Method for improving filtration speed

Abstract

To provide a method for improving the filtration speed in a microfiltration process for producing a useful substance using a microorganism.SOLUTION: This method is for improving the filtration speed in a microfiltration process of the fermentation production of a useful substance using a microorganism, wherein a modified microorganism in which the function of the flagellar gene of the microorganism is suppressed is used as the microorganism.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for improving the filtration rate of microfiltration in the fermentation production of useful substances. [Background technology]

[0002] In the production of useful substances using microorganisms, a process is required in which the fermentation liquid is filtered to remove insoluble matter such as fine particles, cultured cells, and spores in the fermentation liquid in order to recover the desired fermentation products, such as enzymes. However, during the filtration operation, the separation membrane becomes clogged with particles close in size to the pores of the separation membrane, and further, insoluble substances accumulate on the membrane surface, resulting in a decrease in filtration efficiency.

[0003] Conventionally, such physical clogging of separation membranes and accumulation of insoluble substances on the membrane surface have been avoided by performing a procedure such as sweeping the membrane surface while filtering. However, in the case of a fermentation liquid containing a high concentration of bacterial cells, the viscosity of the fermentation liquid is high, so there is a large pressure loss during sweeping, and the filtration pressure increases, resulting in compaction of the insoluble substances on the membrane surface, which in turn leads to a problem of reduced filtration efficiency.

[0004] To improve the microfiltration permeation rate, a solution using a process such as sweeping of undissolved substances on the membrane surface and a solution using additives such as cationic surfactants are also used. For example, Patent Document 1 discloses that the microfiltration permeation flow rate and filtration efficiency can be improved by adding 0.01 to 1 (W / V) of a cationic surfactant to an enzyme-producing fermentation culture solution containing a high concentration of microorganisms during microfiltration.

[0005] However, these solutions had issues such as complicating the fermentation process and lengthening the production time, as well as introducing additives into the final formulation. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 4808725 DISCLOSURE OF THEINVENTION [Problem to be solved by the invention]

[0007] The present invention relates to providing a method for improving the filtration rate in a microfiltration process in the production of useful substances using microorganisms. [Means for solving the problem]

[0008] As a result of studies conducted in consideration of the above problems, the present inventors have found that the filtration rate during microfiltration can be significantly improved by using a microorganism in which the function of the flagellar gene is suppressed.

[0009] That is, the present invention relates to the following. A method for improving the filtration rate in a microfiltration process for the fermentation production of a useful substance using a microorganism, the method comprising using a modified microorganism in which the function of the flagellar gene of the microorganism is suppressed. Effect of the Invention

[0010] According to the present invention, the filtration rate can be improved in the microfiltration step in the fermentation production of a useful substance, and the time and cost required for the production of the useful substance can be reduced. [Brief description of the drawings]

[0011] [Figure 1] Schematic diagram showing introduction of the prsA gene into the nprE locus of the ΔsigF strain by homologous recombination. [Diagram 2] Schematic diagram showing deletion of the ORF sequence of the hag gene by homologous recombination. [Diagram 3] Changes in cell density of modified microbial cultures of the present invention. [Figure 4] The effect of improving the microfiltration permeation flow rate of the modified microbial culture solution of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The method of the present invention is a method for improving the filtration rate in the microfiltration step in the fermentation production of useful substances using microorganisms, and uses a modified microorganism in which the function of the flagellar gene of the microorganism is suppressed.

[0013] The host microorganism for the modified microorganism used in the method of the present invention is not particularly limited as long as it can be used for the fermentative production of useful substances. For example, Bacillus bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus cereus, Bacillus thuringiensis, and Bacillus amyloliquefaciens; Escherichia bacteria such as Escherichia coli; Brevibacillus bacteria such as Brevibacillus choshinensis and Brevibacillus brevis; Clostridium butyricum; Clostridium bacteria such as Corynebacterium butyricum; Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, Corynebacterium alkanolyticum, Corynebacterium crenatum, Corynebacterium crudilactis, Corynebacterium callunae, Examples of bacteria that may be used include bacteria of the genus Corynebacterium such as Corynebacterium callunae; or yeasts. Among these, bacteria of the genus Bacillus, Escherichia, and Brevibacillus are preferred, bacteria of the genus Bacillus are more preferred, and Bacillus subtilis is even more preferred.

[0014] The host microorganism may be a wild-type strain, or may be a mutant strain that has been mutated. Examples of the mutant strain include a Bacillus subtilis mutant strain in which the prsA gene, which is a protein involved in the secretion process of a protein, is overexpressed (JP Patent Publication No. 2007-49987), and a Bacillus subtilis mutant strain in which a sporulation-related gene (e.g., any one of the sigE gene, sigF gene, spoIIE gene, spoIISB gene, sigG gene, and the gene group contained in the region from spoIVCB to spoIIIC, and one or more genes corresponding to the gene or gene group) has been deleted or inactivated (JP Patent Publication No. 2003-47490).

[0015] Details of the group of genes contained in the sigE gene, sigF gene, spoIIE gene, spoIISB gene, sigG gene, and the region from spoIVCB to spoIIIC in Bacillus subtilis are shown in Table 1. Furthermore, genes derived from other microorganisms, preferably from bacteria of the genus Bacillus, which have 70% or more, preferably 80% or more, and more preferably 90% or more identity in base sequence with each of the genes are considered to be genes corresponding to the genes in Table 1, and are included in sporulation-related genes.

[0016] [Table 1]

[0017] "Flagellar gene" refers to a gene that encodes the flagellin protein of a microorganism.

[0018] The flagellum gene is known as the hag gene in Bacillus bacteria, the fliC gene in Escherichia coli, and the flaA gene in Clostridium butyricum. For example, the hag gene of Bacillus subtilis is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:2, and an example of such a gene is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:1; the hag gene of Bacillus licheniformis is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:4, and an example of such a gene is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:3; the hag gene of Brevibacillus brevis is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:6, and an example of such a gene is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:5; the fliC gene of Escherichia coli is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:8, and an example of such a gene is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:7; and the flaA gene of Clostridium butyricum is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:10, and an example of such a gene is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO:9.

[0019] Furthermore, the flagellar gene of the present invention encompasses a polynucleotide that has an amino acid sequence identity of at least 90%, preferably 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, preferably 99% or more to that of a flagellar protein (e.g., a protein consisting of the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10) and encodes a polypeptide that is functionally equivalent to the flagellar protein. Here, a functionally equivalent polypeptide refers to one that has a biological, physiological, or biochemical function equivalent to that of the flagellar protein. The polynucleotide can also be isolated and identified using a gene amplification method based on the base sequence.

[0020] Here, the identity of amino acid sequences is calculated by the Lipman-Pearson method (Science, 1985, 227:1435-1441). Specifically, it is calculated by performing analysis using the search homology program of the genetic information processing software GENETYX Ver. 12 with the unit size to compare (ktup) set to 2.

[0021] Inhibition of flagellar gene function includes inhibition of expression of the gene, for example, reduction in the expression level, inhibition of transcription, and inhibition of translation. The expression level of the flagellar gene can be reduced, for example, by deleting or inactivating the gene, specifically, by deleting or inactivating the coding region, non-coding region, or transcription or translation initiation region of the flagellar gene. Here, inactivation can be, for example, by introducing a mutation that reduces the expression level of the gene. Means for suppressing the transcription of the flagellar gene include introducing a polynucleotide or RNA that has the activity of degrading the transcription product of the flagellar gene, and means for suppressing the translation of the flagellar gene include introducing a polynucleotide or RNA that suppresses the translation of the transcription product into protein.

[0022] In one embodiment, the reduction in gene expression level due to the deletion or inactivation of the flagellar gene can be achieved by removing part or all of the nucleotide sequence of the flagellar gene from the genome or replacing it with another nucleotide sequence, inserting another polynucleotide fragment into the sequence of the flagellar gene, or mutating the transcription or translation initiation region of the flagellar gene, and preferably by deleting or inactivating part or all of the nucleotide sequence of the flagellar gene. More specific examples include a method of specifically deleting or inactivating the flagellar gene on the genome of a microorganism, and a method of randomly deleting or inactivating the gene in a microbial cell, and then evaluating the expression level or activity of the flagellar protein, or performing genetic analysis to select cells having the desired mutation.

[0023] For example, a method using homologous recombination can be used to reduce gene expression by specific deletion or inactivation of the flagellar gene. That is, a DNA fragment of the flagellar gene into which an inactivating mutation has been introduced by substitution or insertion of a polynucleotide, or a DNA fragment containing the outer region of the flagellar gene but not the flagellar gene, is constructed, and then inserted into a parent microorganism to cause homologous recombination in the region of the genome of the parent microorganism that contains the flagellar gene, thereby deleting or inactivating the flagellar gene on the genome. Alternatively, the flagellar gene can be inactivated by inserting a recombinant vector (such as a plasmid) having a DNA fragment containing a partial region of the flagellar gene into a parent microorganism and disrupting a partial region of the flagellar gene in the genome of the parent microorganism by homologous recombination. In particular, when Bacillus subtilis is used as a host microorganism, there have already been several reported methods for deleting or inactivating target genes by homologous recombination (Mol. Gen. Genet., 223, 268, 1990; Genet. Syst., 84(4):315-8, 2009, etc.), and the modified microorganism of the present invention can be obtained by using such methods. Other methods for reducing gene expression by specific deletion or inactivation of flagellar genes include, for example, genome editing, which includes DNA double-strand breaks and associated specific DNA mutations using TALEN or CRISPR-Cas systems, and single-base editing without DNA double-strand breaks.

[0024] Methods for reducing gene expression levels by randomly deleting or inactivating genes in microorganisms include a method of randomly cloning a gene into which an inactivating mutation has been introduced and introducing a DNA fragment into the microorganism to cause homologous recombination between the gene and the genome of the microorganism, and a method of inducing mutation by irradiating the microorganism with ultraviolet light, gamma rays, etc. Gene inactivation mutation refers to a mutation that causes the target gene to lose its original function due to a silence mutation, missense mutation, nonsense mutation, frameshift mutation, etc. For example, a gene into which an inactivating mutation has been introduced does not express a protein, or expresses a protein whose original activity is impaired.

[0025] A method for preparing a DNA fragment containing a flagellar gene into which an inactivating mutation has been introduced includes site-specific mutagenesis. Site-specific mutagenesis can be carried out using a mutagenesis primer containing the nucleotide mutation to be introduced. For example, DNA fragments are prepared by amplifying the upstream and downstream sides of a region containing the flagellar gene by PCR using two pairs of primers containing the nucleotide mutation to be introduced as a template, and then these are linked together by SOE-PCR (splicing by overlap extension PCR) (Gene, 1989, 77(1): p61-68), to construct a DNA fragment containing a desired mutation. Alternatively, site-specific mutagenesis can be carried out by inverse PCR or annealing (Muramatsu et al., eds., "Revised 4th Edition New Genetic Engineering Handbook," Yodosha, p82-88), or by using commercially available site-specific mutagenesis kits such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit.

[0026] The mutation primer can be prepared by a well-known oligonucleotide synthesis method such as the phosphoramidite method (Nucleic Acids Research, 1989, 17:7059-7071). The flagellar gene used as a template may be prepared from a host microorganism by a conventional method or may be chemically synthesized.

[0027] To introduce a DNA fragment or a vector into a host microorganism, known techniques such as the calcium phosphate method, electroporation, lipofection, particle gun method, PEG method, etc. For example, competent cell transformation (J Bacteriol, 1967, 93: 1925-1937), electroporation (FEMS Microbiol Lett, 1990, 55: 135-138), protoplast transformation (Mol Gen Genet, 1979, 168: 111-115), Tris-PEG method (J Bacteriol, 1983, 156: 1130-1134), etc. can be used depending on the host microorganism.

[0028] Furthermore, examples of polynucleotides having an activity of degrading the transcription product of the flagellar gene or polynucleotides that suppress translation of the transcription product into protein include polynucleotides that contain a nucleotide sequence complementary or substantially complementary to the nucleotide sequence of the mRNA of the flagellar gene or a part thereof, such as antisense RNA for the mRNA of the flagellar gene, siRNA for the mRNA of the flagellar gene, and ribozymes for the mRNA of the flagellar gene.

[0029] A microorganism in which the function of the flagellar gene is suppressed can be selected by confirming its genome sequence. Alternatively, a microorganism in which the function of the flagellar gene is suppressed can be selected using the expression level or activity of the flagellar protein as an indicator.

[0030] In the fermentation production of useful substances using the modified microorganism of the present invention, the useful substances are not particularly limited. For example, useful substances include proteins and polypeptides such as enzymes and physiologically active factors useful for food, pharmaceuticals, cosmetics, detergents, textile treatment, medical test drugs, etc., and include various industrial enzymes and physiologically active peptides for detergents, foods, textiles, feed, chemicals, medical treatment, diagnosis, etc., but industrial enzymes are preferred. In addition, industrial enzymes by function include oxidoreductase, transferase, hydrolase, lyase, isomerase, synthetase, etc., but preferably include genes of hydrolases such as cellulase, α-amylase, protease, and lipase. Specifically, α-amylase is exemplified, and among them, α-amylase derived from a microorganism, preferably from a bacterium of the genus Bacillus is exemplified.

[0031] The incorporation of a gene encoding the above protein or polypeptide into the modified microorganism of the present invention can be carried out by introducing a recombinant plasmid to which the gene encoding the above protein or polypeptide has been linked into the host microorganism by a common transformation method such as competent cell transformation, protoplast transformation, electroporation, etc. Alternatively, the incorporation can be carried out by directly integrating a DNA fragment containing such a gene and an appropriate homologous region with the genome of the host microorganism into the genome of the host microorganism by homologous recombination.

[0032] The fermentation production of useful substances is carried out by inoculating the modified microorganism of the present invention, into which a gene encoding the above-mentioned protein or polypeptide has been incorporated, into a medium containing an assimilable carbon source, nitrogen source, and other essential components, culturing it using a conventional microbial culture method, and recovering and purifying the protein or polypeptide after the culture is completed. The protein or polypeptide recovery step is a step of recovering the target substance (protein or polypeptide) from at least one of the cultured transformant and the culture of the transformant, and involves solid-liquid separation (centrifugation or coarse filtration), followed by microfiltration (MF) using a microfiltration membrane, and further purification and concentration by ultrafiltration or the like as necessary, to recover the target substance. Microfiltration is a filtration method using a microfiltration membrane with an average pore size of 0.01 to 10 μm, preferably 0.01 μm to 0.5 μm, more preferably 0.01 μm to 0.1 μm, and more preferably 0.05 to 0.1 μm, by pressure filtration, vacuum filtration, cross-flow filtration, centrifugal filtration, etc. In microfiltration, the filtration membrane may be, for example, an inorganic membrane such as ceramics such as alumina, titania, or zirconia, glass, or metal, or an organic membrane such as a cellulose acetate-based, nitrocellulose-based, aliphatic polyamide-based, polysulfone-based, polyolefin-based, polyacrylonitrile-based, polyethersulfone-based, polyvinyl chloride-based, polyvinyl alcohol-based, or fluorine-based polymer-based membrane.

[0033] As shown in the Examples below, the culture solution in which the modified microorganism of the present invention was cultured was filtered through a microfiltration membrane (effective membrane area 80 cm 2 When microfiltration (outlet pressure: 0.1 MPa, approximately 30 minutes) was performed using a polyethylene hollow fiber microfiltration membrane with a pore size of 0.1 μm, the permeation flow rate (mL / cm 2 ·min) is significantly improved compared to when the parent microorganism before modification is used. That is, in the fermentation production of useful substances using the modified microorganism of the present invention, the filtration rate in the microfiltration step is improved. Here, the filtration rate can be calculated as the membrane permeation flow rate per minute and per square centimeter, and the improvement in the microfiltration rate of the present invention means that the filtration rate is improved by 110% or more, preferably 120% or more, and more preferably 150% or more compared to the case where the parent microorganism before modification is used. This can reduce membrane clogging during microfiltration, making it possible to reduce the time and cost required for the production of useful substances.

[0034] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A method for improving the filtration rate in a microfiltration process for the fermentation production of a useful substance using a microorganism, the method comprising using a modified microorganism in which the function of the flagellar gene of the microorganism is suppressed. <2> Inhibition of the function of the flagellar gene is inhibition of the expression of the gene. <1> How to. <3> The inhibition of the function of the flagellar gene is a decrease in the expression level of the gene. <1> How to. <4> The reduction in the expression level of the flagellar gene is achieved by deleting or inactivating the gene. <3> How to. <5> The microorganism is a bacterium of the genus Bacillus, Escherichia or Brevibacillus. <1> ~ <4> Either way. <6> The microorganism is Bacillus subtilis or Bacillus licheniformis, <1> ~ <4> Either way. <7> The flagellar gene is a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4 or an amino acid sequence having at least 90% identity thereto. <6> How to. <8> The flagella gene is a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or 3. <6> How to. <9> Bacillus subtilis is a mutant strain of Bacillus subtilis in which one or more sporulation-related genes or gene groups selected from any one of the sigE gene, the sigF gene, the spoIIE gene, the spoIISB gene, and the sigG gene, and the gene group contained in the region from spoIVCB to spoIIIC have been deleted or inactivated. <6> ~ <8> Either way. <10> The microfiltration is carried out using a microfiltration membrane having an average pore size of 0.01 μm to 10 μm, preferably 0.01 μm to 0.5 μm, and more preferably 0.01 μm to 0.1 μm. <1> ~ <9> Either way. EXAMPLES

[0035] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

[0036] Example 1: Construction of a hag gene deletion strain A strain in which an excess cassette of the prsA gene was introduced into a strain (ΔsigF strain: JP 2003-47490 A) in which the sigF gene of Bacillus subtilis 168 strain had been deleted was used as the parent strain. The prsA gene overexpression cassette (SEQ ID NO: 11) was introduced into the nprE locus of the ΔsigF strain by homologous recombination (FIG. 1). The DNA fragment in which the prsA overexpression cassette was introduced was constructed by the following method.

[0037] First, the PCR fragment <1> (Primers prsA up F-prsA up R, template DNA: prsA-Ka strain genomic DNA (Patent 4839144)), PCR fragment <2> (Primers prsA mid F-prsA mid R, template DNA: MazF cassette (Genet. Syst., 84(4):315-8, 2009)), PCR fragment <3> (Primers prsA down F-prsA down R, template DNA: genomic DNA of strain 168) were used to construct the PCR fragment. <1> - <3> were linked by SOE-PCR (primers prsA final F-prsA final R). This gene transfer was performed according to the marker-free deletion method developed by Morimoto et al. (Genet. Syst., 84(4):315-8, 2009). This prsA overexpressing ΔsigF strain was used as the parent strain for this experiment.

[0038] The ORF sequence of the hag gene of this parent strain (SEQ ID NO: 1) was deleted by homologous recombination using a hag gene deletion cassette (Figure 2). The DNA fragment for deleting the hag gene was constructed as follows. First, a PCR fragment <4> (Primers hag up F-hag up R, template DNA: genomic DNA of strain 168), PCR fragment <5> (Primers hag mid F-hag mid R, template DNA: genomic DNA of strain 168), PCR fragment <6> (Primers mazF F-mazF R, template DNA: template DNA:MazF cassette (Genet. Syst., 84(4):315-8, 2009)), PCR fragment <7> (Primers hag down F-hag down R, template DNA: genomic DNA of strain 168) were used to construct the PCR fragment. <4> - <7> were linked by SOE-PCR (primers hag final F-hag final R). This gene introduction was performed according to the marker-free deletion method developed by Morimoto et al. (Genet. Syst., 84(4):315-8,2009). Hereafter, this hag gene deletion strain was referred to as the Δhag strain.

[0039] [Table 2]

[0040] Example 2 Introduction of amylase expression vector An amylase expression plasmid (pHY-YR288 (Patent Application No. 2021-112712)) was introduced into the parent strain and Δhag strain obtained in Example 1 by protoplast transformation method.

[0041] Example 3 Culture evaluation A 2L jar fermenter culture tank was used to carry out the culture evaluation according to the following method. The amylase expression vector-introduced strain obtained in Example 2 was shake-cultured overnight at 30°C and 180 rpm in a 500mL pleated Erlenmeyer flask with 30mL of LB medium supplemented with 10ppm of tetracycline hydrochloride. 25mL of this culture was taken and inoculated into 1.2L of 2xL-maltose medium (2% soytone, 1% yeast extract, 1% NaCl, 7.5% maltose, 7.5ppm manganese 4-5hydrate, antifoaming agent). This was cultured for 3 days at 30°C, 850 rpm, and 0.04MPa. Analysis of the cell density of the cultures was measured at a wavelength of 600 nm using a Hitachi spectrophotometer (U-2900) (Hitachi High-Tech Science). The cell density of the Δhag strain was comparable to that of the parent strain, indicating that the deletion of the hag gene does not affect cell growth (Figure 3).

[0042] Example 4 Measurement of MF permeation flow rate Using the culture solution obtained in Example 3, microfiltration (MF) was carried out by the following method. The culture solution used was a sample from the third day (OD600 was about 27 for both the parent strain and the Δhag strain). The MF module had an effective membrane area of ​​80 cm 2 Microfiltration (MF) was performed using a polyethylene hollow fiber microfiltration membrane microsa PSP-013 (Asahi Kasei Corporation) with a pore size of 0.1 μm. The pump used in the microfiltration system was EYELA RP-1100 (Tokyo Rikakikai Co., Ltd.), and the pump rotation speed was set to 100 rpm. The MF module stand used was microsa PX-02001, and the valve was adjusted so that the outlet pressure was 0.1 MPa. Filtration was performed for about 30 minutes under these conditions until the permeation flow rate became constant, and the results of evaluating the permeation flow rate at that time are shown in Figure 4. This result shows that the MF permeation flow rate is significantly improved by deleting the hag gene in culture solutions with the same cell density.

Claims

1. A method for improving the filtration rate in the microfiltration process of fermentation production of useful substances using microorganisms, wherein a modified microorganism is used in which the function of the flagellar gene of the microorganism is suppressed.

2. The method according to claim 1, wherein the suppression of the function of the flagellar gene is the suppression of the expression of said gene.

3. The method according to claim 1, wherein the suppression of the function of the flagellar gene is a decrease in the expression level of the gene.

4. The method according to claim 3, wherein the reduction in the expression level of the flagellar gene is achieved by deletion or inactivation of the gene.

5. The method according to any one of claims 1 to 4, wherein the microorganism is a bacterium of the genus Bacillus, a bacterium of the genus Escherichia, or a bacterium of the genus Brevibacillus.

6. The method according to any one of claims 1 to 4, wherein the microorganism is Bacillus subtilis or Bacillus licheniformis.

7. The method according to claim 5, wherein the flagellar gene is a gene that codes for a protein consisting of the amino acid sequence shown in Sequence ID No. 2, 4, 6, or 8, or an amino acid sequence having at least 90% identity therewith.

8. The method according to claim 6, wherein the flagellar gene is a gene that codes for a protein consisting of the amino acid sequence shown in Sequence ID No. 2 or 4, or an amino acid sequence having at least 90% identity thereto.

9. The method according to claim 6, wherein the Bacillus subtilis mutant strain is one in which any of the sigE gene, sigF gene, spoIIE gene, spoIISB gene, or sigG gene, and one or more spore formation-related genes or gene groups selected from the gene group contained in the region from spoIVCB to spoIIIC, have been deleted or inactivated.

10. The method according to claim 1, wherein microfiltration is performed using a microfiltration membrane having an average pore size of 0.01 μm to 10 μm.

11. A method for producing a useful substance using a modified microorganism, i) A step of providing a modified microorganism in which the function of the flagellar gene is suppressed. ii) A step of culturing the modified microorganism under conditions for producing useful substances, iii) After step iii), a step of fine-filtering the culture solution of the modified microorganism, Methods that include...

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