WO2023032950A1 - 向上したタンパク質生産性を有する微生物及びその利用 - Google Patents

向上したタンパク質生産性を有する微生物及びその利用 Download PDF

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WO2023032950A1
WO2023032950A1 PCT/JP2022/032509 JP2022032509W WO2023032950A1 WO 2023032950 A1 WO2023032950 A1 WO 2023032950A1 JP 2022032509 W JP2022032509 W JP 2022032509W WO 2023032950 A1 WO2023032950 A1 WO 2023032950A1
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protein
microorganism
gene
expression
strain
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寛敬 松原
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Amano Enzyme Inc
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Amano Enzyme Inc
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Priority to CN202280058520.0A priority Critical patent/CN117881785A/zh
Priority to US18/685,949 priority patent/US20250179422A1/en
Priority to JP2023545583A priority patent/JPWO2023032950A1/ja
Priority to EP22864532.1A priority patent/EP4397763A4/en
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
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    • 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
    • C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
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    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
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    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01044—Protein-glutamine glutaminase (3.5.1.44)

Definitions

  • the present invention relates to microorganisms with improved protein productivity.
  • the present invention further relates to a method for producing protein using the microorganisms described above.
  • Microorganisms such as the Bacillus genus are capable of mass secretion and production of enzymes, capable of ultra-high-density culture, rapid growth, many bacteria belonging to BioSafety Level 1, Gram-positive bacteria, and relatively small genome size (4M bp degree), it has excellent performance in the production of substances utilized in food and pharmaceuticals.
  • Microorganisms such as Bacillus are used for the production of enzymes such as ⁇ -amylase, ⁇ -amylase, neutral protease, alkaline protease and cellulase.
  • enzymes such as ⁇ -amylase, ⁇ -amylase, neutral protease, alkaline protease and cellulase.
  • the acquisition of mutant strains by UV irradiation and the acquisition of transformants by genetic recombination techniques are being studied.
  • An ATP Binding Cassette (ABC) transporter is a transmembrane protein that has a region responsible for ATP hydrolase activity that exhibits a highly common amino acid sequence. ABC transporters can actively transport compounds using the energy obtained by self-hydrolysis of ATP.
  • Patent Document 1 describes that ABC transporters are involved in extracellular excretion of cyclic peptides produced by filamentous fungi of the genus Curvularia (paragraph 0142).
  • Patent Document 2 describes a method for measuring the substrate amount of ABC protein, and describes that porphyrins are preferable as the substrate.
  • Patent Document 3 describes an efficient method for producing taurine, and describes that deletion of the tauABC protein gene reduces the activity of the taurine uptake system.
  • ABC transporters are genes involved in the secretion of small molecules and peptides, but were not known to improve protein secretion.
  • the problem to be solved by the present invention is to provide a microorganism with improved protein secretion.
  • a further problem to be solved by the present invention is to provide a method for producing a protein using the above microorganism.
  • the present inventors found that the enzyme productivity was improved by disrupting the ABCtransporter ATP-binding protein gene in a high enzyme-producing strain of Bacillus amyloliquefaciens.
  • the present invention has been completed based on the above findings.
  • ⁇ 1> A microorganism in which the expression of at least one ABC transporter ATP-binding protein is lost or reduced.
  • ⁇ 2> The microorganism according to ⁇ 1>, wherein the ABC transporter ATP-binding protein is a protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:2.
  • ⁇ 3> The microorganism according to ⁇ 1> or ⁇ 2>, which is a microorganism of the genus Bacillus, a microorganism of the genus Cytobacillus, or a microorganism of the genus Paenibacillus.
  • ⁇ 4> The microorganism according to any one of ⁇ 1> to ⁇ 3>, which is Bacillus amyloliquefaciens.
  • ⁇ 5> The microorganism according to any one of ⁇ 1> to ⁇ 4>, wherein expression of at least one ABC transporter ATP-binding protein is lost or reduced, resulting in increased expression of the target protein.
  • ⁇ 6> The microorganism of ⁇ 5>, wherein the target protein is a protein encoded by an endogenous gene or a protein encoded by a foreign gene.
  • ⁇ 7> The microorganism according to ⁇ 5> or ⁇ 6>, wherein the target protein is an enzyme.
  • ⁇ 8> The microorganism according to any one of ⁇ 1> to ⁇ 7>, wherein the gene encoding the ABC transporter ATP-binding protein is disrupted by homologous recombination.
  • a method for producing a protein comprising the steps of culturing the microorganism according to any one of ⁇ 1> to ⁇ 8> to produce the protein, and recovering the obtained protein.
  • protein secretion can be improved.
  • FIG. 1 shows the results of confirming the enzyme productivity of gene-disrupted strains.
  • FIG. 2 shows the results of confirming the disappearance of the effect of enhancing enzyme productivity by gene complementation.
  • FIG. 3 shows the results of confirming the enzyme productivity enhancement effect of a heterologous enzyme by a gene-disrupted strain.
  • the microorganism of the present invention has lost or reduced expression of at least one ABC transporter ATP-binding protein.
  • Microorganisms preferably include Bacillus, Paenibacillus, and Cytobacillus, and more preferably Bacillus.
  • Bacillus genus microorganisms include Bacillus amyloliquefaciens, Bacillus siamensis, Bacillus subtilis, Bacillus velezensis, Bacillus nakamurai, and Bacillus aquiflavi. More preferred are Bacillus amyloliquefaciens, Bacillus siamensis, Bacillus subtilis, and Bacillus velezensis. Especially preferred is Bacillus amyloliquefaciens. As a Paenibacillus genus microorganism, Paenibacillus sp. etc. Examples of Cytobacillus microorganisms include Cytobacillus firmus.
  • the ABC transporter ATP-binding protein is preferably a protein having an amino acid sequence with 70% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, more preferably 75% or more sequence identity, 80% ⁇ 85% sequence identity, ⁇ 90% sequence identity, ⁇ 93% sequence identity, ⁇ 95% sequence identity, ⁇ 97% sequence identity, or ⁇ 99% sequence identity is a protein having an amino acid sequence with the sequence identity of
  • the ABC transporter ATP-binding protein may be any protein that has a structure equivalent to that of the present protein regardless of its function, and includes annotated proteins other than the ABC transporter ATP-binding protein.
  • the ABC transporter ATP binding protein is preferably a protein with ATP binding ability.
  • a protein having an amino acid sequence with 70% or more sequence identity with the amino acid sequence of SEQ ID NO:2 is obtained by making conservative amino acid substitutions with respect to the amino acid sequence of SEQ ID NO:2.
  • conservative amino acid substitution refers to substitution of an amino acid residue with an amino acid residue having a side chain with similar properties.
  • Amino acid residues can have, depending on their side chain, a basic side chain (e.g. lysine, arginine, histidine), an acidic side chain (e.g. aspartic acid, glutamic acid), an uncharged polar side chain (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine).
  • cysteine nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine), aromatic side chains (e.g. tyrosine, phenylalanine, Tryptophan, histidine) are classified into several families. Conservative amino acid substitutions are preferably between amino acid residues within the same family.
  • the identity (%) of two amino acid sequences can be determined, for example, by the following procedure. First, the two sequences are aligned for optimal comparison (eg, gaps may be introduced in the first sequence to optimize alignment with the second sequence). When the molecule (amino acid residue) at a particular position in the first sequence is the same as the molecule at the corresponding position in the second sequence, the molecules at that position are said to be identical.
  • the expression of at least one ABC transporter ATP-binding protein is lost or reduced.
  • Loss or reduction in expression means that the activity per cell of the ABC transporter ATP-binding protein is lost or reduced compared to the unmodified strain, and includes cases where the activity is completely lost.
  • Unmodified strain means a control strain that has not been modified to lose or reduce expression of the ABC transporter ATP binding protein.
  • "Loss or reduction in protein expression” specifically refers to a decrease in the number of protein molecules per cell compared to an unmodified strain, and/or the function of the same protein per molecule. is declining. A reduction in the number of molecules of a protein per cell includes the complete absence of the same protein.
  • a case where the function per molecule of the protein is completely lost is included in the case where the function per molecule of the protein is reduced.
  • the activity of the protein is not particularly limited as long as it is reduced compared to the unmodified strain, for example, 50% or less, 20% or less, 10% or less, 5% or less, or 0 %.
  • a decrease in protein expression means that the expression level of the same protein per cell is reduced compared to unmodified strains such as wild strains and parent strains.
  • “Reduction in gene expression” specifically means that the transcription level (mRNA level) of the gene is decreased and/or the translation level (protein level) of the gene is decreased.
  • “Reduced gene expression” includes cases where the same gene is not expressed at all.
  • “the expression of a gene is decreased” is also referred to as "the expression of a gene is attenuated”. Expression of the gene may be reduced, for example, by 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.
  • a decrease in protein expression may be due to, for example, a decrease in transcription efficiency, a decrease in translation efficiency, or a combination thereof.
  • Reduction of protein expression can be achieved, for example, by altering expression control sequences such as gene promoters, Shine-Dalgarno (SD) sequences (also referred to as ribosome binding sites (RBS)), and spacer regions between RBS and start codons.
  • SD Shine-Dalgarno
  • RBS ribosome binding sites
  • spacer regions between RBS and start codons can be achieved by When modifying an expression regulatory sequence, preferably 1 or more bases, more preferably 2 or more bases, and particularly preferably 3 or more bases are modified. Also, part or all of the expression control sequence may be deleted.
  • Reduction of protein expression can also be achieved, for example, by manipulating factors involved in expression control.
  • Factors involved in expression control include small molecules (inducing substances, inhibitors, etc.), proteins (transcription factors, etc.), nucleic acids (siRNA, etc.), etc., involved in transcription and translation control.
  • a decrease in protein expression can also be achieved, for example, by introducing a mutation that reduces gene expression to the coding region of the gene.
  • expression of a gene can be reduced by replacing codons in the coding region of the gene with synonymous codons that are used less frequently in the host.
  • gene expression itself can be reduced by disruption of the gene as described later.
  • Modifications that reduce the activity of a protein can be achieved, for example, by disrupting the gene that encodes the same protein.
  • disrupted in a gene is meant that the same gene is altered so that it no longer produces a protein that functions normally.
  • “Not producing a protein that functions normally” includes cases where no protein is produced from the same gene, and cases where the same gene produces a protein with reduced or lost function (activity or property) per molecule.
  • Gene disruption can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome.
  • the present invention provides a method of producing a microorganism with improved protein productivity comprising disrupting at least one gene encoding an ABC transporter ATP binding protein in the microorganism.
  • the entire gene may be deleted, including sequences before and after the gene on the chromosome.
  • the region to be deleted may be any region such as the N-terminal region, internal region, C-terminal region, etc., as long as the protein activity can be reduced. Generally, the longer the region to be deleted, the more reliably the gene can be inactivated. Moreover, it is preferable that the sequences before and after the region to be deleted do not have the same reading frame.
  • Gene disruption includes, for example, introducing an amino acid substitution (missense mutation) into the coding region of the gene on the chromosome, introducing a stop codon (nonsense mutation), or frameshifting by adding or deleting 1 to 2 bases. It can also be achieved by introducing a mutation or the like.
  • Gene disruption can also be achieved, for example, by inserting other sequences into the coding region of the gene on the chromosome.
  • the insertion site may be any region of the gene, but the longer the sequence to be inserted, 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 thereof include marker genes such as antibiotic resistance genes and genes useful for the production of target substances.
  • Modifying a gene on a chromosome as described above involves, for example, creating a deletion-type gene modified so as not to produce a protein that functions normally, and transfecting a host with recombinant DNA containing the deletion-type gene. It can be achieved by replacing the wild-type gene on the chromosome with the deletion-type gene by inverting and causing homologous recombination between the deletion-type gene and the wild-type gene on the chromosome.
  • the recombinant DNA may contain a marker gene according to the host's traits such as auxotrophy.
  • Deletion-type genes include genes in which the entire region or a part of the gene is deleted, genes introduced with missense mutations, genes introduced with nonsense mutations, genes introduced with frameshift mutations, transposons, marker genes, etc. Examples include genes introduced with insertion sequences. Even if the protein encoded by the deleted gene is produced, it has a conformation different from that of the wild-type protein, and its function is reduced or lost. Methods for gene disruption by gene replacement using homologous recombination are known to those skilled in the art.
  • a decrease in protein activity can be confirmed by measuring the activity of the same protein. Decreased protein activity can also be confirmed by confirming decreased expression of the gene encoding the same protein. Decreased gene expression can be confirmed by confirming that the amount of transcription of the gene has decreased or by confirming that the amount of protein expressed from the gene has decreased.
  • Confirmation that the transcription level of the gene has decreased can be made by comparing the amount of mRNA transcribed from the same gene with that of the unmodified strain.
  • Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR and the like.
  • the amount of mRNA may be reduced, for example, by 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.
  • Confirmation that the amount of protein has decreased can be performed by Western blot using an antibody.
  • the amount of protein may be reduced, for example, by 50% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the unmodified strain.
  • Gene disruption can be confirmed by determining the base sequence, restriction enzyme map, full length, etc. of part or all of the gene, depending on the means used for disruption.
  • expression of the target protein is increased due to loss or reduction in expression of at least one ABC transporter ATP-binding protein.
  • the protein of interest may be either a protein encoded by an endogenous gene or a protein encoded by a foreign gene.
  • the target protein is not particularly limited, and includes a protein that is secreted by a secretory signal or a protein that becomes secretable by imparting a secretory signal.
  • Specific examples include enzymes, hormones, antibodies, growth factors, receptors and the like.
  • the target protein is preferably an enzyme. Enzymes include various industrial enzymes such as food, detergent, textile, feed, chemical, medical, and diagnostic.
  • Enzymes include amylase ( ⁇ -amylase, ⁇ -amylase, etc.), protein glutaminase, protease, xylanase, lipase, laccase, phenol oxidase, oxidase, cutinase, cellulase, hemicellulase, esterase, peroxidase, catalase, glucose oxidase, phytase, Examples include pectinase, glucosidase, isomerase, transferase, galactosidase, carbohydrase, phosphatase, kinase, and the like, but are not particularly limited.
  • the enzyme is an amylase ( ⁇ -amylase, ⁇ -amylase, etc.) or protein glutaminase.
  • the hormone is not particularly limited, but includes follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone-releasing factor, somatostatin, gonadotropin hormone, vasopressin, oxytocin, erythropoietin, insulin, and the like.
  • Antibodies include, but are not limited to, immunoglobulins.
  • Growth factors include, but are not limited to, platelet-derived growth factor, insulin-like growth factor, epidermal growth factor, nerve growth factor, fibroblast growth factor, transforming growth factor, interleukins (for example, IL-1 to IL- 13), interferons, colony-stimulating factors, and the like.
  • a recombinant vector obtained by combining a DNA fragment encoding the target protein and an appropriate vector is transformed by a general transformation method.
  • a recombinant microorganism can be obtained by incorporating it into a host microorganism.
  • Recombinant vectors can include promoters, signal sequences, terminators, and the like.
  • a recombinant microorganism can also be obtained by directly integrating a DNA fragment, which is a DNA fragment encoding a target protein and an appropriate homologous region with the genome of the host microorganism, into the genome of the microorganism.
  • a protein production method comprising the steps of culturing the microorganism of the present invention to produce a protein, and recovering the obtained protein.
  • the culture method and culture conditions in the process of culturing microorganisms to produce proteins are not particularly limited as long as the target protein is produced. That is, on the condition that the target protein is produced, a method and culture conditions suitable for culturing microorganisms can be appropriately set.
  • the culture method may be either liquid culture or solid culture, but liquid culture is preferred.
  • the medium is not particularly limited as long as it is a medium in which microorganisms can grow.
  • carbon sources such as glucose, maltose, sucrose, gentiobiose, soluble starch, glycerin, dextrin, molasses, organic acids, ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor , gelatin, casein hydrolyzate, bran, meat extract, etc.
  • inorganic salts such as potassium salt, magnesium salt, sodium salt, phosphate, manganese salt, iron salt, zinc salt, etc. can be done. Vitamins, amino acids, etc. may be added to the medium to promote growth.
  • the pH of the medium is, for example, about 3 to 10, preferably about 7 to 8, and the culture temperature is usually about 10 to 50°C, preferably about 20 to 37°C, for 1 to 7 days under aerobic conditions.
  • the culture method includes, for example, a shake culture method and an aerobic submerged culture method using a jar fermenter.
  • the target protein After culturing, the target protein is recovered from the culture solution or the bacterial cells.
  • the culture solution for example, after removing insoluble matter by filtering or centrifuging the culture supernatant, concentration with an ultrafiltration membrane, salting out such as ammonium sulfate precipitation, dialysis, ion exchange resin, etc.
  • the target protein can be obtained by separating and purifying by appropriately combining various chromatographic techniques.
  • the present enzyme when recovering from the cells, can be obtained by, for example, crushing the cells by pressure treatment, ultrasonic treatment, etc., followed by separation and purification in the same manner as described above.
  • the above series of steps may be performed after previously collecting the bacterial cells from the culture solution by filtration, centrifugation, or the like.
  • an immunological analysis method using protein-specific polyclonal or monoclonal antibodies can be used. Examples include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), and fluorescence-activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FFA fluorescence immunoassay
  • FACS fluorescence-activated cell sorting
  • the expression of the protein of interest can also be measured by measuring the activity of the protein of interest in a microorganism.
  • composition of pre-culture medium Polypeptone 3.0 w/v% Yeast extract 1.0w/v% D-glucose 0.1 w/v%
  • Hippuric acid solution (internal standard) Hippuric acid 6.5 mM Dissolve in 10 ml of 0.2 M monopotassium disodium phosphate (pH 6.5)
  • Example 1 Confirmation of enzyme productivity of gene-disrupted strain (strain used)
  • BA strain is a strain obtained as a strain having high ⁇ -amylase-producing ability by breeding Bacillus amyloliquefaciens (NBRC 15535) strain as a parent strain.
  • the BA strain and the Bacillus amyloliquefaciens (NBRC 15535) strain have almost the same genes, and have the same ABC transporter ATP-binding protein gene. It is inferred that the effect can be obtained.
  • the BA ⁇ 30 strain is the genome number of the BA strain. It is a strain in which 30 genes have been disrupted by homologous recombination. No. above. 30 genes are genes encoding ABC transporter ATP-binding proteins. No. The base sequences of the 30 genes are shown in SEQ ID NO: 1 of the Sequence Listing. The amino acid sequence encoded by the 30 genes is shown in SEQ ID NO: 2 of the Sequence Listing.
  • SEQ ID NO: 1 SEQ ID NO: 2
  • SEQ ID NO: 2 MHAIELKQLTKHYKETAAVDRLEFSIEKGEFFALLGENGAGKTTLIRMLCGLLSPDEGDASVLGHSILTDLDKIKPKMNMSPQETAIAPNLTVRENLEFIAGVYGIPKKEGKKRTDEMLELFQLKEKEREKTKTLSGGMQRRLSIAMGMITKPDIYFLDEPTLGLDVRSRYARELWKNLESLKGDMTIILTTHYLEEAEALADRICILENGTLKALGEAEDLKKQDGSATFEDAFLAIC
  • Example 2 Confirmation of Absence of Enzyme Productivity Enhancement Effect by Gene Complementation
  • the genome number of the BA ⁇ 30 strain was examined. Thirty genes were restored by homologous recombination. No. 4 by homologous recombination.
  • Confirmation of the 30-gene complementation strain BA ⁇ 30::30 strain was confirmed by whole-genome sequencing to be truly complemented without gaps. No. In the same manner as in Example 1, the effect of enhancing enzyme production was confirmed for the BA ⁇ 30::30 strain, which is a genomic complementation strain of 30 genes.
  • Example 3 Confirmation of Enzyme Productivity Enhancement Effect of Heterologous Enzyme by Gene Disruption Strain It was confirmed that the disruption of the 30 genes improved the secretory productivity of endogenous ⁇ -amylase. This No. The following experiment was performed to confirm whether the effect of the 30 gene disruption was limited to the secretory production of ⁇ -amylase.
  • BA ⁇ amyA strain in which the ⁇ -amylase gene on the genome of BA strain was disrupted was used.
  • a disrupted strain BA ⁇ amyA ⁇ 30 was constructed by homologous recombination of 30 genes.
  • pUBCM21 is an E. coli/Bacillus subtilis shuttle vector that combines a pUC-based plasmid commonly used in E. coli and a pUB-based plasmid commonly used in Bacillus subtilis.
  • a ⁇ -amylase/pUBCM21 vector was generated by integrating into .
  • the ⁇ -amylase/pUBCM21 vector was introduced into each of the BA ⁇ amyA strain and the BA ⁇ amyA ⁇ 30 strain by the protoplast-PEG method. After culturing in the same manner as in Example 1, the expression level of ⁇ -amylase was confirmed by SDS-PAGE to determine the amount of the enzyme secreted.
  • a protein glutaminase/pLAM1 vector was prepared by inserting the Chryseobacterium proteolyticum-derived protein glutaminase mature sequence into an expression vector (pLAM1) having an amyA-type expression cassette consisting of an endogenous ⁇ -amylase-derived promoter, signal sequence and terminator.
  • the protein glutaminase/pLAM1 vector was introduced into each of the BA ⁇ amyA strain and the BA ⁇ amyA ⁇ 30 strain by the protoplast-PEG method. After culturing in the same manner as in Example 1, the amount of the enzyme secreted and produced was confirmed from the protein glutaminase activity.
  • the secretion amount of useful proteins such as enzymes can be improved.
  • INDUSTRIAL APPLICABILITY The present invention is useful in producing useful proteins such as enzymes.

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