 7, 9, or 11) as a query sequence. Can be acquired.
  • the gene encoding the homologue of the RpsA protein can be obtained by PCR using, for example, a coryneform bacterium chromosome as a template and oligonucleotides prepared based on these known gene sequences as primers.
  • the wild-type ribosomal protein S1 does not have the “mutation” described later and has a function as the ribosomal protein S1, one or several amino acids at one or several positions in the amino acid sequence are substituted. It may be a protein having an amino acid sequence deleted, inserted or added.
  • the above “one or several” differs depending on the position of the amino acid residue in the three-dimensional structure of the protein and the kind of amino acid residue, but specifically, preferably 1 to 20, more preferably 1 to 10 More preferably, it means 1 to 5.
  • substitution, deletion, insertion, or addition of one or several amino acids described above is a conservative mutation that maintains the protein function normally.
  • a typical conservative mutation is a conservative substitution.
  • Conservative substitution is a polar amino acid between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. In this case, between Gln and Asn, when it is a basic amino acid, between Lys, Arg, and His, when it is an acidic amino acid, between Asp and Glu, when it is an amino acid having a hydroxyl group Is a mutation that substitutes between Ser and Thr.
  • substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Trp to Phe or Tyr, substitution
  • the wild-type ribosomal protein S1 does not have a “mutation” described later and has a function as the ribosomal protein S1, it is 80% or more, preferably 90% or more, more than the entire amino acid sequence. It may be a protein having a homology of preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more. In the present specification, “homology” may refer to “identity”.
  • the wild-type ribosomal protein S1 gene does not have a “mutation” described later, and a probe that can be prepared from a known gene sequence as long as it encodes a protein having a function as the ribosomal protein S1, for example, the above base sequence It may be a DNA that hybridizes under stringent conditions with a complementary sequence for all or a part thereof. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more DNAs having homology.
  • the probe used for the hybridization may be a part of a complementary sequence of a gene.
  • a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template.
  • hybridization washing conditions include 50 ° C., 2 ⁇ SSC, and 0.1% SDS.
  • the wild-type ribosomal protein S1 gene may be obtained by substituting an arbitrary codon with a codon equivalent thereto as long as it encodes the wild-type ribosomal protein S1 as described above.
  • the wild-type ribosomal protein S1 gene may be modified to have an optimal codon depending on the codon usage frequency of the host to be used.
  • the mutant ribosomal protein S1 has a “mutation” described later in the amino acid sequence of the wild-type ribosomal protein S1 as described above.
  • mutant ribosomal protein S1 may be the RpsA protein of the coryneform bacterium described above or a conservative variant thereof except that it has a “mutation” described later.
  • mutant ribosomal protein S1 may be a protein having the amino acid sequence shown in SEQ ID NO: 4, 8, 10, or 12 except that it has a “mutation” described later.
  • mutant ribosomal protein S1 is substituted with one or several amino acids in the amino acid sequence shown in SEQ ID NO: 4, 8, 10, or 12 except that it has a “mutation” described later.
  • the mutant ribosomal protein S1 is 80% or more, preferably 80% or more with respect to the amino acid sequence shown in SEQ ID NO: 4, 8, 10, or 12, except that it has a “mutation” described later. It may be a protein having an amino acid sequence having a homology of 90% or more, more preferably 95% or more, more preferably 97% or more, and particularly preferably 99% or more.
  • mutant ribosomal protein S1 is a variant having the “mutation” described later in the RpsA protein of the above-mentioned coryneform bacterium, and further containing a conservative mutation at a place other than the “mutation”. It's okay.
  • the mutant ribosomal protein S1 has a “mutation” to be described later in the amino acid sequence shown in SEQ ID NO: 4, 8, 10, or 12, and is further provided at a place other than the “mutation”. It may be a protein having an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions or additions.
  • the mutant ribosomal protein S1 gene is not particularly limited as long as it encodes the mutant ribosomal protein S1 as described above.
  • the “mutation” is not particularly limited as long as the amino acid sequence of the wild-type ribosomal protein S1 changes as described above, but is preferably a mutation that improves the secretory production amount of a heterologous protein. “Improving the secretory production amount of a heterologous protein” means that a coryneform bacterium modified to have a mutant ribosomal protein S1 gene can secrete and produce a larger amount of a heterologous protein than an unmodified strain.
  • non-modified strain is a control strain having no mutation in the ribosomal protein S1 gene, ie, a control strain having no mutant ribosomal protein S1 gene, and may be, for example, a wild strain or a parent strain.
  • “Secretly produce a heterologous protein in an amount larger than that of an unmodified strain” is not particularly limited as long as the amount of secretory production of the heterologous protein is increased as compared with that of the unmodified strain.
  • the amount accumulated on the surface layer is preferably 1.1 times or more, more preferably 1.2 times or more, particularly preferably 1.3 times or more, and even more preferably 2 times or more of that of non-modified strains. It may be the production of proteins secreted.
  • heterologous protein cannot be detected when the culture supernatant of the unmodified strain that has not been concentrated is subjected to SDS-PAGE and stained with CBB.
  • a mutation is a mutation that improves the production of secreted heterologous proteins, for example, based on a strain belonging to coryneform bacteria, a strain modified to have a gene encoding ribosomal protein S1 having the mutation is prepared. And quantifying the amount of the heterologous protein secreted and produced when the modified strain is cultured in the medium, and comparing it with the amount of the heterologous protein secreted and produced when the unmodified strain (non-modified strain) is cultured in the medium. This can be confirmed.
  • the “mutation” is preferably one in which any amino acid residue of the wild-type ribosomal protein S1 is substituted with another amino acid residue.
  • the “mutation” is preferably one in which the amino acid residue at position 173 of the wild-type ribosomal protein S1 is replaced with another amino acid residue.
  • the “mutation” is preferably one in which the amino acid residue at position 173 of the wild-type ribosomal protein S1 is replaced with a glycine residue.
  • the “mutation” is preferably one in which the glutamic acid residue at position 173 of the wild-type ribosomal protein S1 is replaced with a glycine residue.
  • amino acid residue at the X position of the wild-type ribosomal protein S1 means an amino acid residue corresponding to the X position in SEQ ID NO: 4.
  • the “X position” in the amino acid sequence means the X position from the N terminal of the amino acid sequence, and the amino acid residue at the N terminal is the first amino acid residue. That is, the position of the amino acid residue indicates a relative position, and the position may be moved back and forth by amino acid deletion, insertion, addition, or the like.
  • amino acid residue at position 173 of wild-type ribosomal protein S1 means an amino acid residue corresponding to position 173 in SEQ ID NO: 4, and one amino acid residue on the N-terminal side from position 173 has been deleted.
  • the 172nd amino acid residue from the N-terminal is “the amino acid residue at position 173 of wild-type ribosomal protein S1”.
  • the 174th amino acid residue from the N-terminal is assumed to be “the 173rd amino acid residue of the wild-type ribosomal protein S1”.
  • amino acid residue in any amino acid sequence is the “amino acid residue corresponding to the X position in SEQ ID NO: 4” can be determined by aligning the arbitrary amino acid sequence with the amino acid sequence of SEQ ID NO: 4. .
  • the alignment can be performed using, for example, known gene analysis software. Specific software includes DNA Solutions from Hitachi Solutions and GENETYX from Genetics (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).
  • the mutant ribosomal protein S1 gene can be obtained by modifying the wild-type ribosomal protein S1 gene so that the encoded ribosomal protein S1 has the “mutation” described above.
  • Modification of DNA can be performed by a known method. Specifically, for example, as a site-specific mutation method for introducing a target mutation into a target site of DNA, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds. , Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, H. J., Meth.
  • the mutant ribosomal protein S1 gene can also be obtained by chemical synthesis.
  • Altering coryneform bacteria to have a mutant ribosomal protein S1 gene can be achieved by introducing the mutant ribosomal protein S1 gene into the coryneform bacterium. Further, modifying the coryneform bacterium so as to have a mutant ribosomal protein S1 gene can also be achieved by introducing a mutation into the ribosomal protein S1 gene by natural mutation or mutagen treatment.
  • the method of introducing the mutant ribosomal protein S1 gene into coryneform bacteria is not particularly limited.
  • the mutant ribosomal protein S1 gene only needs to be retained so that it can be expressed under the control of a promoter that functions in the coryneform bacterium.
  • the mutant ribosomal protein S1 gene may be present on a vector that autonomously proliferates outside the chromosome, such as a plasmid, or may be integrated on the chromosome.
  • the bacterium of the present invention may have only one copy of the mutant ribosomal protein S1 gene, or may have two or more copies.
  • the bacterium of the present invention may have only one type of mutant ribosomal protein S1 gene or may have two or more types of mutant ribosomal protein S1 genes.
  • the introduction of the mutant ribosomal protein S1 gene can be performed, for example, in the same manner as the introduction of the gene construct used in the present invention described later.
  • the bacterium of the present invention may or may not have the wild-type ribosomal protein S1 gene, but preferably does not have it.
  • Coryneform bacteria that do not have the wild-type ribosomal protein S1 gene can be obtained by disrupting the wild-type ribosomal protein S1 gene on the chromosome.
  • the disruption of the wild-type ribosomal protein S1 gene can be performed by a known method. Specifically, for example, the wild-type ribosomal protein S1 gene can be disrupted by deleting part or all of the promoter region and / or coding region of the wild-type ribosomal protein S1 gene.
  • the ribosomal protein S1 gene is an essential gene, it is preferable to first introduce the mutant ribosomal protein S1 gene and then destroy the wild-type ribosomal protein S1 gene.
  • the wild-type ribosomal protein S1 gene has been modified to have no wild-type ribosomal protein S1 gene and a mutant ribosomal protein S1 gene.
  • Coryneform bacteria can be obtained.
  • a method for performing such gene replacement for example, a method called “Red-driven integration” (Datsenko, K. A, and Wanner, B. L. Proc. Natl. Acad. Sci. U S A. 97: 6640-6645 (2000)), Red driven integration method and ⁇ phage-derived excision system (Cho, E. H., Gumport, R.
  • I., Gardner, J. F. J. Bacteriol. 184 A method using linear DNA such as a method combined with 5200-5203 (2002)) (see WO2005 / 010175), a method using a plasmid containing a temperature-sensitive replication origin, a method using a plasmid capable of conjugation transfer
  • a method using a suicide vector that does not have a replication origin and functions in a host US Pat. No. 6,303,383, Japanese Patent Laid-Open No. 05-007491.
  • Enhancement of Tat secretion apparatus The bacterium of the present invention is modified so that expression of one or more genes selected from genes encoding the Tat secretion apparatus is increased. Also good. In the present invention, such modification is also referred to as “enhancement of the Tat secretion apparatus”.
  • a technique for increasing the expression of a gene encoding a Tat secretion apparatus is described in Patent Document 6 (Patent No. 4730302).
  • genes encoding the Tat 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 are sequences of positions 1571065 to 1581382 in the genome sequence registered as GenBank accession NC_003450 (VERSION NC_003450.3 GI: 58036263) in the NCBI database, respectively. It corresponds to the complementary sequence, the sequence of positions 1167110 to 1167580, and the complementary sequence of the sequences of positions 1569929 to 1570873.
  • the TatA protein, TatB protein, and TatC protein of C are sequences of positions 1571065 to 1581382 in the genome sequence registered as GenBank accession NC_003450 (VERSION NC_003450.3 GI: 58036263) in the NCBI database, respectively. It corresponds to the complementary sequence, the sequence of positions 1167110 to 116
  • locus_tag "NCgl1077”
  • 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: 13-18.
  • genes encoding the Tat secretion apparatus include the tatA gene, tatB gene, tatC gene, and tatE gene of E. coli.
  • the tatA gene, tatB gene, tatC gene, and tatE gene of E.coli K-12 MG1655 are respectively included in the genome sequence registered as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990) in the NCBI database. This corresponds to the sequence at position 4020237, the sequence at positions 4020241 to 4020756, the sequence at positions 4020759 to 4021535, and the sequence at positions 658170 to 658373.
  • the gene encoding the Tat secretion apparatus may be a variant of the above base sequence as long as it encodes a protein having a function as a Tat secretion apparatus. That is, for example, “tatA gene”, “tatB gene”, “tatC gene”, and “tatE gene” each include a variant thereof in addition to the above base sequence.
  • the above description concerning the wild-type ribosomal protein S1 and the variant of the gene encoding it can be applied mutatis mutandis to the Tat secretion apparatus and the gene encoding the same.
  • the “function as a Tat system secretion device” may be a function of secreting a protein having a Tat system-dependent signal peptide added to the N-terminus to the outside of a cell.
  • “gene expression is increased” means that the expression of a target gene is increased relative to an unmodified strain such as a wild strain or a parent strain.
  • the expression of the gene is not particularly limited as long as it is higher than that of the non-modified strain, but is preferably 1.5 times or more, more preferably 2 times or more, more preferably 3 times or more compared to the non-modified strain.
  • “increasing gene expression” means not only increasing the expression level of a target gene in a strain that originally expresses the target gene, but also in a strain that originally does not express the target gene. Including expressing a gene. That is, “increasing gene expression” includes, for example, introducing the gene into a strain that does not hold the target gene and expressing the gene.
  • An increase in gene expression can be achieved, for example, by increasing the copy number of the gene.
  • Increase in gene copy number can be achieved by introducing the target gene onto the chromosome of the host microorganism.
  • Genes can be introduced into chromosomes by transposon or Mini-Mu random introduction onto chromosomes (Japanese Patent Laid-Open No. 2-109985, US5,882,888 EP805867B1), or sequences that exist in multiple copies on chromosomal DNA. This can be achieved by utilizing the target for homologous recombination.
  • repetitive DNA inverted DNA
  • inverted repeats present at both ends of the transposon can be used.
  • a gene onto a chromosome by using the Red driven integration method (WO2005 / 010175). It is also possible to introduce a gene onto a chromosome by transduction using a phage or a conjugation transfer vector. Further, as described in WO03 / 040373, it is also possible to introduce a gene by targeting a gene unnecessary for secretory production of a heterologous protein on a chromosome. In this way, one or more copies of the gene can be introduced into the target sequence.
  • the increase in the copy number of the gene can also be achieved by introducing a vector containing the target gene into the host bacterium.
  • a DNA fragment containing a target gene is linked to a vector that functions in the host bacterium to construct an expression vector for the gene, and the host bacterium is transformed with the expression vector to increase the copy number of the gene.
  • the vector a vector capable of autonomous replication in a host bacterial cell can be used.
  • the vector is preferably a multicopy vector.
  • the vector preferably has a marker such as an antibiotic resistance gene.
  • the vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid.
  • vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol.
  • plasmids having improved drug resistance genes plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21 and pCRY2KE described in JP-A-2-72876 and US Pat. No. 5,185,262.
  • the increase in gene expression can be achieved by improving the transcription efficiency of the gene.
  • Improvement of gene transcription efficiency can be achieved, for example, by replacing a promoter of a gene on a chromosome with a stronger promoter.
  • strong promoter is meant a promoter that improves transcription of the gene over the native wild-type promoter.
  • a stronger promoter for example, a known high expression promoter such as T7 promoter, trp promoter, lac promoter, tac promoter, PL promoter and the like can be used.
  • a highly active promoter of a conventional promoter may be obtained by using various reporter genes.
  • the activity of the promoter can be increased by bringing the ⁇ 35 and ⁇ 10 regions in the promoter region closer to the consensus sequence (WO 00/18935).
  • Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotickpromoters in biotechnology. Biotechnol. Annu. Rev.,. 1, 105-128 (1995)).
  • the increase in gene expression can be achieved by improving the translation efficiency of the gene.
  • Improvement of gene translation efficiency can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also referred to as ribosome binding site (RBS)) of the gene on the chromosome with a stronger SD sequence.
  • SD Shine-Dalgarno
  • RBS ribosome binding site
  • a stronger SD sequence is meant an SD sequence in which the translation of mRNA is improved over the originally existing wild-type SD sequence.
  • RBS of gene 10 derived from phage T7 can be mentioned (Olins P. O. et al, Gene, 1988, 73, 227-235).
  • substitution of several nucleotides in the spacer region between the RBS and the start codon, particularly the sequence immediately upstream of the start codon (5'-UTR), or insertion or deletion contributes to mRNA stability and translation efficiency. It is known to have a great influence, and the translation efficiency of a gene can be improved by modifying them.
  • a site that affects gene expression such as a promoter, an SD sequence, and a spacer region between the RBS and the start codon is also collectively referred to as an “expression control region”.
  • the expression regulatory region can be determined using a promoter search vector or gene analysis software such as GENETYX.
  • GENETYX gene analysis software
  • These expression control regions can be modified by, for example, a method using a temperature sensitive vector or a Red driven integration method (WO2005 / 010175).
  • the increase in gene expression can be achieved by amplifying a regulator that increases the expression of the target gene, or by deleting or weakening a regulator that decreases the expression of the target gene.
  • the method of transformation is not particularly limited, and a conventionally known method can be used.
  • a method for increasing the permeability of DNA by treating recipient cells with calcium chloride (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162) and methods for introducing competent cells from proliferating cells and introducing DNA as reported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young, F. E .., 1997. Gene 1: 153-167) can be used.
  • DNA-receptive cells such as those known for Bacillus subtilis, actinomycetes, and yeast, can be made into protoplasts or spheroplasts that readily incorporate recombinant DNA into recombinant DNA.
  • Introduction method (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, M. J., Ward, J. M. and Hopwood, O. A. 1978. Nature 274: 398-400; Hinnen, A., Hicks, J. B. and Fink, G. R. 1978. Proc. Natl.Acad. Sci. USA 75: 1929-1933) can also be applied.
  • transformation of coryneform bacteria can also be performed by an electric pulse method (Japanese Patent Laid-Open No. 2-207791).
  • the increase in the expression of the target gene can be confirmed, for example, by confirming that the activity of the target protein expressed from the same gene has increased.
  • An increase in the activity of the target protein can be confirmed by measuring the activity of the protein.
  • the increase in the activity of the Tat secretion apparatus can be confirmed, for example, by confirming that the amount of secretory production of a protein having a Tat-dependent signal peptide added to the N-terminus has increased.
  • the amount of secretory production of a protein with a Tat-dependent signal peptide added to the N-terminus is increased by, for example, 1.5 times or more, 2 times or more, or 3 times or more compared to the unmodified strain. preferable.
  • the increase in the expression of the target gene can be confirmed, for example, by confirming that the transcription amount of the gene has increased, or by confirming that the amount of the target protein expressed from the gene has increased. it can.
  • the transcription amount of the target gene has increased by comparing the amount of mRNA transcribed from the same gene with an unmodified strain such as a wild strain or a parent strain.
  • Methods for assessing the amount of mRNA include Northern hybridization, RT-PCR, 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 is preferably increased 1.5 times or more, 2 times or more, or 3 times or more, compared to the unmodified strain, for example.
  • the amount of protein is preferably increased by 1.5 times or more, 2 times or more, or 3 times or more, for example, as compared to the unmodified strain.
  • secretory proteins are generally translated as preproteins (also referred to as prepeptides) or preproproteins (also referred to as prepropeptides), and then It is known that it becomes a mature protein by processing.
  • secreted proteins are generally translated as preproteins or preproproteins, and then the signal peptide, which is the prepart, is cleaved by a protease (commonly called signal peptidase) to be converted into a mature protein or proprotein.
  • protease commonly called signal peptidase
  • signal peptide is used for secretory production of a heterologous protein.
  • preproteins and preproproteins of secretory proteins are sometimes collectively referred to as “secreted protein precursors”.
  • signal peptide refers to an amino acid sequence that is present at the N-terminus of a secretory protein precursor and is not normally present in a natural mature protein.
  • the gene construct used in the present invention includes, in the 5 ′ to 3 ′ direction, a promoter sequence that functions in coryneform bacteria, a nucleic acid sequence that encodes a signal peptide that functions in coryneform bacteria, and a nucleic acid sequence that encodes a heterologous protein. .
  • 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 the heterologous protein may be linked downstream of the nucleic acid sequence encoding the signal peptide so that the heterologous protein is expressed as a fusion protein with the signal peptide.
  • the gene construct used in the present invention has control sequences (operators, terminators, etc.) effective for expressing heterologous protein genes in coryneform bacteria at appropriate positions so that they can function. Also good.
  • the promoter used in the present invention is not particularly limited as long as it is a promoter that functions in coryneform bacteria, and may be a promoter derived from coryneform bacteria or a promoter derived from a different species.
  • the “promoter that functions in coryneform bacteria” refers to a promoter having promoter activity in coryneform bacteria.
  • Specific examples of heterologous promoters include promoters derived from E. coli such as tac promoter, lac promoter, trp promoter, and araBAD promoter. Among them, a strong promoter such as tac promoter is preferable, and an inducible promoter such as araBAD promoter is also preferable.
  • promoters derived from coryneform bacteria include promoters for cell surface proteins PS1, PS2 (also referred to as CspB), SlpA (also referred to as CspA), and promoters of various amino acid biosynthesis genes.
  • Specific promoters for various amino acid biosynthesis genes include, for example, glutamate biosynthesis glutamate dehydrogenase gene, glutamine synthesis glutamine synthetase gene, lysine biosynthesis aspartokinase gene, threonine biosynthesis Homoserine dehydrogenase gene, isoleucine and valine biosynthesis acetohydroxy acid synthase gene, leucine biosynthesis 2-isopropylmalate synthase gene, proline and arginine biosynthesis glutamate kinase gene, histidine biosynthesis Phosphoribosyl-ATP pyrophosphorylase gene, aromatic amino acid biosynthetic deoxyarabinohepturonic acid phosphate (DAHP) synthase genes such as tryptophan, tyrosine and phenylalanine, such as inosinic acid and guanylic acid Examples include phosphoribosyl pyrophosphate (PRPP) amide transferase gene, ino
  • a high activity type of a conventional promoter may be obtained and used by using various reporter genes.
  • the activity of the promoter can be increased by bringing the ⁇ 35 and ⁇ 10 regions in the promoter region closer to the consensus sequence (WO 00/18935).
  • Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotickpromoters in biotechnology. Biotechnol. Annu. Rev.,. 1, 105-128 (1995)).
  • substitution of several nucleotides in the spacer region between the ribosome binding site (RBS) and the start codon, especially in the sequence immediately upstream of the start codon (5'-UTR), insertion or deletion makes the mRNA stable. It is known to greatly affect sex and translation efficiency, and these can be modified.
  • the signal peptide used in the present invention is not particularly limited as long as it is a signal peptide that functions in a coryneform bacterium, and may be a signal peptide derived from a coryneform bacterium or a signal peptide derived from a different species.
  • the “signal peptide that functions in coryneform bacteria” refers to a peptide that can be secreted by coryneform bacteria when linked to the N-terminus of the target protein.
  • Whether or not a signal peptide functions in coryneform bacteria can be confirmed by, for example, expressing the target protein by fusing it with the signal peptide and confirming whether the protein is secreted.
  • the signal peptide used in the present invention is preferably a Tat system-dependent signal peptide.
  • Tat system-dependent signal peptide refers to a signal peptide recognized by the Tat system.
  • the “Tat system-dependent signal peptide” may be a peptide in which the protein is secreted by the Tat system secretion apparatus when linked to the N-terminus of the target protein.
  • Tat-dependent signal peptides examples include E. coli TorA protein (trimethylamine-N-oxide reductase) signal peptide, E. coli SufI protein (ftsI suppressor) signal peptide, Bacillus subtilis PhoD protein (phosphodiesterase) Signal peptide of LipA protein (lipoic acid synthase) of Bacillus subtilis, signal peptide of IMD protein (isomaltodextranase) of Arthrobacter globiformis.
  • the amino acid sequences of these signal peptides are as follows.
  • TorA signal peptide MNNNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 19)
  • SufI signal peptide MSLSRRQFIQASGIALCAGAVPLKASA (SEQ ID NO: 20)
  • the Tat-dependent signal peptide has a twin arginine motif.
  • twin arginine motif include S / T-R-X-F-L-K (SEQ ID NO: 24) and R-R-X-#-# (#: hydrophobic residue) (SEQ ID NO: 25).
  • the Tat-dependent signal peptide may be a variant of the above signal peptide as long as it has a twin arginine motif and has a function as a Tat-dependent signal peptide.
  • the above description regarding the wild-type ribosomal protein S1 and the variant of the gene encoding the same can be applied mutatis mutandis to the signal peptide and the gene encoding the same.
  • the variant of the signal peptide is an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted or added in the amino acid sequence of the signal peptide. It may be a peptide having.
  • the “one or several” in the signal peptide variant is specifically preferably 1 to 7, more preferably 1 to 5, further preferably 1 to 3, particularly preferably 1 to 2. Means individual.
  • “TorA signal peptide”, “SufI signal peptide”, “PhoD signal peptide”, “LipA signal peptide”, and “IMD signal peptide” have SEQ ID NOs: 19, 20, 21, 22 respectively. In addition to the peptides described in, and 23, variants thereof are included.
  • “Function as a Tat-dependent signal peptide” refers to recognition by the Tat system. Specifically, the protein is secreted by the Tat secretion apparatus when linked to the N-terminus of the target protein. It may be a function.
  • Whether a certain peptide has a function as a Tat-dependent signal peptide can be confirmed, for example, by confirming that the amount of secreted protein produced by adding the peptide to the N-terminus is increased by enhancement of the Tat-based secretion apparatus, This can be confirmed by confirming that the amount of secreted protein produced by adding a peptide to the N-terminus decreases due to a defect in the Tat secretion apparatus.
  • the signal sequence is generally cleaved by a signal peptidase when the translation product is secreted outside the cell.
  • the gene encoding the signal peptide can be used in its natural form, but may be modified so as to have an optimal codon according to the codon usage frequency of the host to be used.
  • heterologous proteins secreted and produced by the method of the present invention include bioactive proteins, receptor proteins, antigen proteins used as vaccines, and enzymes.
  • transglutaminase examples include transglutaminase, protein glutaminase, isomalt dextranase, protease, endopeptidase, exopeptidase, aminopeptidase, carboxypeptidase, collagenase, and chitinase.
  • transglutaminase examples include actinomycetes such as Streptoverticillium mobaraense IFO 13819 (WO01 / 23591), Streptoverticillium cinnamoneum IFO 12852, Streptoverticillium griseocarneum IFO 12776, Streptomyces lydicus (WO9606931), etc. Examples include transglutaminase.
  • Examples of the protein glutaminase include Chryseobacterium proteolyticum protein glutaminase (WO2005 / 103278). Examples of isomalt dextranase include Arthrobacter globiformis isomaltdextranase (WO2005 / 103278).
  • physiologically active proteins include growth factors (growth factors), hormones, cytokines, and antibody-related molecules.
  • growth factors specifically, for example, epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), transforming growth factor (Transforming growth factor; TGF), Nerve ⁇ growtherfactor (NGF), Brain-derived ; neurotrophic factor (BDNF), Vascular endothelial growth factor (Vesicular endothelial growth factor; VEGF), Granulocyte Colony-stimulating factor (G-CSF), granulocyte-macrophage-colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (Erythropoietin; EPO), Thrombopoietin (TPO), Acidic fibroblast growth factor (aFGF or FGF1) Basic fibroblast growth factor (basic fibroblast growth factor; bFGF or FGF2), keratinocyte growth factor (keratinocyto growth factor; KGF-1 or FGF7, KGF-2 or FGF10)
  • hormones include insulin, glucagon, somatostatin, human growth hormone (hGH), parathyroid hormone (PTH), and calcitonin.
  • cytokines include interleukin, interferon, and tumor necrosis factor (TNF).
  • growth factors growth factors
  • hormones and cytokines
  • the bioactive protein may belong to any one group selected from growth factors (growth factors), hormones, and cytokines, and belongs to a plurality of groups selected from them. Also good.
  • physiologically active protein may be the whole protein or a part thereof.
  • the part which has physiological activity is mentioned, for example.
  • Specific examples of the physiologically active moiety include a physiologically active peptide Teriparatide comprising the N-terminal 34 amino acid residues of a mature parathyroid hormone (PTH).
  • the antibody-related molecule refers to a protein containing a molecular species consisting of a single domain selected from domains constituting a complete antibody or a combination of two or more domains. Domains constituting a complete antibody include VH, CH1, CH2, and CH3, which are heavy chain domains, and VL and CL, which are light chain domains.
  • the antibody-related molecule may be a monomeric protein or a multimeric protein as long as it contains the above-described molecular species. When the antibody-related molecule is a multimeric protein, it may be a homomultimer composed of a single type of subunit or a heteromultimer composed of two or more types of subunits. Also good.
  • antibody-related molecules include, for example, complete antibodies, Fab, F (ab ′), F (ab ′) 2 , Fc, dimer consisting of heavy chain (H chain) and light chain (L chain) , Fc fusion protein, heavy chain (H chain), light chain (L chain), single chain Fv (scFv), sc (Fv) 2 , disulfide bond Fv (sdFv), Diabody.
  • the receptor protein is not particularly limited, and may be, for example, a receptor protein for a physiologically active protein or other physiologically active substance. Examples of other physiologically active substances include neurotransmitters such as dopamine.
  • the receptor protein may also be an orphan receptor for which the corresponding ligand is not known.
  • the antigenic protein used as a vaccine is not particularly limited as long as it can elicit an immune response, and may be appropriately selected according to the target of the assumed immune response.
  • genes encoding these proteins can be modified depending on the host used and to obtain the desired activity.
  • the genes encoding these proteins may be modified so that these proteins include additions, deletions, substitutions, etc. of one or several amino acids.
  • the above description concerning the wild-type ribosomal protein S1 and the variant of the gene encoding it can be applied mutatis mutandis to the heterologous protein secreted and produced by the method of the present invention and the gene encoding the same.
  • the genes encoding these proteins may be those obtained by replacing an arbitrary codon with an equivalent codon.
  • the genes encoding these proteins may be converted into optimal codons depending on the codon usage frequency of the host, if necessary.
  • the N-terminal region of the heterologous protein finally obtained by the method of the present invention may or may not be the same as the natural protein.
  • the N-terminal region of the finally obtained heterologous protein may have one or several extra amino acids added or deleted compared to the natural protein.
  • the “one or several” may differ depending on the total length, structure, etc. of the target heterologous protein, but specifically preferably 1-20, more preferably 1-10, and even more preferably 1-5. Means individual.
  • the heterologous protein that is secreted and produced may be a protein added with a pro-structure part (pro-protein).
  • the finally obtained heterologous protein may or may not be a proprotein. That is, the proprotein may be cleaved from the prostructure to become a mature protein.
  • the cleavage can be performed by, for example, a protease.
  • a protease from the viewpoint of the activity of the finally obtained protein, it is generally preferable that the proprotein is cleaved at almost the same position as the natural protein, and cleaved at the same position as the natural protein. More preferably, a mature protein identical to the natural one is obtained.
  • proteases that cleave proproteins at positions that produce proteins identical to naturally occurring mature proteins are most preferred.
  • the N-terminal region of the finally obtained heterologous protein may not be the same as the natural protein.
  • Proteases that can be used in the present invention include those obtained from a culture solution of microorganisms, such as a culture solution of actinomycetes, in addition to those commercially available such as Dispase (manufactured by Boehringer Mannheim). Such a protease can be used in an unpurified state, or may be used after being purified to an appropriate purity as required.
  • the method for introducing the gene construct used in the present invention into coryneform bacteria is not particularly limited.
  • the gene construct used in the present invention may be present on a vector that autonomously proliferates outside the chromosome, such as a plasmid, or may be integrated on the chromosome.
  • the introduction of the genetic construct used in the present invention, the introduction of the mutant ribosomal protein S1 gene, and other modifications can be performed in any order.
  • the gene construct used in the present invention can be introduced into a host using, for example, a vector containing the gene construct.
  • the vector is not particularly limited as long as it can autonomously replicate in coryneform bacteria, and may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid.
  • a plasmid derived from coryneform bacteria is preferable.
  • Specific examples of vectors capable of autonomous replication in coryneform bacteria include pHM1519 (Agric, Biol.
  • plasmids having improved drug resistance genes plasmid pCRY30 described in JP-A-3-210184; plasmids pCRY21 and pCRY2KE described in JP-A-2-72876 and US Pat. No. 5,185,262.
  • artificial transposons can be used.
  • a transposon When a transposon is used, a heterologous protein gene is introduced onto the chromosome by homologous recombination or its own ability to transfer.
  • homologous recombination for example, linear DNA, a plasmid containing a temperature-sensitive replication origin, a plasmid capable of conjugation transfer, or a suspension vector that does not have a replication origin that functions in the host is used. A method is mentioned.
  • any of a promoter sequence and a nucleic acid sequence encoding a signal peptide contained in the gene construct may be present on the host chromosome.
  • a nucleic acid sequence encoding a signal peptide connected to the downstream of the promoter sequence that is originally present on the host chromosome is used as it is, and downstream of the nucleic acid sequence encoding the signal peptide.
  • the gene construct for secretory expression of each protein may be retained in the bacterium of the present invention so that the secretory expression of the target heterologous protein can be achieved.
  • the gene construct for secretory expression of each protein may be all retained on a single expression vector, or all may be retained on a chromosome.
  • the gene construct for secretory expression of each protein may be separately held on a plurality of expression vectors, or may be separately held on a single or a plurality of expression vectors and on a chromosome. “When two or more types of proteins are expressed” means, for example, when two or more types of heterologous proteins are secreted and heteromultimeric proteins are secreted and produced.
  • the method of introducing the gene construct used in the present invention into the coryneform bacterium is not particularly limited, and a commonly used method such as a protoplast method (Gene, 39, 281-286 (1985)), an electroporation method (Bio / Technology, 7, 1067-1070) (1989)).
  • the bacterium of the present invention can be cultured according to commonly used methods and conditions.
  • the bacterium of the present invention can be cultured in a normal medium containing a carbon source, a nitrogen source, and inorganic ions.
  • organic micronutrients such as vitamins and amino acids can be added as necessary.
  • carbon source carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and the like can be used.
  • nitrogen source ammonia gas, aqueous ammonia, ammonium salt, and others can be used.
  • inorganic ions calcium ions, magnesium ions, phosphate ions, potassium ions, iron ions and the like are appropriately used as necessary. Cultivation is carried out under aerobic conditions in an appropriate range of pH 5.0 to 8.5 and 15 ° C. to 37 ° C., and cultured for about 1 to 7 days.
  • culture conditions for L-amino acid production of coryneform bacteria and other conditions described in the method for producing proteins using Sec and Tat signal peptides can be used (see WO01 / 23591 and WO2005 / 103278).
  • the culture can be performed by adding a promoter inducer to the medium. By culturing the bacterium of the present invention under such conditions, the target protein is produced in large quantities in the microbial cells and efficiently secreted outside the microbial cells.
  • the produced heterologous protein is secreted outside the cell body, for example, a protein that is generally lethal when accumulated in a large amount in a cell body of a microorganism such as transglutaminase has a lethal effect. Can be continuously produced without receiving
  • the protein secreted into the medium by the method of the present invention can be separated and purified from the cultured medium according to a method well known to those skilled in the art. For example, after removing cells by centrifugation, etc., salting out, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium / high pressure liquid chromatography, reverse phase chromatography And can be separated and purified by a known appropriate method such as hydrophobic chromatography, or a combination thereof. In some cases, the culture or culture supernatant may be used as it is.
  • the protein secreted into the cell surface by the method of the present invention is the same as that secreted into the medium after being solubilized by methods well known to those skilled in the art, for example, by increasing the salt concentration or using a surfactant. And can be separated and purified.
  • the protein secreted into the surface of the bacterial cell may be used as, for example, an immobilized enzyme without solubilizing the protein.
  • the target heterologous protein is secreted and produced by SDS-PAGE using the fraction containing the culture supernatant and / or cell surface as a sample and confirming the molecular weight of the separated protein band. Can do.
  • the fraction containing the culture supernatant and / or cell surface layer can be used as a sample for confirmation by Western blotting using an antibody (Molecular cloning (Cold spring Harbor Laboratory Press,) Cold spring Harbor (USA), 2001)). It can also be confirmed by determining the N-terminal amino acid sequence of the target protein using a protein sequencer. It can also be confirmed by determining the mass of the target protein using a mass spectrometer.
  • the target heterologous protein has an enzyme or some measurable physiological activity
  • the fraction containing the culture supernatant and / or cell surface layer is used as a sample, and the enzyme activity or physiological activity of the target heterologous protein is measured. By measuring the activity, it can be confirmed that the target heterologous protein is secreted and produced.
  • Coryneform bacterium having a specific mutation also relates to a coryneform bacterium modified to have a mutation in the ribosomal protein S1 gene, wherein the mutation is the amino acid at position 173 of the wild-type ribosomal protein S1.
  • a coryneform bacterium is provided that is a mutation that replaces a residue with another amino acid residue.
  • the coryneform bacterium may or may not have the ability to produce a heterologous protein. That is, the coryneform bacterium may or may not have a gene construct for secretory expression of a heterologous protein.
  • coryneform bacterium With respect to the coryneform bacterium, the description about the “coryneform bacterium used in the method of the present invention” described above can be applied mutatis mutandis, except that it does not have to have a gene construct for secretory expression of a heterologous protein.
  • the coryneform bacterium can be suitably used for secretory production of a heterologous protein, for example, by retaining a gene construct for secretory expression of the heterologous protein.
  • Example 1 Construction of RpsA (E173G) mutant derived from C. glutamicum YDK010 (1) Acquisition of natural mutant having mutation in rpsA gene Using protransglutaminase expression plasmid pPKT-PTG1 described in WO2005 / 103278 C. glutamicum YDK010 strain described in WO2004 / 029254 was transformed.
  • PPKT-PTG1 is a vector for secretory expression of protransglutaminase (transglutaminase with prostructure), a promoter derived from the PS2 gene of C. glutamicum ATCC13869 strain, and an Escherichia coli operably linked downstream of the promoter.
  • coli is a plasmid having a TorA signal peptide-derived DNA and a protransglutaminase gene derived from S. mobaraense linked to be expressed as a fusion protein with the signal peptide (WO2005 / 103278),
  • the C. glutamicum YDK010 strain is a strain lacking the cell surface protein PS2 of C. glutamicum AJ12036 (FERM BP-734) (WO2004 / 029254).
  • MM liquid medium containing 25 mg / l kanamycin (glucose 60 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 450 ⁇ g, biotin 450 ⁇ g, DL-methionine 0.15 g, calcium carbonate 50 g, adjusted to pH 7.0 with 1 L with water) Cultured for 48 hours.
  • THM1 strain After culture, a natural mutant strain in which a mutation was introduced into the rpsA gene was selected and named THM1 strain.
  • the nucleotide sequence of the mutant rpsA gene possessed by the THM1 strain and the amino acid sequence of the mutant RpsA protein encoded by the same gene are shown in ⁇ SEQ ID NO: 01> and ⁇ SEQ ID NO: 02>.
  • a at position 518 in the base sequence ⁇ SEQ ID NO: 03> of the rpsA gene of the YDK010 strain is mutated to G.
  • the amplified DNA fragment of about 2.4 kbp was recovered by agarose gel electrophoresis using WizardR SV Gel and PCR Clean-Up System (manufactured by Promega) and inserted into the Sma5I site of pBS5T described in WO2006 / 057450 Then, it was introduced into a competent cell of E. coli JM109 (manufactured by Takara Bio Inc.).
  • a strain holding a plasmid in which a DNA fragment containing the mutant rpsA gene was cloned was obtained, and the plasmid was recovered from this to obtain a plasmid pBS5T-RpsA (E173G) in which the mutant rpsA gene was cloned.
  • E173G plasmid pBS5T-RpsA
  • the nucleotide sequence was determined using BigDyeR Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3130 Genetic Analyzer (Applied Biosystems).
  • Example 2 Secretion expression of protransglutaminase using TorA signal sequence by RpsA (E173G) mutant strain YDK010 strain and Example 1 (3) using protransglutaminase expression plasmid pPKT-PTG1 described in WO2005 / 103278
  • the YDK010 :: RpsA (E173G) strain obtained in (1) was transformed to obtain YDK010 / pPKT-PTG1 and YDK010 :: RpsA (E173G) / pPKT-PTG1.
  • Each transformant obtained was treated with MM liquid medium (glucose 60 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin. 0.03 g of Japanese product, 0.03 g of manganese sulfate pentahydrate, 450 ⁇ g of thiamine hydrochloride, 450 ⁇ g of biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with water and adjusted to pH 7.0) at 30 °C And cultured for 48 hours.
  • MM liquid medium glucose 60 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin.
  • the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE and then stained with CBB R250 (Bio-Rad).
  • CBB R250 Bio-Rad
  • the amount of protransglutaminase secretion in the YDK010 :: RpsA (E173G) strain was significantly improved as compared with the YDK010 strain (FIG. 1). From this, it became clear that the RpsA (E173G) mutation is an effective mutation that leads to an increase in secretion amount in protransglutaminase secretion using the TorA signal sequence derived from E. coli.
  • FIG. 2 shows the alignment of the amino acid sequences of RpsA protein of C. glutamicum THM1, C. glutamicum YDK010, C. glutamicum ATCC13869, C. glutamicum ATCC13032, C. efficiens YS-314 and C. stationis ATCC6872
  • the amino acid residue at position 173 is shown in a box.
  • the rpsA gene is considered to be an essential gene in E. licoli and the like, and it has been difficult to predict that the mutation of amino acid residues having high conservation of such a gene will lead to an increase in the secretion amount of heterologous proteins.
  • Example 3 Secretory expression of pro-structured protein glutaminase using TorA signal sequence by RpsA (E173G) mutant strain YDK010 strain and Example using the pro-structured protein glutaminase expression plasmid pPKT-PPG described in WO2005 / 103278
  • Each of the YDK010 :: RpsA (E173G) strains obtained in 1 (3) was transformed to obtain YDK010 / pPKT-PPG and YDK010 :: RpsA (E173G) / pPKT-PPG.
  • pPKT-PPG is a vector for secretory expression of protein glutaminase with a pro-structure, and is a promoter derived from the PS2 gene of C. glutamicum ATCC13869 strain, and E. coli ligated to express downstream of the promoter.
  • a plasmid having a pro-structured protein glutaminase gene derived from Chryseobacterium proteolyticum linked to be expressed as a fusion protein with the DNA encoding the TorA signal peptide derived therefrom (WO2005 / 103278).
  • Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin. 0.03 g of Japanese product, 0.03 g of manganese sulfate pentahydrate, 450 ⁇ g of thiamine hydrochloride, 450 ⁇ g of biotin, 0.15 g of DL-methionine, 50 g of calcium carbonate, adjusted to 1 L with water and adjusted to pH 7.0) at 30 °C And cultured for 72 hours.
  • MM liquid medium glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin.
  • YDK010 :: RpsA (E173G) strain significantly improved the amount of secreted protein glutaminase secreted compared to the YDK010 strain (FIG. 3). From this, it was revealed that the RpsA (E173G) mutation is an effective mutation that leads to an increase in the secretion amount even in the secretion of pro-structured protein glutaminase using the TorA signal sequence derived from E. coli.
  • Example 4 Secretion expression of isomaltdextranase using IMD signal sequence by RpsA (E173G) mutant strain YDK010 strain and examples using isomaltdextranase expression plasmid pPKI-IMD described in WO2005 / 103278
  • RpsA E173G mutant strain YDK010 strain and examples using isomaltdextranase expression plasmid pPKI-IMD described in WO2005 / 103278
  • Each of the YDK010 :: RpsA (E173G) strains obtained in 1 (3) was transformed to obtain YDK010 / pPKI-IMD and YDK010 :: RpsA (E173G) / pPKI-IMD.
  • PPKI-IMD is a vector for secretory expression of isomaltdextranase, a promoter derived from the PS2 gene of C. glutamicum ATCC13869 strain, an IMD gene derived from Arthrobacter globiformis that is operably linked downstream of the promoter ( (Including the coding region of the IMD signal sequence) (WO2005 / 103278).
  • Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin.
  • the YDK010 :: RpsA (E173G) strain had a significantly improved isomaltdextranase secretion amount compared to the YDK010 strain (FIG. 4). From this, it was revealed that the RpsA (E173G) mutation is an effective mutation that leads to an improvement in the amount of isomaltdextranase secretion using an IMD signal sequence derived from A. globiformis.
  • Example 5 Combined effect of RpsA (E173G) mutation and Tat secretion apparatus amplification YDK010 / pPKT-PTG1 constructed in Example 2 using pVtatABC which is an amplification plasmid of the Tat secretion apparatus described in WO2005 / 103278 YDK010 :: RpsA (E173G) / pPKT-PTG1, YDK010 / pPKT-PPG and YDK010 :: RpsA (E173G) / pPKT-PPG constructed in Example 3, YDK010 / pPKI-IMD and YDK010 constructed in Example 4: : Transform each of RpsA (E173G) / pPKI-IMD, Tat secretion apparatus amplification strain YDK010 / pPKT-PTG1 / pVtatABC, YDK010 :
  • Each transformant obtained was treated with MM liquid medium (glucose 120 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, phosphate, containing 25 mg / l kanamycin and 5 mg / l chloramphenicol. Potassium dihydrogen 1.5 g, iron sulfate heptahydrate 0.03 g, manganese sulfate pentahydrate 0.03 g, thiamine hydrochloride 450 ⁇ g, biotin 450 ⁇ g, DL-methionine 0.15 g, calcium carbonate 50 g, 1 L with water Each of the cells was cultured at 30 ° C. for 72 hours.
  • the culture supernatant obtained by centrifuging each culture solution was subjected to reducing SDS-PAGE and then stained with CBB R250 (Bio-Rad). After staining, the band intensity of the target protein was quantified using image analysis software “Multi Gauge Ver3.0” (FUJIFILM). The band intensity of each sample was calculated as a relative value to the band intensity when the target protein was expressed in the YDK010 strain introduced with pVC7. As a result, in each of the expression strains, the secretion amount of each target protein was significantly improved in the strain introduced with pVtatABC as compared with the control strain introduced with pVC7.
  • Example 6 Effect of RpsA (E173G) Mutation on Protein Secretion by Sec System Using the protransglutaminase expression plasmid pPKSPTG1 described in WO01 / 23591, C. glutamicum YDK010 strain and YDK010 :: RpsA (E173G) strain were Transformation was performed to obtain YDK010 / pPKSPTG1 and YDK010 :: RpsA (E173G) / pPKSPTG1.
  • PPKSPTG1 is a vector for secretory expression of protransglutaminase (transglutaminase with prostructure), a promoter derived from the PS2 gene of C.
  • glutamicum ATCC13869 strain and C. ammoniagenes operably linked downstream of the promoter.
  • This is a plasmid having a DNA encoding a signal peptide derived from SlpA of the ATCC6872 strain and a protransglutaminase gene derived from S. mobaraense ligated to be expressed as a fusion protein with the signal peptide (WO01 / 23591).
  • Each transformant obtained was treated with MM liquid medium (glucose 60 g, magnesium sulfate heptahydrate 3 g, ammonium sulfate 30 g, potassium dihydrogen phosphate 1.5 g, iron sulfate heptahydrate containing 25 mg / l kanamycin.
  • the amount of protransglutaminase secretion was decreased as compared to the parent strain YDK010 (FIG. 8). From this, it was found that the RpsA (E173G) mutation is a mutation having a negative effect of decreasing the secretion amount in protein secretion by the Sec system. From this, it was confirmed that the RpsA (E173G) mutation is an effective mutation that specifically improves the secretion amount in protein secretion by the Tat system.
  • heterologous proteins can be efficiently secreted and produced.
  • SEQ ID NO: 1 Nucleotide sequence of mutant rpsA gene of C. glutamicum THM1 strain
  • SEQ ID NO: 2 Amino acid sequence of mutant RpsA protein of C. glutamicum THM1 strain
  • SEQ ID NO: 3 Nucleotide sequence of rpsA gene of C. glutamicum YDK010 strain
  • No. 4 Amino acid sequence of RpsA protein of C. glutamicum YDK010 strain
  • SEQ ID NO: 5 6: Primer SEQ ID NO: 7: Nucleotide sequence of rpsA gene of C. glutamicum ATCC13032
  • SEQ ID NO: 8 Amino acid of RpsA protein of C.
  • glutamicum ATCC13032 strain SEQ ID NO: 9 nucleotide sequence of rpsA gene of C. efficiens YS-314 strain SEQ ID NO: 10: amino acid sequence of RpsA protein of C. efficiens YS-314 strain SEQ ID NO: 11: nucleotide sequence of rpsA gene of C. stationis ATCC6872 strain SEQ ID NO: 12: Amino acid sequence of RpsA protein of C. stationis ATCC6872 SEQ ID NO: 13: Base sequence of tatA gene of C. glutamicum ATCC13032 SEQ ID NO: 14: TatA protein of C.
  • glutamicum ATCC13032 Mino acid sequence SEQ ID NO: 15: nucleotide sequence of tatB gene of C. glutamicum ATCC13032 sequence
  • SEQ ID NO: 16 amino acid sequence of TatB protein of C.
  • SEQ ID NO: 17 nucleotide sequence sequence number of tatC gene of C.
  • glutamicum ATCC13032 strain 18 amino acid sequence of TatC protein of C.

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Abstract

La présente invention concerne une nouvelle technique pour l'amélioration de la sécrétion/production d'une protéine exogène en utilisant une bactérie coryneforme et un procédé de sécrétion/production de la protéine exogène. La présente invention décrit une protéine exogène pouvant être sécrétée/produite par la culture d'une bactérie coryneforme pouvant sécréter/produire ladite protéine exogène et est modifiée de manière à avoir une mutation dans un gène de la protéine ribosomale s21.
PCT/JP2013/050681 2012-02-08 2013-01-16 Procédé de sécrétion/production de protéine WO2013118544A1 (fr)

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WO2015060391A1 (fr) 2013-10-23 2015-04-30 味の素株式会社 Procédé de production d'une substance cible
WO2015115612A1 (fr) 2014-01-31 2015-08-06 味の素株式会社 Glutamate-cystéine ligase mutante et procédé de fabrication de γ-glutamyl-valyl-glycine
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WO2018179834A1 (fr) 2017-03-28 2018-10-04 Ajinomoto Co., Inc. Procédé de production d'arn
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WO2016171224A1 (fr) * 2015-04-24 2016-10-27 味の素株式会社 Procédé de production d'une protéine par sécrétion
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JPWO2018074579A1 (ja) * 2016-10-21 2019-08-08 味の素株式会社 タンパク質の分泌生産法
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JP7159867B2 (ja) 2016-10-21 2022-10-25 味の素株式会社 タンパク質の分泌生産法
WO2018179834A1 (fr) 2017-03-28 2018-10-04 Ajinomoto Co., Inc. Procédé de production d'arn
WO2019163827A1 (fr) 2018-02-20 2019-08-29 味の素株式会社 Procédé d'induction de silençage d'arn
EP3530749A1 (fr) 2018-02-27 2019-08-28 Ajinomoto Co., Inc. Synthétase de glutathione mutant et procédé de production de gamma-glu-val-gly
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