Method for evaluating hemolysin production capability

Abstract

To provide a new method to efficiently evaluate hemolysin production capability of hemolysin-producing bacteria including Bacillus species.SOLUTION: Provided is a method for evaluating hemolysin production capability of a hemolysin-producing test bacterium, the method comprising: an inoculation step of inoculating, at fixed intervals, a plurality of hemolysin-producing bacterial strains including the hemolysin-producing test bacterium and a hemolysin-producing control bacterium onto on a solid medium containing blood; a culturing step of culturing the plurality of hemolysin-producing bacterial strains following the inoculation step; and a comparison step of comparing sizes of hemolysis zones between colonies of the hemolysin-producing test bacterium and the hemolysin-producing control bacterium formed after the culturing step.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating hemolysin-producing ability, a method for screening a mutant strain with enhanced hemolysin-producing ability, and the like. [Background technology]

[0002] Cyclic lipopeptides, such as surfactin and iturin, are amphiphilic substances derived from microorganisms and consist of a peptide moiety and a fatty acid moiety.

[0003] It is known that Bacillus bacteria produce various types of cyclic lipopeptides, which are mainly classified into the iturin family, surfactin family, and fengicin family depending on the amino acid sequence and fatty acid chain type in the peptide portion (Non-patent Document 1).

[0004] Currently, cyclic lipopeptides are used as safe and biodegradable biosurfactants in a wide range of fields, including medicine, food production, agriculture, and environmental sanitation, and further applications are expected.

[0005] For example, cyclic lipopeptides such as surfactin are widely used in pharmaceuticals, cosmetics, foods, etc. due to their surfactant properties. Surfactin can form a stable D phase (bicontinuous phase) that can encapsulate both water and oil in extremely small amounts, making it suitable for producing cleansing agents such as oil gels and fine particle emulsions.

[0006] Furthermore, cyclic lipopeptides such as iturin not only have a so-called surfactant effect, but also exhibit excellent antibacterial activity against a wide range of bacteria and fungi, based on their ability to destroy the membrane structures of bacteria and fungi that are plant pathogens (Non-Patent Document 2). Therefore, they are expected to be used in a variety of technical fields as antibacterial agents, antifungal agents, infectious disease treatment agents, biopesticides (biological pesticides), plant disease control agents, etc., and are expected to become safe pesticides that can replace conventional synthetic chemical pesticides and reduce their impact on the environment, ecosystems, and human health.

[0007] Improving productivity is an important issue in the industrial production of useful substances by microorganisms. Because cyclic lipopeptides such as surfactin and iturin are produced by Bacillus bacteria, industrial production of cyclic lipopeptides is mainly carried out by culturing Bacillus bacteria. Therefore, there is a need for a method for improving the productivity of cyclic lipopeptides in Bacillus bacteria and a method for efficiently evaluating the productivity of cyclic lipopeptides in Bacillus bacterial strains. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ongena M. and Jacques P.,Trends in Microbiology,2007,16(3):115-125. [Non-patent document 2] Dunlap CA, Bowman MJ, and Rooney AP, Frontiers in Microbiology, 2019, Vol. 10, Article 1794. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a new method for efficiently evaluating the hemolysin-producing ability of hemolysin-producing bacteria such as Bacillus bacteria. [Means for solving the problem]

[0010] When Bacillus bacteria are cultured on blood-containing solid media such as sheep blood agar to form colonies, the cyclic lipopeptides produced by the Bacillus bacteria destroy red blood cells in the medium, forming a transparent ring structure called a hemolytic ring around the colony (Figure 1). Because large hemolytic rings can form around colonies with high cyclic lipopeptide production, it is thought that the amount of cyclic lipopeptide produced by a specific Bacillus strain can be evaluated based on the size of the hemolytic ring.

[0011] To actually examine the effectiveness of the above-mentioned evaluation method, the present inventors inoculated a culture solution of Bacillus bacteria onto sheep blood agar medium to form single colonies, and measured the hemolytic ring diameter of each single colony. As a result, it was found that colonies were small in areas where colonies were densely formed on the solid medium, and large in areas where colonies were sparsely formed, resulting in large variations in hemolytic ring diameter even for the same bacterial strain cultured for the same period of time. Therefore, it became clear that it is difficult to evaluate the production amount of cyclic lipopeptides simply based on the size of the hemolytic ring.

[0012] Next, the present inventors inoculated Bacillus bacteria onto sheep blood agar medium at points arranged in a grid pattern so that the size of the colonies that grew would be constant regardless of their position on the medium, and found that this resulted in stable colony size and suppressed fluctuations in the hemolytic ring diameter.

[0013] Furthermore, the present inventors inoculated and cultured a high-surfactin-producing strain, a standard surfactin strain, and a high-iturin-producing strain, and a standard iturin strain in a grid pattern on sheep blood agar medium. As a result, in the case of both surfactin and iturin, they found that the hemolytic ring diameter and the ratio of the hemolytic ring diameter to the colony diameter were significantly larger in the high-producing strain than in the standard strain, and that the coefficient of variation within the same strain was kept low.

[0014] From the above results, it has been possible to efficiently evaluate the hemolysin-producing ability of hemolysin-producing bacteria based on the size of the hemolytic ring by inoculating the bacteria into points arranged in a grid pattern on a blood-containing medium, and a technique has been established for efficiently screening mutant strains with enhanced hemolysin-producing ability using the size of the hemolytic ring as an index. The present invention is based on the above findings and provides the following:

[0015] (1) A method for evaluating the hemolysin-producing ability of a test bacterium, comprising: an inoculation step of inoculating a plurality of strains including the test bacterium and the control bacterium onto a blood-containing solid medium at regular intervals; a culturing step of culturing the plurality of strains after the inoculation step; and a comparison step of comparing the size of the hemolytic ring between the colonies of the test bacterium and the control bacterium formed after the culture step; The method comprising: (2) The method according to (1), wherein the hemolysin is a lipopeptide. (3) The method according to (2), wherein the lipopeptide is a cyclic lipopeptide. (4) The method according to (3), wherein the cyclic lipopeptide is a surfactin family lipopeptide, an iturin family lipopeptide, a fengicin family lipopeptide, or a combination of any two or more thereof. (5) The method according to any one of (1) to (4), wherein the inoculation step inoculates the strain in a grid pattern. (6) The method according to (5), wherein the lattice is an orthorhombic lattice, a hexagonal lattice, a square lattice, a rectangular lattice, or a parallelepiped lattice. (7) The method according to (1), wherein the constant interval is 10 mm to 30 mm. (8) The method according to (1), wherein the blood content in the blood-containing solid medium is 1% by volume to 10% by volume. (9) The method according to (1), wherein the glucose concentration in the blood-containing solid medium is 2% to 6%. (10) The method according to (1), wherein the comparison step compares the diameter of the hemolytic ring, the ratio of the diameter of the hemolytic ring to the diameter of the colony, and / or the difference between the diameter of the hemolytic ring and the diameter of the colony between colonies. (11) The method according to (1), wherein the test bacterium is a strain obtained by genetic manipulation of the control bacterium. (12) The method according to (1), wherein the test bacterium and / or the control bacterium are derived from a hemolysin-producing bacterium after random mutagenesis. (13) The method according to (12), wherein the random mutation is introduced by mutagen treatment or error-prone PCR. (14) The method according to (1), wherein the test bacterium and / or the control bacterium are derived from a mutant library for a specific base or a specific region on the genome. (15) A method for screening a mutant strain with enhanced hemolysin-producing ability derived from a hemolysin-producing bacterium, comprising the steps of: a mutation introduction step of introducing a mutation into the hemolysin-producing bacterium; a first inoculation step of inoculating the hemolysin-producing bacterium after the mutation introduction step onto a solid medium; a single colony formation step of culturing the hemolysin-producing bacteria after the inoculation step to form a plurality of single colonies; a second inoculation step in which bacterial cells are collected from the plurality of single colonies after the single colony formation step and inoculated onto a blood-containing solid medium at regular intervals; a culturing step of culturing the bacterial cells after the second inoculation step; and an identification step in which the size of the hemolytic rings is compared among the colonies of the hemolysin-producing bacteria formed after the culture step, and a bacterial strain contained in a colony having a larger hemolytic ring than other colonies is identified as a mutant strain with enhanced hemolysin-producing ability; The method comprising: (16) The method according to (15), wherein the mutagenesis is random mutagenesis. (17) The method according to (15), wherein the step of introducing mutations comprises constructing a mutant library for a specific base or a specific region on the genome. (18) The method according to (15), wherein the second inoculation step inoculates the hemolysin-producing bacteria in a rhombic or square lattice pattern with intervals of 10 to 30 mm. (19) The method according to (18), wherein the orthorhombic lattice or the square lattice consists of 48 or more points. (20) The method according to (18) or (19), wherein the identification step compares the diameter of the hemolytic ring and / or the ratio of the diameter of the hemolytic ring to the diameter of the colony between colonies, and identifies the strain of a colony having a higher diameter and / or ratio than other colonies as the mutant strain with enhanced hemolysin production ability. [Effects of the Invention]

[0016] According to the present invention, a new method for efficiently evaluating the hemolysin-producing ability of hemolysin-producing bacteria such as Bacillus bacteria is provided. [Brief explanation of the drawings]

[0017] [Figure 1] 1A and 1B show a method for inoculating a culture solution of a hemolysin-producing bacterium onto a sheep blood agar medium to form a single colony and a hemolytic ring. Fig. 1A shows a schematic diagram of each step of inoculating a culture solution onto a sheep blood agar medium and culturing it. Fig. 1B shows a schematic diagram of a hemolytic ring formed around a colony. [Figure 2] These are diagrams showing colonies and hemolytic rings after culturing when a culture solution of a mycostilin-producing strain is inoculated and when the mycostilin-producing strain is inoculated in a grid pattern. Figure 2A shows colonies after culturing by spreading a culture solution of a mycostilin-producing strain. Figure 2B shows colonies after culturing by inoculating a mycostilin-producing strain in a grid pattern. Figure 2C shows the results of measuring the colony diameter and hemolytic ring diameter. The diameters shown in the figure are expressed as the mean value ± standard deviation. CV indicates the coefficient of variation. [Figure 3] FIG. 1 is a diagram showing the surfactin production amounts (SF production amounts) of a surfactin standard strain (SF standard strain) and a surfactin high-producing strain (SF high-producing strain), with the SF production amount of the SF standard strain set at 100%. [Figure 4] FIG. 1 shows colonies and hemolytic rings of the SF standard strain and the SF high-producing strain cultured in a grid pattern. [Figure 5]5A and 5B show the colony diameter, hemolytic ring diameter, and ratio of hemolytic ring diameter to colony diameter of the SF standard strain and the SF high-producing strain. FIG. 5A shows the results of colony diameter measurement. FIG. 5B shows the results of hemolytic ring diameter measurement. FIG. 5C shows the ratio of hemolytic ring diameter to colony diameter. CV indicates coefficient of variation. Error bars indicate standard deviation. [Figure 6] This figure shows the iturin production (ITU production) of an iturin standard strain (ITU standard strain) and an iturin high-producing strain (ITU high-producing strain), with the ITU production of the ITU standard strain set at 100%. [Figure 7] FIG. 1 shows colonies and hemolytic rings of the ITU standard strain and the ITU high-producing strain cultured in a grid pattern. [Figure 8] 8A and 8B show the colony diameter, hemolytic ring diameter, and the ratio of hemolytic ring diameter to colony diameter for the ITU standard strain and the ITU high-producing strain. Figure 8A shows the results of colony diameter measurement. Figure 8B shows the results of hemolytic ring diameter measurement. Figure 8C shows the ratio of hemolytic ring diameter to colony diameter. CV indicates coefficient of variation. Error bars indicate standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0018] 1. Method for evaluating hemolysin production Overview A first aspect of the present invention is a method for evaluating the hemolysin-producing ability of a test bacterium (hereinafter, sometimes abbreviated as a "method for evaluating hemolysin-producing ability" or "evaluation method"). The evaluation method of this aspect includes, as essential steps, an inoculation step, a culture step, and a comparison step. According to the evaluation method of this aspect, the hemolysin-producing ability of a test bacterium can be efficiently evaluated, and for example, a strain with high hemolysin-producing ability can be efficiently identified from among a large number of candidate bacterial strains.

[0019] 1-2.Definition of Terms The following terms frequently used in this specification are defined below.

[0020] As used herein, the term "hemolysin" refers to any substance having hemolytic activity. The type of substance is not particularly limited and may be either a natural or non-natural molecule. Specific types of hemolysin include, for example, peptides, oligopeptides, polypeptides, proteins, lipopeptides, lipids, glycopeptides, carbohydrates (sugars, etc.), steroids, nucleic acids, and low-molecular-weight compounds. The term "hemolysin" as used herein may refer to any substance produced by a hemolysin-producing bacterium (described below) or its degradation products, or may be a substance produced by the reaction of the substance or its degradation products with medium components, etc.

[0021] As used herein, the term "lipopeptide" refers to a substance comprising a peptide moiety and a fatty acid moiety. The specific structures of the peptide moiety and fatty acid moiety constituting the lipopeptide are not limited, and the bonding mode between the peptide moiety and the fatty acid moiety is also not limited. Lipopeptides having a cyclic molecular structure are called "cyclic lipopeptides." Lipopeptides other than cyclic lipopeptides are called "linear lipopeptides." As used herein, it is preferable that the lipopeptide has hemolytic activity based on surfactant activity or the like.

[0022] As used herein, "hemolysin-producing ability" refers to the ability of bacteria, fungi, and other microorganisms to produce hemolysin. More specifically, hemolysin-producing ability can be understood as the amount of hemolysin produced by a certain number of microorganisms under specific culture conditions within a certain period of time, e.g., the amount of hemolysin accumulated in the culture supernatant and / or the microorganisms after culturing in a liquid medium for a certain period of time. Bacteria with high hemolysin-producing ability can produce more hemolysin and / or hemolysin with high hemolytic activity, compared to, for example, standard strains such as wild-type strains or strains before transformation or mutagen treatment. Colonies formed by bacteria with high hemolysin-producing ability can form large hemolytic rings around the colonies. Therefore, hemolysin-producing ability can be evaluated by inoculating the microorganisms in a grid pattern, culturing them, and then measuring the size of the hemolytic ring, as described below.

[0023] As used herein, "hemolytic activity" refers to the activity of acting on and disrupting erythrocyte membranes. When erythrocyte membranes are disrupted by hemolytic activity, hemoglobin in erythrocytes is released from the erythrocytes. Hemolytic activity can be quantified by adding hemolysin to blood or erythrocytes obtained from any animal, such as a mammal (e.g., human or sheep), incubating with shaking at 37°C for several hours, and then measuring the absorbance of the supernatant in a wavelength range, such as the red range, corresponding to erythrocytes. Hemolysin-producing ability and activities such as surfactant activity of hemolysin can be evaluated based on the measured value of hemolytic activity. The method of the present invention for evaluating the hemolysin-producing ability of a test bacterium can also be used to evaluate the hemolytic activity of a test bacterium.

[0024] As used herein, the term "hemolysin-producing bacteria" refers to bacteria, fungi, archaea, or other bacteria that produce the above-mentioned hemolysins. Hemolysin-producing bacteria may be wild-type or mutant strains, and may be known strains or strains obtained by any genetic manipulation or random mutagenesis of known strains. Examples of hemolysin-producing bacteria that produce cyclic lipopeptides include Bacillus bacteria, Paenibacillus bacteria, Brevibacillus bacteria, Streptomyces bacteria, and Pseudomonas bacteria. The hemolysin-producing bacteria may produce one or more types of hemolysin; for example, they may produce multiple types of cyclic lipopeptides and / or linear lipopeptides. As described below, many Bacillus bacteria are known to simultaneously produce multiple cyclic lipopeptides. In this specification, the hemolysin-producing bacterium to be evaluated for its hemolysin-producing ability, or its strain, colony, or bacterial body, etc., is referred to as the "hemolysin-producing test bacterium." Furthermore, the hemolysin-producing bacterium to be used as a control when evaluating the hemolysin-producing ability of the hemolysin-producing test bacterium, or its strain, colony, or bacterial body, etc., is referred to as the "hemolysin-producing control bacterium."

[0025] As used herein, the term "hemolytic ring" refers to a region of hemolysis that forms around a colony when hemolysin-producing bacteria are cultured on a blood-containing solid medium to form colonies. The hemolytic ring is observed as a region in which the intensity of a color such as red derived from hemoglobin is reduced due to the breakdown of the membrane of blood-derived red blood cells. Note that, as used herein, the hemolytic ring is not limited to a transparent region in which the red color or other color has been completely lost, but may be any region in which the intensity of a color such as red has been reduced to a level distinguishable by the naked eye or image processing software, compared to the original red color or other color derived from the blood-containing solid medium. For example, the hemolytic ring may be a region in which the intensity of a color such as red has been reduced to a certain threshold or below.

[0026] As used herein, the term "cyclic lipopeptide" refers to a substance having a cyclic molecular structure containing a peptide portion and a fatty acid portion. Cyclic lipopeptides typically have at least a portion of the ring structure composed of a peptide chain, with at least a portion of the fatty acid chain extending from the ring structure. Examples of cyclic lipopeptides include cyclic lipopeptides in which a fatty acid chain is attached to the side chain of the peptide chain constituting the ring structure, and cyclic lipopeptides in which a portion of the peptide chain is connected to a portion of the fatty acid chain and the other portion of the fatty acid chain extends from the ring structure. Examples of cyclic lipopeptides that can be produced by bacteria of the genus Bacillus include iturin family lipopeptides, surfactin family lipopeptides, and fengicin family lipopeptides. Cyclic lipopeptides may be amphiphilic. For example, at least a portion of the peptide chain may be hydrophilic, and the fatty acid portion may be hydrophobic.

[0027] As used herein, "iturin family lipopeptides" (Iturinic lipopeptides) is a general term for iturins and cyclic lipopeptides similar to iturin. Iturin family lipopeptides have a structure in which seven amino acids and a portion of a β-amino fatty acid are linked in a ring, and can be classified into iturins, mycostilins, bacillomycins, mohavensins, etc. based on the amino acid sequence of the seven amino acid portion (heptapeptide). Iturins, mycostilins, bacillomycins, and mohavensins are similar in that, in principle, the amino acid residue at position 1 of the heptapeptide is L-Asn or L-Asp, and the amino acid residues at positions 2 and 3 are D-Tyr-D-Asn, but the amino acid residues at positions 4 to 7 vary.

[0028] As used herein, "iturin" refers to an iturin family lipopeptide containing the amino acid sequence L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser (SEQ ID NO: 1) or an amino acid sequence similar thereto. Specific examples of iturin include iturin A and iturin A, which contain the amino acid sequence shown in SEQ ID NO: 1. L Iturin C, which contains the amino acid sequence shown in SEQ ID NO: 3 (L-Asp-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser), iturin W, which contains the amino acid sequence shown in SEQ ID NO: 4 (L-Asn-D-Ser-D-Asn-L-Pro-L-Tyr-D-Asn-L-Gln), and iturin V are known, and the iturin referred to herein may be any of these.

[0029] As used herein, "mycosubtilin" refers to an iturin family lipopeptide containing the amino acid sequence L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Ser-L-Asn (SEQ ID NO: 2).

[0030] As used herein, "surfactin family lipopeptides" is a general term for surfactin and cyclic lipopeptides similar to surfactin. Surfactin family lipopeptides have a structure in which seven amino acids and a portion of a β-hydroxy fatty acid are linked in a ring, and can be classified into surfactin, esperin, lichenysin, pumilacidin, etc. based on the amino acid sequence of the seven amino acid portion (heptapeptide).

[0031] As used herein, "fengycin family lipopeptides" is a general term for fengycin and cyclic lipopeptides similar to fengycin. Fengycin family lipopeptides have a structure in which 10 amino acids and a portion of a β-hydroxy fatty acid are linked in a ring, and can be classified into fengycin A, fengycin B, plipastatin A, plipastatin B, and the like based on the amino acid sequence of the 10 amino acid portion (decapeptide).

[0032] Bacillus bacteria produce a variety of lipopeptides depending on the species, subspecies, and strain (Dunlap CA, Bowman MJ, and Rooney AP, Frontiers in Microbiology, 2019, Vol. 10, Article 1794.).

[0033] Examples of Bacillus species that produce iturin include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus nakamurai, and Bacillus siamensis. An example of a Bacillus subtilis strain that produces iturin is the Bacillus subtilis RB14 strain.

[0034] Examples of species of Bacillus bacteria that produce mycostilin include Bacillus subtilis and Bacillus atrophaeus. An example of a subspecies of Bacillus that produces mycostilin is Bacillus subtilis subsp. spizizenii, and an example of a strain belonging to this subspecies is Bacillus subtilis ATCC 6633.

[0035] Examples of Bacillus species that produce bacillomycin include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus siamensis, and Bacillus velezensis. An example of a subspecies of Bacillus that produces bacillomycin is Bacillus subtilis subsp. inaquosorum.

[0036] Examples of species of Bacillus bacteria that produce mohavencin include Bacillus swezeyi, Bacillus halotolerans, Bacillus genomospecies #1, and Bacillus tequilensis.

[0037] Species of Bacillus bacteria that produce surfactin include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, Bacillus siamensis, Bacillus atrophaeus, Bacillus nakamurai, Bacillus halotolerans, Bacillus megaterium, Bacillus mojavensis, Bacillus tequilensis, Bacillus cereus, and Bacillus thuringiensis. thuringiensis) etc. Examples of Bacillus strains that produce surfactin include Bacillus subtilis RB14, Bacillus subtilis ATCC 6633, Bacillus subtilis subsp. inaquosorum, and Bacillus subtilis ATCC 21332 strains, as well as Bacillus bacterial strains (e.g., Bacillus subtilis 168 strain) into which a surfactin synthase gene and / or a gene encoding its modifying enzyme (e.g., 4-phosphopantetheinyl transferase) have been introduced.

[0038] Examples of species of Bacillus bacteria that produce esperin include Bacillus mesentericus.

[0039] Examples of Bacillus species that produce lichenisin include Bacillus licheniformis and Bacillus megaterium.

[0040] An example of a species of Bacillus bacteria that produces pumilacidin is Bacillus pumilus.

[0041] Examples of Bacillus species that produce fengicin A and / or fengicin B include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, and Bacillus mojavensis. Examples of Bacillus subtilis strains that produce fengicin A and / or fengicin B include Bacillus subtilis F-29-3 and Bacillus subtilis strain S499.

[0042] Examples of species of Bacillus bacteria that produce plipastatin A and / or plipastatin B include Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus cereus. Examples of Bacillus subtilis strains that produce plipastatin A and / or fendicin B include Bacillus subtilis RB14 and Bacillus subtilis pB2-L strains, as well as Bacillus bacterial strains (e.g., Bacillus subtilis 168 strain) into which the plipastatin synthase gene and / or a gene encoding its modifying enzyme have been introduced. In addition, since the modification enzyme gene in Bacillus subtilis 168 strain is a pseudogene, it does not produce lipopeptides by itself. However, since it has the surfactin synthase gene and the plipastatin synthase gene on its genome, it can produce surfactin and plipastatin by introducing the modification enzyme gene.

[0043] As used herein, the term "linear lipopeptide" refers to a substance that contains a peptide portion and a fatty acid portion and does not have a cyclic molecular structure. Specific examples of linear lipopeptides include gageopeptide, gageotetrin, brevibacillin, bogorol, and paenipeptin. Specific examples of gageopeptides include gageopeptide A, gageopeptide B, gageopeptide C, and gageopeptide D, which are produced by the Bacillus subtilis strain 109GGC020. Specific examples of gageotetrins include gageotetrin B, which is produced by the Bacillus subtilis strain 109GGC020. Specific examples of brevibacillins include brevibacillin, brevibacillin I, brevibacillin V, and brevibacillin 2V, which are produced by the Brevibacillus laterosporus strain DSM25. Specific examples of Bogorol include Bogorol A, Bogorol B, Bogorol C, Bogorol D, and Bogorol E produced by Brevibacillus laterosporus PNG-276 strain. Specific examples of Paenipeptin include Paenipeptin A, Paenipeptin B, and Paenipeptin C produced by Paenibacillus sp. OSY-N strain.

[0044] The bacterial strains targeted by the evaluation method of the present invention (referred to as "test bacteria" in this embodiment) are any strains of bacteria, fungi, archaea, or the like. The test bacteria may be, for example, hemolysin-producing strains that are known to produce hemolysin but whose hemolysin-producing ability is unknown, but are not limited to such hemolysin-producing bacteria. For example, the test bacteria may be strains whose hemolysin-producing ability is unknown (e.g., strains in which a lipopeptide synthetase gene has been introduced into a strain that does not originally have the ability to produce hemolysin). For example, the test bacteria may be non-hemolysin-producing bacteria such as Escherichia coli into which a gene or operon encoding cyclic lipopeptide synthetase has been introduced, and such non-hemolysin-producing bacteria may also have a gene encoding a modification enzyme additionally introduced. For example, the modification enzyme gene may be placed within the operon encoding cyclic lipopeptide synthetase so that the two constitute a single operon. Furthermore, the test bacteria may be non-hemolysin-producing strains, since the evaluation method of the present invention can also be used to evaluate whether the bacteria have (substantially) no hemolysin-producing ability. The test bacterium may be the lipopeptide-producing strain described above or a strain derived from the lipopeptide-producing strain described above. Strains derived from the lipopeptide-producing strain described above may be genetically modified strains (e.g., recombinant bacteria) of Bacillus bacteria or the like into which a gene and / or operon encoding a cyclic lipopeptide synthase has been introduced. A gene encoding a modification enzyme may also be introduced into the genetically modified strain. The modification enzyme may be, for example, 4-phosphopantetheinyl transferase, and the Bacillus bacterium may be Bacillus subtilis 168. Specific examples of strains derived from Bacillus subtilis 168 include strains obtained by introducing a mycostilin synthase gene and / or a modification enzyme gene into Bacillus subtilis 168, as described below, and strains obtained by introducing an iturin synthase gene and / or a modification enzyme gene into Bacillus subtilis 168. The test bacterium may be a strain of the same genus or species as the strain used as a comparison control for hemolysin production in the evaluation method of the present invention (referred to as the "control bacterium" in this embodiment), or it may be a strain of a different genus or species.

[0045] The type of hemolysin evaluated by the evaluation method of the present invention is not particularly limited and may be, for example, any lipopeptide, either a cyclic or linear lipopeptide. Examples of cyclic lipopeptides include surfactin family lipopeptides, iturin family lipopeptides, fengicin family lipopeptides, or a combination of two or more thereof. Many Bacillus bacteria are known to simultaneously produce multiple iturin family lipopeptides, surfactin family lipopeptides, and / or fengicin family lipopeptides. Therefore, the evaluation method and screening method of the present invention also target bacteria that produce hemolysins consisting of a combination of two or more selected from iturin family lipopeptides, surfactin family lipopeptides, and fengicin family lipopeptides. For example, when the evaluation method of the present invention is performed on a hemolysin-producing bacterial strain that produces iturin family lipopeptides and surfactin family lipopeptides, the combined hemolytic activity of the iturin family lipopeptides and the surfactin family lipopeptides can be evaluated.

[0046] As used herein, the term "plant disease" refers to a general term for illnesses that occur in plants. Known plant diseases include those caused by infectious pathogens such as fungi, bacteria, and viruses, as well as those caused by non-infectious pathogens such as a lack or excess of nutrients or water, or chemical damage. Unless otherwise specified, plant diseases in this specification refer to diseases caused by bacteria, fungi, and / or viruses.

[0047] As used herein, "control" refers to prevention or treatment (eradication) (from the Japan Agricultural Chemicals Association website). Therefore, as used herein, "plant disease control" refers to the prevention of plant diseases caused by bacteria, fungi, and / or viruses, or the treatment of plant diseases caused by bacteria, fungi, and / or viruses.

[0048] As used herein, "inoculate" or "seed" refers to adding and / or placing bacterial cells in a solid or liquid medium by any method. "Inoculation" includes placing bacterial cells on a solid medium in a dotted or linear pattern, spreading bacterial cells by streaking them using an inoculation loop or toothpick, and spreading bacterial cells over a partial area or the entire surface of a solid medium, as in the "spreading" method described below.

[0049] As used herein, the term "spread" refers to spreading bacterial cells on the surface of a solid medium. For example, this refers to spreading bacteria cultured in a culture medium over a partial area or the entire surface of a solid medium using a spreader or the like.

[0050] In this specification, the term "plurality" is not limited to a number equal to or greater than two, and refers to, for example, 2 to 480, 4 to 384, 8 to 288, 16 to 192, 32 to 96, or 48.

[0051] 1-3. Method The evaluation method of this embodiment includes an inoculation step, a culture step, and a comparison step as essential steps, and includes a single colony formation step and / or a recovery step as optional steps. Each step in the evaluation method of this embodiment will be specifically described below.

[0052] (Single colony formation process) In the method of the present invention, the "single colony formation step" refers to a step of forming single colonies by inoculating a test bacterium (e.g., a hemolysin-producing test bacterium) and / or a control bacterium (e.g., a hemolysin-producing control bacterium) onto a solid medium and culturing them. The purpose of this step is to ensure that the bacteria to be inoculated in the inoculation step described below are derived from single colonies.

[0053] The method for forming single colonies in this step is not particularly limited. For example, a bacterial solution diluted to an appropriate bacterial cell concentration may be spread over a portion or the entire surface of the solid medium using a spreader or the like so that several to several hundred colonies appear after cultivation. Single colony isolation can also be performed by streaking and spreading the bacterial cells using an inoculation loop or toothpick.

[0054] The method for culturing the test bacteria and / or control bacteria in this step is not particularly limited and can be appropriately selected depending on the types of bacterial strains of the test bacteria, control bacteria, etc. For example, a culture method known in the art, such as that described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, may be used.

[0055] In principle, the medium used for inoculating and culturing the strain (e.g., hemolysin-producing strain) in this step is a solid medium. Usable media include, for example, solid media such as agar medium containing one or more components selected from the group consisting of proteolytic enzyme hydrolysates such as peptone and tryptone, biological extracts such as potato dextrose, yeast extract, soybean hydrolysate, ground beans, and extracts thereof, amino acids such as glutamic acid or salts thereof, sugars such as glucose and sucrose, and inorganic salts such as sodium chloride, magnesium chloride, and potassium dihydrogen phosphate. Furthermore, if necessary, substances required for the growth of the strain (e.g., required amino acids for amino acid-requiring strains, required vitamins for vitamin-requiring strains) can be added. Specific media and compositions include NY medium (1 g / L Nutrient Broth, 1 g / L Yeast Extract, 40 g / L LD(+)-glucose, 3 g / L sodium chloride), LB medium (tryptone, yeast extract, sodium chloride), soy flour medium (40 g / L soy flour, 5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.18 g / L calcium chloride dihydrate, 0.025 g / L ferrous sulfate heptahydrate, 0.022 g / L manganese chloride tetrahydrate), YPG medium (yeast extract, peptone, glucose), PD medium (potato dextrose), and Suwa medium with peptone (sucrose, glutamic acid, peptone).

[0056] Furthermore, when the strain used in this step contains a drug resistance gene in a vector, etc., this step can be carried out in the presence of an antibiotic, such as ampicillin, kanamycin, tetracycline, spectinomycin, erythromycin, lincomycin, or chloramphenicol.

[0057] The culture conditions in this step can be appropriately selected depending on the type of strain. For example, the culture in this step can be carried out at 25 to 45°C, 30 to 40°C, 33 to 37°C, or 35°C.

[0058] The culture time in this step is not limited as long as it allows the growth of a quantity of bacterial cells that can be harvested in the inoculation step described below, and may be, for example, 4 hours or more, 12 hours or more, 24 hours or more, 2 days or more, or 3 days or more, and / or 7 days or less, 6 days or less, 5 days or less, or 4 days or less.

[0059] (Inoculation process) In the method of the present invention, the "inoculation step" refers to a step of inoculating multiple strains of bacteria, including test bacteria (e.g., hemolysin-producing test bacteria) and / or control bacteria (e.g., hemolysin-producing control bacteria), onto a blood-containing solid medium at regular intervals.

[0060] As used herein, a "blood-containing solid medium" refers to a solid medium prepared by adding blood to the above-mentioned medium at a desired concentration. The blood contained in the blood-containing solid medium is not limited as long as it is blood for which the hemolytic activity of hemolysin can be measured, and blood derived from any animal species can be used. For example, blood derived from mammals such as humans or sheep may be used. Furthermore, the type of blood may be whole blood, or a liquid obtained by removing any components other than red blood cells from whole blood, as long as it contains red blood cells. Examples of suitable blood include preserved blood, defibrinated blood, and whole blood. The blood content in the blood-containing solid medium is not limited as long as the hemolytic ring formed around the colony formed by the bacterial strain can be sufficiently distinguished from the surrounding non-hemolytic region, and may be, for example, 0.5% or more by volume, 1% or more by volume, 2% or more by volume, 3% or more by volume, 4% or more by volume, or 5% or more by volume, and / or 20% or less by volume, 15% or less by volume, 12% or less by volume, 10% or less by volume, 9% or less by volume, 8% or less by volume, 7% or less by volume, or 6% or less by volume, with an exemplary range being 1% to 10% by volume. Note that "% by volume" here refers to the percentage of the volume of blood components relative to the volume of the blood-containing solid medium.

[0061] In one embodiment, the blood-containing solid medium used in this step contains glucose. The glucose concentration in the blood-containing solid medium is not limited, but may be, for example, 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more, and / or 20% or less, 15% or less, 12% or less, 10% or less, 9% or less, 8% or less, 7% or less, or 6% or less, with an exemplary range being 2% to 6%. Note that "%" here indicates the percentage (% weight) of the weight of the glucose component relative to the weight of the blood-containing solid medium.

[0062] As used herein, "inoculating at regular intervals" means that the positions at which the bacterial cells of a strain (e.g., a hemolysin-producing strain) are placed on a blood-containing solid medium are spaced apart at regular intervals. More specifically, when the bacterial cells are inoculated at three or more positions on the blood-containing solid medium, the distance from each position to the nearest other position is constant, and preferably the distance from all the inoculated positions on the blood-containing solid medium to the nearest other position is constant. For example, the bacterial cells may be inoculated at regular intervals in a straight line, or at regular intervals along a circumference or arc. The specific distance of the "constant interval" is not limited as long as the colonies formed by the strains do not fuse with each other, but may be, for example, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, or 15 mm or more, and / or 50 mm or less, 40 mm or less, 35 mm or less, 30 mm or less, 25 mm or less, 20 mm or less, or 18 mm or less, with a preferred range being 10 to 30 mm.

[0063] In this step, the amount of bacterial cells to be inoculated at one location of a bacterial strain is not limited, as long as it is a constant amount or an amount that can be considered to be substantially constant among multiple bacterial strains, including the test bacteria and / or the control bacteria (for example, within a range of 2 to 10 times). For example, approximately 1 μL or more, 2 μL or more, or 3 μL or more, and / or 10 μL or less, 8 μL or less, 7 μL or less, 6 μL or less, or 5 μL or less of bacterial cells can be collected from each colony formed in the above-mentioned single colony formation step and inoculated into each location on the blood-containing solid medium.

[0064] In one embodiment, this step involves inoculating both a test bacterium (e.g., a hemolysin-producing test bacterium) and a control bacterium (e.g., a hemolysin-producing control bacterium) onto a blood-containing solid medium. In this embodiment, the number of positions at which the test bacterium and the control bacterium are inoculated is not limited, as long as they are at least one or more. For example, one control bacterium may be inoculated for two or more test bacterium, or the same number of test bacterium and control bacterium may be inoculated. In the method of this embodiment, some of the test bacterium may be used as control bacteria for comparison in the comparison step described below, or multiple test bacterium may be compared with each other as control bacteria. Therefore, in this specification, such cases are also considered to be encompassed by the inoculation of both the test bacterium and the control bacterium. This method is convenient when it is expected that the hemolysin-producing ability of more than half or the majority of the hemolysin-producing test bacterium, such as the hemolysin-producing test bacterium obtained after mutagen treatment, is unchanged, or when comparing the relative hemolysin-producing abilities of multiple hemolysin-producing test bacteria, because it eliminates the need to inoculate a standard strain, such as a strain before mutagen treatment.

[0065] In one embodiment of the evaluation method of the present invention, the inoculation step involves inoculating the strains in a grid pattern. As used herein, "inoculating in a grid pattern" refers to inoculating the strains at the vertices of a periodically arranged polygon (e.g., a rectangle such as a square, a parallelogram such as a rhombus, or a hexagon such as a regular hexagon). Inoculating in a grid pattern satisfies the rule that when strains are inoculated at three or more positions on a blood-containing solid medium, the distance between any of the positions and the nearest other positions is constant. This constant distance may be, for example, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, or 15 mm or more, and / or 50 mm or less, 40 mm or less, 35 mm or less, 30 mm or less, 25 mm or less, 20 mm or less, or 18 mm or less, preferably 10 to 30 mm. In this embodiment, it is preferable that all of the inoculation positions in this step are in a grid pattern. The specific type of lattice is not limited, and may be, for example, a rhombic lattice (also called a rhombic lattice), a hexagonal lattice, a square lattice, a rectangular lattice, or a parallelepiped lattice, with a rhombic lattice or a square lattice being preferred. The number of inoculation positions in the lattice is not limited as long as it is two or more, and may be, for example, 2 to 480, 4 to 384, 8 to 288, 16 to 192, 32 to 96, or 48. For example, a rhombic lattice or a square lattice can be composed of 48 or more points.

[0066] The specific inoculation method used in this step is not limited as long as it allows for spot or linear inoculation of the bacterial strain. For example, stickers or paper indicating inoculation positions at regular intervals, such as a grid pattern, can be attached to the culture vessel, and the bacterial cells can be inoculated into these positions using sterilized tip tips or toothpicks. Alternatively, tips with bacterial cells attached can be attached to all or part of the tip attachment positions of a multi-pipette such as an 8-channel pipettor, allowing simultaneous inoculation. Furthermore, the culture medium after pre-culture or a suspension of bacterial cells collected from colonies after the single colony formation step can be diluted as necessary and then dispensed onto solid medium in an amount of several μL to several tens of μL using a micropipette or the like. Alternatively, an automatic colony picker can be used to automate the process from collecting bacterial cells from colonies to inoculation. Automatic colony pickers are available with pin shapes and tip diameters suitable for each target bacterial strain. For example, pins with diameters of 0.5 mm to 2.0 mm and lengths of 40 mm to 70 mm can be used.

[0067] (Culture process) In the method of the present invention, the "culturing step" refers to a step of culturing multiple strains (e.g., hemolysin-producing strains) after the inoculation step described above. The purpose of this step is to allow the multiple strains to form colonies and hemolytic rings before the comparison step described below.

[0068] The method for culturing the strain in this step is not particularly limited, and can be appropriately selected depending on the type of strain, as in the above-mentioned single colony formation step.

[0069] The culture conditions for this step can be appropriately selected depending on the type of transformed microorganism. For example, the culture can be carried out at 25 to 45°C, 30 to 40°C, 33 to 37°C, or 35°C.

[0070] Furthermore, the culture time in this step is not limited as long as it is a time that allows colonies to form around them a hemolytic ring of a size that can be compared in the comparison step described below, and may be, for example, 4 hours or more, 12 hours or more, 24 hours or more, 2 days or more, or 3 days or more, and / or 7 days or less, 6 days or less, 5 days or less, or 4 days or less.

[0071] This step results in the formation of hemolytic rings around at least some or all of the colonies on the blood-containing solid medium, for example, around at least the colonies formed by the bacteria with high hemolysin-producing ability and / or the control bacteria.

[0072] (Comparison process) In the method of the present invention, the "comparison step" is a step of comparing the size of the hemolytic ring between the colonies of the test bacterium and the control bacterium formed after the culture step.

[0073] The method for comparing the size of the hemolytic ring in this step is not limited, and may be a visual comparison, or a comparison of the numerical values obtained by photographing the hemolytic ring and quantifying the size of the hemolytic ring using image analysis software.

[0074] Furthermore, the number of colonies compared in this step may be two or more, including at least one colony of the test bacterium and at least one colony of the control bacterium, and some or all of the colonies on the blood-containing solid medium may be compared. For example, this step may only compare colonies of similar size, and more specifically, comparison may be performed on colonies whose colony diameters fall within a range of ±10 to 30% of the average.

[0075] Furthermore, the specific indicator, such as a measured value, used to compare the size of the hemolytic ring in this step is not limited as long as it is an indicator of the hemolysin-producing ability of a colony. Examples include the diameter of the hemolytic ring, the ratio of the hemolytic ring diameter to the colony diameter, the difference between the hemolytic ring diameter and the colony diameter, the area of the hemolytic ring (e.g., the area of the hemolytic ring excluding the colony area; the difference between the hemolytic ring area and the colony area), and / or the ratio of the hemolytic ring area to the colony area. For example, to eliminate the influence of differences in the growth rate of bacterial strains, the diameter of the hemolytic ring may be compared between colonies of similar size (e.g., colonies whose colony diameter falls within a range of ±10 to 30% of the average). Alternatively, the ratio of the hemolytic ring diameter to the colony diameter may be compared for all colonies on the blood-containing solid medium. The diameters of hemolytic rings and colonies can be measured by any method, including, for example, performing grayscaling on an image containing hemolytic rings and colonies using image analysis software such as Image J, setting a specific threshold value so that colonies or hemolytic rings are selected, and then performing binarization, assuming that the binarized figure is a perfect circle and calculating the perimeter by pi (π). Alternatively, the diameter of the hemolytic ring or colony can be determined by the diameter of a circle fitted to the binarized figure. Alternatively, the distance between the two most distant points on the binarized figure or the maximum length of a straight line contained in the binarized figure can be determined as the diameter of the hemolytic ring or colony. The area can be measured by any method, including, for example, performing binarization in the same manner as above and determining the area of a circle fitted to the binarized figure as the area of the hemolytic ring or colony. Alternatively, the area of the hemolytic ring or colony can be determined by quantifying the number of pixels in the image whose brightness is above or below a threshold.

[0076] The method for comparing hemolytic rings based on measured values is not limited, and examples include a method in which a cutoff value is determined based on the measured value of the hemolytic rings of a control bacterium, and the hemolytic rings of a test bacterium are determined to be large based on the cutoff value.

[0077] The cutoff value refers to a boundary value for classifying a measured value as high or low. The cutoff value can usually be calculated based on the sensitivity and specificity calculated from an ROC curve, but the method for setting the cutoff value is not particularly limited in this specification.

[0078] For example, if the average value of the measurement values of the hemolytic rings of the control bacteria is used as a cutoff value, and the measurement value of the hemolytic rings of the test bacteria is higher than the cutoff value, the hemolytic rings of the test bacteria can be determined to be large.

[0079] Alternatively, the cutoff value can be set to 1.5 times or more, 2.0 times or more, 3.0 times or more, 4 times or more, 5 times or more, or 6 times or more the average value of the hemolytic ring measurement value of the control bacteria, and when the measurement value of the hemolytic ring of the test bacteria is higher than the cutoff value, the hemolytic ring of the test bacteria can be determined to be large.

[0080] Alternatively, the measured values obtained from the control group can be classified by percentile, and the percentile value used for the classification can be used as the cutoff value. For example, if the 95th percentile of the measured values of the hemolytic rings of the control bacteria is used as the cutoff value and values above that value are considered large, then if the measured value of the hemolytic rings of the test bacteria is above the 95th percentile, the hemolytic rings of the test bacteria can be determined to be large.

[0081] In this step, the size of the hemolytic rings is compared between colonies of the test bacteria and the control bacteria. If the hemolytic ring of the test bacteria is larger than that of the control bacteria, it indicates that the hemolysin-producing ability of the test bacteria is higher than that of the control bacteria. Conversely, if the hemolytic ring of the test bacteria is smaller than that of the control bacteria, it indicates that the hemolysin-producing ability of the test bacteria is lower than that of the control bacteria. Furthermore, the colony with the largest hemolytic ring size among multiple test bacteria is indicated to have the highest hemolysin-producing ability.

[0082] (Recovery process) In the method of the present invention, the "recovery step" refers to a step of recovering test bacteria from colonies determined to have large hemolytic rings in the comparison step. The test bacteria recovered in this step are bacteria with high hemolysin-producing ability, and can be further cultured or stored.

[0083] In one embodiment of the evaluation method of this aspect, the test bacterium is a strain obtained by genetic manipulation of a control bacterium. As used herein, "genetic manipulation" refers to any genetic modification. The target region of genetic modification is not limited, and may be, for example, a protein-coding region, an intragenic region such as a 5'-untranslated region or a 3'-untranslated region, or an intergenic region such as a promoter region or an enhancer region. The genetic modification method is also not limited, and examples include gene knockout, gene knock-in, random mutagenesis (described below), gene editing (genome editing), introduction of a genetic vector such as a plasmid, and introduction of an antisense nucleic acid. Note that, as used herein, "introduction" of a gene may refer to any of the genetic modifications described above, such as introduction into the genome or introduction of a genetic vector such as a plasmid, or a combination thereof. For example, the test bacterium may be a strain into which a cyclic lipopeptide synthase gene has been introduced into its genome and a plasmid containing a modification enzyme gene has been introduced. Alternatively, the test bacterium may be a strain into which a modification enzyme gene has been introduced into its genome and a plasmid containing a cyclic lipopeptide synthase gene has been introduced.

[0084] In one embodiment, the test bacterium and / or the control bacterium are derived from a strain (e.g., a hemolysin-producing bacterium) after random mutagenesis. As used herein, "random mutagenesis" refers to randomly inducing mutations in the entire genome or a specific genome region, or in the entire vector or a specific vector sequence region. For example, mutagen treatment can randomly induce mutations throughout the genome. The specific method of mutagen treatment is not limited, but examples include irradiation with electromagnetic waves or radiation such as X-rays or ultraviolet rays, contact with mutagens such as nitrosoguanidine, nitrosamine, bromodeoxyuridine, N-ethyl-N-nitrosourea, methyl ethanesulfonate, benzopyrene, and ethidium bromide, and treatment using viruses, transposons, or the like to introduce nucleic acids into random positions in the genome. Mutation induction using these mutagens can be performed by known methods. Mutagen treatment induces point mutations, deletion mutations, insertion mutations, and other mutations. Furthermore, error-prone PCR involves amplifying specific genomic regions (such as target genes or promoter regions) or vector sequence regions to introduce mutations, and then transforming the amplified products to induce random mutations limited to those specific genomic regions or vector sequence regions.

[0085] In a further embodiment, the test bacterium and / or the control bacterium are derived from a mutant library for a specific base or a specific region on the genome. The term "mutant library for a specific base" as used herein refers to a group of mutants in which specific bases in a gene promoter region or a protein-coding region that may affect hemolysin-producing ability have been mutated, preferably comprehensively, to bases other than the wild-type base. The term "mutant library for a specific region" also refers to a group of mutants in which a base sequence consisting of two or more bases that may affect hemolysin-producing ability has been mutated, preferably comprehensively, to a sequence other than the wild-type base sequence. Specific examples include a group of mutants in which a specific codon has been substituted, preferably comprehensively, with a codon encoding a different amino acid; and a group of mutants in which each amino acid residue in the entire amino acid sequence or a specific domain constituting a protein has been substituted, preferably comprehensively, with a different amino acid (e.g., a specific amino acid residue such as an alanine residue, or a corresponding amino acid residue in an orthologous protein). Further specific examples include a group of mutants in which specific bases in the promoter region are mutated to bases other than the wild-type base, and a group of mutants in which mutations such as addition, deletion, and / or substitution have been introduced into the sequences constituting the promoter region, for example, consensus sequences in the -35 region and / or the -10 region, or sequences between the consensus sequences. A mutant library for a specific base or a specific region can be efficiently constructed by using, for example, site-directed mutagenesis methods such as the QuikChange method.

[0086] 1-4.Effects According to the evaluation method of this embodiment, the hemolysin-producing ability of a test bacterium (e.g., a hemolysin-producing test bacterium) can be efficiently evaluated. For example, by comparing the hemolysin-producing ability of a bacterial strain before and after a specific genetic manipulation, the effect of the genetic manipulation on the hemolysin-producing ability can be evaluated.

[0087] 2. Screening method for mutant strains with enhanced hemolysin production ability 2-1. Overview A second aspect of the present invention is a method for screening mutant strains with enhanced hemolysin-producing ability derived from hemolysin-producing bacteria (hereinafter, sometimes abbreviated as "screening method for mutant strains with enhanced hemolysin-producing ability" or "screening method"). The screening method of this aspect comprises, as essential steps, a mutation introduction step, a first inoculation step, a single colony formation step, a second inoculation step, a culture step, and an identification step. According to the screening method of this aspect, it is possible to efficiently identify strains with high hemolysin-producing ability from among a large number of candidate strains, and to efficiently create mutant strains with enhanced hemolysin-producing ability.

[0088] 2-2. Method The screening method of this embodiment includes, as essential steps, a mutation introduction step, a first inoculation step, a single colony formation step, a second inoculation step, a culture step, and an identification step, and includes, as an optional step, a recovery step. Each step in the screening method of this embodiment will be specifically described below.

[0089] (Mutation introduction step) In the screening method of the present invention, the "mutation introduction step" refers to a step of introducing a mutation into a hemolysin-producing bacterium. The mutagenesis method used in this step is not limited as long as it is a method that can generate candidate mutant strains with enhanced hemolysin-producing ability.

[0090] In this embodiment, the hemolysin-producing bacterial strain to be subjected to mutation introduction may be a wild-type strain or a mutant strain, and may be, for example, a known hemolysin-producing bacterium, a strain obtained by subjecting a known hemolysin-producing bacterium to arbitrary genetic manipulation or random mutagenesis, or a hemolysin-producing bacterium derived from a non-hemolysin-producing bacterium such as Escherichia coli (for example, a strain obtained by introducing a gene or operon encoding a hemolysin synthase such as a cyclic lipopeptide synthase gene into a non-hemolysin-producing bacterium, and which has been confirmed to produce hemolysin as needed, or a strain obtained by subjecting such a strain to arbitrary genetic manipulation or random mutagenesis, for example, a strain into which a modification enzyme gene has been additionally introduced). For example, by subjecting a mutant strain having higher hemolytic activity than the wild-type strain to mutation introduction, it is also possible to screen for a mutant strain with even enhanced hemolysin-producing ability.

[0091] In this embodiment, an example of a hemolysin-producing bacterium to be subjected to mutation introduction is a strain obtained by introducing a mycostilin synthase gene and / or a modification enzyme gene into Bacillus subtilis 168. This strain may be one that produces only mycostilin by replacing the promoter region that controls the expression of the mycostilin synthase gene with a high-expression promoter and / or disrupting surfactin synthase.

[0092] Another example is a strain obtained by introducing the iturin synthase gene and / or modifying enzyme genes into Bacillus subtilis 168. This strain may be one that produces iturin alone by replacing the promoter region that controls the expression of the iturin synthase gene with a high-expression promoter and / or disrupting the surfactin synthase. Furthermore, the strain may be one in which the fatty acid chain composition of the iturin produced has been modified by introducing a mutation, such as an amino acid substitution mutation, into the region of the iturin synthase gene that encodes the acyl ligase domain.

[0093] Another example is a strain obtained by introducing a modification enzyme gene into Bacillus subtilis 168. The strain may be a strain to which any genetic modification has been added in order to improve surfactin productivity.

[0094] In one embodiment, the mutagenesis in this step is random mutagenesis, which can be carried out by, for example, mutagen treatment or error-prone PCR.

[0095] In another embodiment, this step involves constructing a mutant library for a specific base or a specific region on the genome. To construct the mutant library, a site-directed mutagenesis method such as the QuikChange method can be used.

[0096] (1st inoculation process) In the screening method of the present invention, the "first inoculation step" is a step of inoculating the hemolysin-producing bacteria after the mutation introduction step onto a solid medium.

[0097] This step can be performed by inoculating the hemolysin-producing bacteria into which the mutations have been introduced after the above-mentioned mutagenesis step directly, or after culturing as necessary, onto the surface of a solid medium. For example, a bacterial solution diluted to an appropriate bacterial cell concentration so that several to several hundred colonies appear after culturing can be applied over a wide area or the entire surface of the solid medium using a spreader or the like. Alternatively, single colony isolation can be performed by placing the hemolysin-producing bacteria strain into which the mutations have been introduced after the above-mentioned mutagenesis step on a solid medium and then spreading it by streaking using an inoculation loop or toothpick.

[0098] (Single colony formation process) In the screening method of the present invention, the "single colony formation step" refers to a step of culturing the hemolysin-producing bacteria after the first inoculation step to form multiple single colonies. The purpose of this step is to isolate clones containing the same mutation after the above-mentioned mutagenesis step as candidate strains for mutants with enhanced hemolysin-producing ability, so that they can be inoculated separately in the second inoculation step described below. The culture method, medium, culture temperature, culture time, etc. used in this step are similar to those in the single colony formation step of the first embodiment, and detailed explanations thereof will be omitted here.

[0099] (Second inoculation process) In the screening method of the present invention, the "second inoculation step" refers to a step in which bacterial cells are collected from multiple single colonies after the single colony formation step and inoculated onto a blood-containing solid medium at regular intervals. The specific configuration of this step is similar to the configuration of the inoculation step of the first embodiment, and detailed description thereof will be omitted here.

[0100] In one embodiment, in this step, the hemolysin-producing bacterial strain is inoculated in a lattice pattern. The lattice may be, for example, an orthorhombic, hexagonal, square, rectangular, or parallelepiped lattice, and is preferably an orthorhombic or square lattice. Each inoculation position has a constant distance from its nearest neighbor, and this distance may be, for example, 3 mm or more, 4 mm or more, 5 mm or more, 6 mm or more, 7 mm or more, 8 mm or more, 9 mm or more, 10 mm or more, 11 mm or more, 12 mm or more, 13 mm or more, 14 mm or more, or 15 mm or more, and / or 50 mm or less, 40 mm or less, 35 mm or less, 30 mm or less, 25 mm or less, 20 mm or less, or 18 mm or less, and preferably 10 to 30 mm. The number of inoculation positions in a lattice pattern is not limited as long as it is two or more, and may be, for example, 2 to 480, 4 to 384, 8 to 288, 16 to 192, 32 to 96, or 48. For example, an orthorhombic lattice or a square lattice can be composed of 48 or more points.

[0101] In a further embodiment, in this step, the hemolysin-producing bacteria are inoculated in a rhombic or square lattice pattern with 10 to 30 mm intervals. The number of points constituting the rhombic or square lattice is, for example, 12 or more, 24 or more, or 48 or more.

[0102] The strains inoculated onto the blood-containing solid medium in this step may all be hemolysin-producing bacteria after the above-mentioned mutagenesis step, or a control strain may be inoculated onto the same medium. The control strain may be the strain before mutagenesis in the above-mentioned mutagenesis step and / or an existing mutant strain with enhanced hemolysin-producing ability that serves as a standard for evaluating the degree of enhancement of hemolysin-producing ability.

[0103] (Culture process) In the screening method of the present invention, the "culturing step" refers to a step of culturing the bacterial cells after the second inoculation step. The specific configuration of this step is similar to the configuration of the culturing step of the first embodiment, and detailed description thereof will be omitted here.

[0104] (Identification step) In the screening method of the present invention, the "identification step" refers to a step of comparing the size of hemolytic rings among multiple colonies of hemolysin-producing bacteria formed after the culture step, and identifying a bacterial strain contained in a colony having a larger hemolytic ring than other colonies as a mutant strain with enhanced hemolysin-producing ability. The comparison method in this step is similar to the comparison method in the comparison step of the first embodiment, and detailed explanations thereof will be omitted here.

[0105] In one embodiment, this step involves comparing the diameter of the hemolytic ring and / or the ratio of the diameter of the hemolytic ring to the diameter of the colony between colonies, and identifying the strain of the colony whose diameter and / or ratio is larger than that of other colonies as the mutant strain with enhanced hemolysin production ability.

[0106] Alternatively, this step can be performed on colonies excluding those whose growth rate has decreased and / or increased. For example, the diameter of the hemolytic ring may be compared between colonies of similar size (e.g., colonies whose colony diameter falls within ±10 to 30% of the average).

[0107] (Recovery process) In the screening method of the present invention, the "recovery step" refers to a step of recovering bacterial cells from colonies identified as mutant strains with enhanced hemolysin-producing ability in the identification step. The hemolysin-producing test bacteria recovered in this step can be further cultured or stored.

[0108] The mutant strain with enhanced hemolysin-producing ability recovered in this step may be further confirmed as a hemolysin-producing test bacterium by the evaluation method of the first aspect to determine whether it has enhanced hemolysin-producing ability, or by using other known methods for measuring hemolytic activity.

[0109] Alternatively, mutant strains with enhanced hemolysin-producing ability recovered in this step can be used as a starting strain and the above-mentioned steps from mutagenesis to identification can be repeated to screen for mutant strains with enhanced hemolysin-producing ability. The number of repetitions may be, for example, once, twice, or three or more times.

[0110] 2-3.Effects The screening method of this embodiment allows the hemolysin-producing abilities of many candidate strains to be compared simultaneously on the same blood-containing solid medium, and allows efficient identification of mutant strains with enhanced hemolysin-producing ability, and therefore has higher throughput than conventional methods of measuring the hemolytic activity of candidate strains one by one. [Example]

[0111] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0112] Comparative Example 1: Evaluation of hemolysin-producing ability of mycostilin-producing strains inoculated onto a medium (the purpose) The culture medium of the mycostilin-producing strain is inoculated onto sheep blood agar medium to form single colonies, and the colony diameter and hemolytic ring diameter of each single colony are measured.

[0113] (Methods and Results) A glycerol stock of the mycostilin-producing strain was inoculated into LB liquid medium (10 g / L Tryptone (Becton Dickinson and Company), 5 g / L Yeast Extract (Becton Dickinson and Company), 5 g / L sodium chloride; the same composition of LB liquid medium is used in the following examples) and cultured overnight at 37°C with shaking at 200 rpm. This culture was diluted 100,000 times and 100 μL was spread onto sheep blood agar medium (1 g / L Nutrient Broth (Becton Dickinson and Company), 1 g / L Yeast Extract (Becton Dickinson and Company), 40 g / L L D(+)-glucose, 3 g / L sodium chloride, 40 mL / L defibrinated sheep blood, 15 g / L agar; the same composition of sheep blood agar is used in the following examples) prepared in an 8.6 cm round Petri dish and cultured at 35°C for 48 hours (Figure 1A). The defibrinated sheep blood used in the sheep blood agar medium was Japan Bioserum Co., Ltd. (product number 027-00313-01). After incubation, hemolytic rings formed around the colonies, indicating hemolysis of the blood components in the medium due to hemolysin released by the mycostilin-producing strain (Figure 1B, Figure 2A). The colony diameter and hemolytic ring diameter were measured for a total of 48 colonies (Figure 2C), and the mean, standard deviation, and coefficient of variation of each measurement were calculated.

[0114] The results are shown in Figure 2C. The mean ± standard deviation of the colony diameter was 0.26 ± 0.06 cm, with a coefficient of variation (CV) of 21.69%. The mean ± standard deviation of the hemolytic ring diameter was 0.47 ± 0.09 cm, with a coefficient of variation (CV) of 19.44%.

[0115] The above results showed that the coefficient of variation for both colony diameter and hemolytic ring diameter reached approximately 20%. This result is thought to be due to the fact that the method of inoculating culture medium to form colonies does not control the distance between colonies, so colonies are small in areas where colonies form densely on solid medium and large in areas where colonies form sparsely, resulting in unstable colony size. Using this method, it was found to be difficult to compare the amount of mycostilin produced by each colony based on the size of the hemolytic ring.

[0116] Example 1: Evaluation of hemolysin-producing ability of mycostilin-producing strains inoculated in a grid pattern (the purpose) The mycostilin-producing strain is inoculated onto a sheep blood agar medium in a grid pattern, and the colony diameter and hemolytic ring diameter of each colony obtained after incubation are measured.

[0117] (Methods and Results) A glycerol stock of the same mycostilin-producing strain used in Comparative Example 1 was inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 200 rpm. This culture was diluted 100,000-fold, and 100 μL was spread onto NY agar medium (1 g / L Nutrient Broth (Becton, Dickinson and Company), 1 g / L Yeast Extract (Becton, Dickinson and Company), 40 g / LD(+)-glucose, 3 g / L sodium chloride, 15 g / L agar) prepared in an 8.6 cm round Petri dish and cultured at 35°C for 24 hours. Colonies formed on the NY agar medium were arrayed and inoculated onto sheep blood agar medium prepared in a 9.9 cm x 12.2 cm square Petri dish using a colony picker (QPix460, Molecular Devices). For alignment, 48 pins (Molecular Devices, X4006G), half of the 96 pins arranged at approximately 9 mm intervals in all directions, were used. The 48 pins were aligned at 13 mm intervals. The mycostilin-producing strains inoculated in a grid pattern were cultured at 35°C for 48 hours. After incubation, the diameters of the formed colonies and the diameters of the hemolytic rings were measured, and the mean, standard deviation, and coefficient of variation of the measured values for a total of 48 colonies were calculated.

[0118] The results are shown in Figure 2C. The mean value ± standard deviation of the colony diameter was 0.97 ± 0.05 cm, with a coefficient of variation (CV) of 5.52%. The mean value ± standard deviation of the hemolytic ring diameter was 1.25 ± 0.04 cm, with a coefficient of variation (CV) of 3.44%. The coefficients of variation for the colony diameter and hemolytic ring diameter in Example 1 were significantly reduced compared to those in Comparative Example 1. These results demonstrate that arranging the colonies in a grid pattern stabilizes colony size and reduces inter-colony variation in colony diameter and hemolytic ring diameter.

[0119] <Example 2: Evaluation of hemolysin production ability of surfactin-producing strains inoculated in a grid pattern> (the purpose) A surfactin high-producing strain (hereinafter referred to as "SF high-producing strain") and a surfactin standard strain (hereinafter referred to as "SF standard strain") are inoculated in a grid pattern onto sheep blood agar medium and cultured. After culture, the colony diameter and hemolytic ring diameter of each colony of the above two strains are measured.

[0120] (Methods and Results) (1) Measurement of surfactin production (SF production) Glycerol stocks of the SF high-producing strain and the SF standard strain were inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 300 rpm. This culture was then inoculated into a production medium (40 g / L soybean flour, 5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.18 g / L calcium chloride dihydrate, 0.025 g / L iron sulfate heptahydrate, 0.022 g / L manganese chloride tetrahydrate, and 30 g / L maltose monohydrate) and cultured at 35°C with shaking at 300 rpm for 72 hours. Surfactin was extracted from the resulting culture using methanol and subjected to the following HPLC analysis.

[0121] HPLC analysis was performed using a Shimadzu HPLC Prominence column, with an NPS ODS-IIIE 1.5 μm (4.6 × 33 mm) (EPROGEN). Liquid chromatography conditions were 10 μL of sample, 40°C column temperature, and 0.7 mL / min flow rate. The mobile phases were 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The gradient conditions for mobile phase B were 10% (0 min), 51% (5 min), 51% (20 min), 10% (20.1 min), and 10% (35 min). UV detection was performed at 205 nm. Quantitation was performed using a calibration curve prepared using a surfactin standard sample (Sigma-Aldrich).

[0122] The results of measuring SF production are shown in Figure 3. The high SF-producing strain produced approximately 1.6 times more SF than the standard SF strain.

[0123] (2) Evaluation of hemolysin production Glycerol stocks of the SF high-producing strain and the SF standard strain were inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 200 rpm. The culture was diluted 100,000-fold, and 100 μL of each was applied to NY agar plates in 8.6 cm round Petri dishes. Cultures were then cultured at 35°C for 24 hours. Bacteria were harvested from colonies formed on the NY agar plates to prepare suspensions. 2 μL of the suspension was then inoculated in a grid pattern onto sheep blood agar plates in 8.6 cm round Petri dishes. Specifically, sterile tips were attached to four of the eight tip attachment positions of an 8-channel pipettor, spaced 18 mm apart (corresponding to the center-to-center distance between adjacent colonies in Figure 4). The inoculated bacteria were cultured at 35°C for 48 hours. After culture, the colony diameters and hemolytic ring diameters of four colonies were measured for each of the SF high-producing strain and the SF standard strain, and the ratio of the hemolytic ring diameter to the colony diameter was calculated. The coefficient of variation was also calculated.

[0124] Figure 4 shows colonies of the SF high-producing strain and the SF standard strain after cultivation. Figures 5A to 5C also show the colony diameter, hemolytic ring diameter, and ratio of hemolytic ring diameter to colony diameter, as well as the coefficient of variation for each parameter. The colony diameter of the SF high-producing strain was smaller than that of the SF standard strain, but the hemolytic ring diameter and ratio of hemolytic ring diameter to colony diameter of the SF high-producing strain were significantly larger than those of the SF standard strain. Furthermore, the low coefficient of variation indicated that the hemolytic ring diameter and ratio of hemolytic ring diameter to colony diameter were stable between colonies.

[0125] <Example 3: Evaluation of hemolysin production ability of iturin-producing strains inoculated in a grid pattern> (the purpose) The high-producing iturin strain (hereafter referred to as the "ITU high-producing strain") and the standard iturin strain (hereafter referred to as the "ITU standard strain") are inoculated onto sheep blood agar medium in a grid pattern and cultured. After culture, the colony diameter and hemolytic ring diameter of each colony of the two strains are measured.

[0126] (Methods and Results) (1) Measurement of Iturin Production (ITU Production) Glycerol stocks of the ITU high-producing strain and the ITU standard strain were inoculated into LB liquid medium and cultured overnight at 37°C with shaking at 300 rpm. This culture was then inoculated into a production medium (40 g / L soybean flour, 5 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.18 g / L calcium chloride dihydrate, 0.025 g / L ferrous sulfate heptahydrate, and 0.022 g / L manganese chloride tetrahydrate) and cultured at 30°C with shaking at 300 rpm for 72 hours. Iturin was extracted from the resulting culture using methanol and subjected to HPLC analysis similar to that used to measure SF production.

[0127] Quantitation was performed by creating a calibration curve using a standard sample of iturin A (manufactured by Sigma-Aldrich).

[0128] The results of measuring ITU production are shown in Figure 6. The ITU high-producing strain showed approximately 1.8 times the ITU production of the ITU standard strain.

[0129] (2) Evaluation of hemolysin production The ITU high-producing strain and the ITU standard strain were inoculated onto sheep blood agar medium in a grid pattern and cultured in the same manner as in Example 1. The colony spacing (the center-to-center distance between diagonally adjacent colonies in Figure 7) was 13 mm. After culture, the colony diameter and hemolytic ring diameter of four selected colonies were measured, and the ratio of the hemolytic ring diameter to the colony diameter was calculated. The coefficient of variation was also calculated.

[0130] Figure 7 shows colonies of the ITU high-producing strain and the ITU standard strain after cultivation. Figures 8A to 8C show the colony diameter, hemolytic ring diameter, and ratio of hemolytic ring diameter to colony diameter, as well as the coefficient of variation for each parameter. The colony diameter of the ITU high-producing strain was comparable to that of the ITU standard strain, but the hemolytic ring diameter and ratio of hemolytic ring diameter to colony diameter of the ITU high-producing strain were significantly larger than those of the ITU standard strain. Furthermore, the low coefficient of variation indicated that the hemolytic ring diameter and ratio of hemolytic ring diameter to colony diameter were stable between colonies.

Claims

1. A method for evaluating the hemolysin-producing ability of a test bacterium, comprising the steps of: an inoculation step of inoculating a plurality of strains including the test bacterium and the control bacterium onto a blood-containing solid medium at regular intervals; a culturing step of culturing the plurality of strains after the inoculation step; and a comparison step of comparing the size of the hemolytic ring between the colonies of the test bacterium and the control bacterium formed after the culture step; The method comprising:

2. The method of claim 1 , wherein the hemolysin is a lipopeptide.

3. 3. The method of claim 2, wherein the lipopeptide is a cyclic lipopeptide.

4. The method of claim 3, wherein the cyclic lipopeptide is a surfactin family lipopeptide, an iturin family lipopeptide, a fengicin family lipopeptide, or a combination of any two or more thereof.

5. The method according to any one of claims 1 to 4, wherein the inoculation step inoculates the strain in a grid pattern.

6. The method of claim 5 , wherein the lattice is an orthorhombic lattice, a hexagonal lattice, a square lattice, a rectangular lattice, or a parallelepiped lattice.

7. The method of claim 1, wherein the regular interval is between 10 mm and 30 mm.

8. 2. The method of claim 1, wherein the blood content in the blood-containing solid medium is 1% to 10% by volume.

9. 2. The method of claim 1, wherein the glucose concentration in the blood-containing solid medium is 2% to 6%.

10. The method of claim 1, wherein the comparing step compares the diameter of the hemolytic ring, the ratio of the diameter of the hemolytic ring to the diameter of the colony, and / or the difference between the diameter of the hemolytic ring and the diameter of the colony between colonies.

11. The method of claim 1, wherein the test bacterium is a strain obtained by genetic manipulation of the control bacterium.

12. The method of claim 1, wherein the test bacterium and / or the control bacterium is derived from a hemolysin-producing bacterium after random mutagenesis.

13. The method according to claim 12, wherein the random mutation is carried out by mutagen treatment or error-prone PCR.

14. The method according to claim 1 , wherein the test bacterium and / or the control bacterium are derived from a mutant library for a specific base or a specific region on the genome.

15. A method for screening a mutant strain with enhanced hemolysin-producing ability derived from a hemolysin-producing bacterium, comprising the steps of: a mutation introduction step of introducing a mutation into the hemolysin-producing bacterium; a first inoculation step of inoculating the hemolysin-producing bacterium after the mutation introduction step onto a solid medium; a single colony formation step of culturing the hemolysin-producing bacteria after the first inoculation step to form a plurality of single colonies; a second inoculation step in which bacterial cells are collected from the plurality of single colonies after the single colony formation step and inoculated onto a blood-containing solid medium at regular intervals; a culturing step of culturing the bacterial cells after the second inoculation step; and an identification step in which the size of the hemolytic rings is compared among the colonies of the hemolysin-producing bacteria formed after the culture step, and a bacterial strain contained in a colony having a larger hemolytic ring than other colonies is identified as a mutant strain with enhanced hemolysin-producing ability; The method comprising:

16. The method of claim 15, wherein the mutagenesis is random mutagenesis.

17. The method according to claim 15, wherein the step of introducing mutations constructs a mutant library for a specific base or a specific region on the genome.

18. The method according to claim 15, wherein the second inoculation step inoculates the hemolysin-producing bacteria in a rhombic or square lattice pattern with intervals of 10 to 30 mm.

19. 19. The method of claim 18, wherein the orthorhombic lattice or the square lattice consists of 48 or more points.

20. The method described in claim 18 or 19, wherein the identification step compares the diameter of the hemolytic ring, the ratio of the diameter of the hemolytic ring to the diameter of the colony, and / or the difference between the diameter of the hemolytic ring and the diameter of the colony between colonies, and identifies the strain of the colony whose diameter of the hemolytic ring, the ratio, and / or the difference is higher than that of other colonies as the mutant strain with enhanced hemolysin production ability.

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