Iturin-containing composition

Abstract

To provide an iturin-containing composition that exhibits increased antimicrobial activity.SOLUTION: An iturin-containing composition is provided in which 20% or more of a total weight of iturin is iturin that contains a fatty acid chain having 17 or more carbon atoms.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an iturin-containing composition containing iturin with a fatty acid chain having 17 or more carbon atoms, and an iturin-containing composition that is an extract of Bacillus bacteria. [Background technology]

[0002] Biopesticides (biological pesticides) are pesticides that contain biological cultures, fermentation products, extracts, or the organisms themselves as active ingredients for plant pathogen control. In recent years, expectations have been growing for biopesticides as safe pesticides that can replace conventional synthetic chemical pesticides and reduce their impact on the environment, ecosystems, and human health.

[0003] Bacillus bacteria, which are currently being used as biopesticides, are capable of producing more than several dozen types of antibacterial substances, and the components they contain are known to be effective in controlling various plant pathogens such as bacteria and fungi.

[0004] In particular, cyclic lipopeptides produced by bacteria of the genus Bacillus are amphipathic antibacterial substances composed of a peptide moiety and a fatty acid moiety, and have the activity of destroying the membrane structure of bacteria and fungi that are plant pathogens (Non-Patent Document 1). It is known that various types of cyclic lipopeptides are produced by bacteria of the genus Bacillus, and these can be 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 moiety.

[0005] For example, cyclic lipopeptides of the iturin family (often abbreviated herein as "iturin family lipopeptides") have a structure in which seven amino acids and a β-amino fatty acid are linked in a ring, and exhibit potent antibacterial activity based on their ability to destroy the membrane structure of plant pathogens and to induce a defense response in plants (Non-Patent Document 2).

[0006] Iturin family lipopeptides are further classified into iturins, mycostilins, bacillomycins, mohavensins, etc. based on the amino acid sequence of the heptaamino acid moiety. Each of these members can be produced as a mixture of lipopeptides with different carbon numbers and branching structures in the β-amino fatty acid moiety. For example, isomers of iturin are known to have different carbon numbers in the β-amino fatty acid moiety and different branching structures.

[0007] In order to advance the application of cyclic lipopeptides to plant disease control, a method for further enhancing the antibacterial activity of cyclic lipopeptides produced by Bacillus bacteria is needed. [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] It is an object of the present invention to provide an iturin-containing composition with enhanced antibacterial activity. [Means for solving the problem]

[0010] In Bacillus bacteria, iturin family lipopeptides are synthesized by an iturin family lipopeptide synthase complex consisting of four enzyme units, including iturin family lipopeptide synthase A. Iturin family lipopeptide synthase A is known as a modular enzyme consisting of multiple domains that catalyze different reactions in the initial stages of iturin synthesis.

[0011] To achieve this, the present inventors conceived the idea that substituting the acyl ligase domain (also referred to herein as the "AL domain") of iturin A synthase A (also referred to herein as "ituA"), an iturin family lipopeptide synthase A involved in the biosynthesis of iturin A, with the AL domain of mycostylen synthase subunit A (also referred to herein as "mycA") would alter the β-amino fatty acid moiety and produce iturin A with altered antibacterial activity. Based on this idea, they introduced an ituA mutant with a substituted AL domain into the iturin A operon of Bacillus subtilis and found that iturin A with dramatically enhanced antibacterial activity compared to wild-type ituA could be produced. The proportion of fatty acid chains with 17 or more carbon atoms in the resulting iturin A was found to be significantly increased compared to iturin A produced by wild-type Bacillus subtilis, accounting for more than 20% of the total weight of iturin A. Furthermore, the inventors prepared iturin A compositions containing fatty acid chains with 17 or more carbon atoms at different ratios and evaluated their antibacterial activity. As a result, they found that the higher the ratio of fatty acid chains with 17 or more carbon atoms, the stronger the antibacterial activity.

[0012] The present invention is based on the above findings and provides the following. (1) An iturin-containing composition, wherein 20% or more of the total weight of iturin is iturin containing a fatty acid chain having 17 or more carbon atoms. (2) The composition described in (1), in which 30% or more of the total weight of iturin is iturin containing a fatty acid chain having 17 or more carbon atoms. (3) The composition described in (1), in which 50% or more of the total weight of iturin is iturin containing a fatty acid chain having 17 or more carbon atoms. (4) The composition according to any one of (1) to (3), wherein the number of carbon atoms is 17. (5) The composition according to any one of (1) to (3), which contains iturin containing a fatty acid chain having 18 carbon atoms. (6) An iturin-containing composition, wherein 1% or more of the total weight of iturin is iturin containing a fatty acid chain having 18 carbon atoms. (7) The composition according to any one of (1) to (6), which is an extract of a bacterium belonging to the genus Bacillus. (8) The composition according to any one of (1) to (6), which is a culture solution or a supernatant thereof, a precipitate derived from the culture solution or a suspension thereof, a disruption solution or a supernatant thereof, or a precipitate derived from the disruption solution or a suspension thereof, of a Bacillus bacterium. (9) A method for controlling plant diseases, comprising a contacting step of contacting a target plant with the composition according to any one of (1) to (8). [Effects of the Invention]

[0013] According to the present invention, there is provided an iturin-containing composition with enhanced antibacterial activity. [Brief explanation of the drawings]

[0014] [Figure 1] The structure of the iturin family lipopeptide operon is shown in Figure 1A. Figure 1B shows the structure of the iturin A operon (ituO-ituAL) containing ituAL. Figure 1C shows the structure of the iturin A operon (ituO-mycAL) in which ituAL has been replaced with mycAL. [Figure 2] FIG. 1 shows the fatty acid chain length composition of iturin A produced using each mutant strain, such as the mycAL strain, the ituAL strain, and the F208A strain. In the figure, C14 to C19 indicate the number of carbon atoms contained in the fatty acid chain. [Figure 3]This figure shows the results of analyzing the fatty acid chain length (C14 to C19) composition of iturin A in each iturin A solution. Iturin A solution (mycAL), iturin A solution (75:25), iturin A solution (50:50), iturin A solution (25:75), and iturin A solution (ituAL) were analyzed. In the figure, C14 to C19 indicate the number of carbon atoms contained in the fatty acid chain. [Figure 4] This figure shows the results of evaluating the antibacterial activity of each iturin A solution. The horizontal axis shows the concentration of iturin A added to the culture medium when culturing the test bacteria. The vertical axis shows the OD620 measured in the culture medium after culturing. DETAILED DESCRIPTION OF THE INVENTION

[0015] 1. Mutant iturin family lipopeptide synthase A or an active fragment thereof Overview A first aspect of the present invention is a mutant iturin family lipopeptide synthetase A or an active fragment thereof. The mutant iturin family lipopeptide synthetase A or an active fragment thereof of the present invention comprises an acyl ligase domain, and in this acyl ligase domain, the amino acid residue corresponding to position 208 in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than Phe, Arg, Trp, or Tyr. An iturin family lipopeptide synthetase complex comprising the mutant iturin family lipopeptide synthetase A of this aspect or an active fragment thereof can efficiently produce iturin family lipopeptides containing a fatty acid chain having 17 or more carbon atoms.

[0016] 1-2.Definition of Terms The following terms frequently used in this specification are defined below. 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. In cyclic lipopeptides, at least a portion of the ring structure typically consists of a peptide chain, and at least a portion of the fatty acid chain may protrude from the ring structure. Examples of cyclic lipopeptides include cyclic lipopeptides in which a fatty acid chain is bonded to the side chain of the peptide chain constituting the ring structure, and cyclic lipopeptides in which a portion of the fatty acid chain is linked to a ring structure in which the other portion of the fatty acid chain protrudes from the ring structure. Examples of cyclic lipopeptides that can be produced by bacteria of the genus Bacillus include the iturin family lipopeptides, the surfactin family lipopeptides (e.g., esperin, lichenysin, pumilacidin, surfactin), and the fengycin family lipopeptides (e.g., fengycin A, fengycin B, plipastatin A, plipastatin B). 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.

[0017] 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. In this specification, the seven amino acid residues in a heptapeptide contained in an iturin family lipopeptide (hereinafter referred to as an "iturin family heptapeptide") are designated as positions 1 to 7, starting from the N-terminus, with the N-terminal amino acid residue bonded to the carbonyl group of the β-amino fatty acid being designated position 1. Therefore, the C-terminal amino acid residue at position 7 of the heptapeptide is bonded to the β-amino group of the β-amino fatty acid. In addition, in iturin family lipopeptides, three carbon atoms of the β-amino fatty acid form a ring structure with the heptapeptide. The remaining portion of the β-amino fatty acid moiety protrudes outside the ring structure, and isomers of this branched structure exist, including linear (n), isoform (i), and anteisoform (ai). In this specification, when it is said that an iturin family lipopeptide "contains a fatty acid chain with N or more carbon atoms," it is understood that three of the N carbon atoms are contained in the ring structure of the iturin family lipopeptide, and the remaining (N-3) or more carbon atoms protrude outside the ring structure.

[0018] As used herein, "iturin" refers to an iturin family lipopeptide having the amino acid sequence L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Ser (SEQ ID NO: 3) 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: 3. L Iturin C, which contains the amino acid sequence shown in SEQ ID NO: 16 (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: 17 (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. The number of carbon atoms in the fatty acid chain contained in iturin is not limited herein. It is known that the majority of iturins produced by wild-type Bacillus subtilis have fatty acid chains with 14 to 16 carbon atoms, and that iturins with fatty acid chains with 17 or more carbon atoms are rarely contained.

[0019] Iturin A and Iturin A containing fatty acid chains of carbon number N L The structure of iturin C, which contains a fatty acid chain with N carbon atoms, is shown in formula (II) below, and the structure of iturin W, which contains a fatty acid chain with N carbon atoms, is shown in formula (III) below. In each iturin, three of the N carbon atoms contained in the β-amino fatty acid form a ring structure together with the heptapeptide, and the remaining carbon atoms (N-3) are located outside the ring structure.

[0020] [ka] [ka] [ka]

[0021] Furthermore, the present specification does not limit the branched structure of the fatty acid chain contained in iturin. Known branched structures of the fatty acid chain contained in iturin include linear (n), isoform (i), and anteisoform (ai).

[0022] As used herein, "mycosubtilin" refers to an iturin family lipopeptide having the amino acid sequence L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Ser-L-Asn (SEQ ID NO: 4). The number of carbon atoms in the fatty acid chain contained in mycosubtilin is not limited herein. The branched structure of the fatty acid chain is also not limited, and may be, for example, a linear type (n), an isotype (i), or an anteisotype (ai). The structure of mycosubtilin containing a fatty acid chain with N carbon atoms is shown in formula (IV) below.

[0023] [ka]

[0024] As used herein, "bacillomycin" includes bacillomycin D, which has the amino acid sequence L-Asn-D-Tyr-D-Asn-L-Pro-L-Glu-D-Ser-L-Thr (SEQ ID NO: 5); bacillomycin F, which has the amino acid sequence L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Thr (SEQ ID NO: 6); bacillomycin L, which has the amino acid sequence L-Asp-D-Tyr-D-Asn-L-Ser-L-Gln-D-Ser-L-Thr (SEQ ID NO: 7); and bacillomycin LC (also known as bacillopeptin), which has the amino acid sequence L-Asn-D-Tyr-D-Asn-L-Ser-L-Glu-D-Ser-L-Thr (SEQ ID NO: 8). As used herein, the number of carbon atoms in the fatty acid chain contained in bacillomycin is not limited. Furthermore, the branched structure of the fatty acid chain is not limited, and may be, for example, a linear type (n), an isotype (i), or an anteisotype (ai).

[0025] As used herein, "mojavensin" refers to an iturin family lipopeptide having the amino acid sequence L-Asn-D-Tyr-D-Asn-L-Gln-L-Pro-D-Asn-L-Asn (SEQ ID NO: 18). The number of carbon atoms in the fatty acid chain contained in mojavensin is not limited herein. The branched structure of the fatty acid chain is also not limited, and may be, for example, a linear (n), isoform (i), or anteisoform (ai).

[0026] As used herein, the term "iturin family lipopeptide synthase complex" refers to a complex that is essentially composed of four enzyme units: iturin family lipopeptide synthase A, acyl carrier protein-S-malonyltransferase, iturin family lipopeptide synthase B, and iturin family lipopeptide synthase C, and that can catalyze the biosynthesis of iturin family lipopeptides. In the biosynthesis of iturin family lipopeptides by the iturin family lipopeptide synthase complex, the three enzyme units, iturin family lipopeptide synthase A, iturin family lipopeptide synthase B, and iturin family lipopeptide synthase C, catalyze the incorporation of amino acid residues at positions 1, 2-5, and 6-7, respectively, in the heptapeptide that constitutes the iturin family lipopeptide. In the wild-type Bacillus subtilis genome, the four ORFs encoding these four enzyme units constitute a single operon (referred to herein as the "iturin family lipopeptide operon"). In this specification, each of the four enzyme units constituting an iturin family lipopeptide synthetase complex is not limited to a full-length enzyme protein, but may be a fragment (active fragment) having the activity of synthesizing an iturin family lipopeptide. Furthermore, the combination of the four enzyme units constituting an iturin family lipopeptide synthetase complex is not limited to a combination constituting a native complex (hereinafter referred to as a "non-chimeric iturin family lipopeptide synthetase complex"), but may also be a combination containing at least one enzyme unit corresponding to a different iturin family lipopeptide (hereinafter referred to as a "chimeric iturin family lipopeptide synthetase complex"). Examples of non-chimeric iturin family lipopeptide synthetase complexes include the iturin synthetase complex, mycostilin synthetase complex, bacillomycin synthetase complex, and mohavencin synthetase complex.Examples of chimeric iturin family lipopeptide synthetase complexes include hybrid iturin family lipopeptide synthetase complexes containing one to three enzyme units constituting an iturin synthase complex and at least one enzyme unit constituting a mycostylen synthase complex. In this specification, an iturin family lipopeptide synthetase complex composed of wild-type enzyme units is referred to as a "natural iturin family lipopeptide synthetase complex," and an iturin family lipopeptide synthetase complex containing a mutant enzyme unit and the above-mentioned chimeric iturin family lipopeptide synthetase complex are collectively referred to as a "non-natural iturin family lipopeptide synthetase complex."

[0027] As used herein, "iturin family lipopeptide synthase A" refers to an enzyme that catalyzes a series of reactions that produce an amino acid thioester linked to a β-amino fatty acid during the biosynthesis of iturin family lipopeptides. Iturin family lipopeptide synthase A is classified as K15661 in the ORTHOLOGY section of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and contains an acyl ligase domain (AL domain), an acyl carrier protein domain (ACP domain), a β-ketoacyl synthase domain (KS domain), an aminotransferase domain (AMT domain), a condensation domain (C domain), a peptidyl carrier protein domain (PCP domain), and an adenylation domain (A domain). The series of reactions catalyzed by iturin family lipopeptide synthase A in the biosynthesis of iturin family lipopeptides can be explained using iturin A synthetase A (ituA) as an example. First, a fatty acid activated by the AL domain of ituA binds to a 4-phosphopantetheine cofactor bound to the N-terminal ACP domain of ituA to generate an acyl thioester. Second, an asparagine activated by the A domain of ituA binds to a 4-phosphopantetheine cofactor bound to the PCP domain of ituA to generate an asparagine thioester. Finally, malonyl-CoA binds to a 4-phosphopantetheine cofactor bound to the C-terminal ACP domain of ituA to generate a malonyl thioester (this reaction is catalyzed by acyl carrier protein S-malonyltransferase, as described below). Condensation of the malonyl thioester with the acyl thioester is then catalyzed by the KS domain of ituA to generate a β-ketoacyl thioester, which is converted to a β-amino fatty acid by a transamination reaction catalyzed by the AMT domain of ituA.Next, the β-amino fatty acid binds to the PCP domain through the activity of the C domain of ituA. Finally, the β-amino fatty acid binds to an asparagine thioester through the activity of the C domain of ituA. The asparagine thioester with the β-amino fatty acid bound thereto, produced by ituA, is linked to the remaining six amino acids by the action of other enzyme units of the iturin A synthase complex to produce iturin A. Specific examples of iturin family lipopeptide synthase A include ituA, mycA, and bmyA, which will be described later.

[0028] As used herein, the term "mutant iturin-family lipopeptide synthetase A" refers to a mutant polypeptide derived from iturin-family lipopeptide synthetase A of any biological species. The amino acid sequence of the mutant iturin-family lipopeptide synthetase A has at least one mutation relative to the amino acid sequence of wild-type iturin-family lipopeptide synthetase A. The mutation may be, for example, an addition, deletion, and / or substitution of an amino acid. Examples of mutant iturin-family lipopeptide synthetase A include mutant ituA, mutant mycA, and mutant bmyA.

[0029] As used herein, the term "iturin synthase complex" refers to a complex that is composed of four enzyme units, i.e., iturin synthase D, iturin synthase A, iturin synthase B, and iturin synthase C, and that can catalyze the biosynthesis of iturin. For example, the iturin A synthase complex is composed of four enzyme units, i.e., iturin A synthase D (ituD), iturin A synthase A (ituA), iturin A synthase B (ituB), and iturin A synthase C (ituC), and is a complex that can catalyze the biosynthesis of iturin A. In the biosynthesis of iturin A, the three enzyme units, i.e., iturin A synthase A, iturin A synthase B, and iturin A synthase C, catalyze the incorporation of amino acid residues at positions 1, 2-5, and 6-7, respectively, of the heptapeptide contained in iturin (hereinafter referred to as "iturin heptapeptide" herein). The four ORFs encoding iturin synthase D, iturin synthase A, iturin synthase B, and iturin synthase C are known to form a single operon (herein referred to as the "iturin operon") in the Bacillus subtilis genome; for example, the iturin A operon is composed of four ORFs encoding iturin A synthase D, iturin A synthase A, iturin A synthase B, and iturin A synthase C.

[0030] As used herein, "iturin A synthetase A (ituA)" refers to an enzyme that catalyzes a series of reactions that produce an asparagine thioester bound to a β-amino fatty acid in the biosynthesis of iturin A. ituA contains an AL domain, an ACP domain, a KS domain, an AMT domain, a C domain, a PCP domain, and an A domain. The full-length amino acid sequence of wild-type ituA from Bacillus subtilis is shown in SEQ ID NO: 1.

[0031] As used herein, the term "mycostilin synthase complex" refers to a complex that can catalyze the biosynthesis of mycostilin and is composed of four enzyme units: fenF, mycostilin synthase subunit A (mycA), mycostilin synthase subunit B (mycB), and mycostilin synthase subunit C (mycC). fenF is an enzyme belonging to the malonyl-CoA-acyl carrier protein transacylases, and is classified as K00645 in the KEGG ORTHOLOGY along with ituD (described above) and bmyD (described below). In the synthesis of mycostilin by the mycostilin synthase complex, the three enzyme units mycA, mycB, and mycC catalyze the incorporation of amino acid residues at positions 1, 2-5, and 6-7, respectively, of the heptapeptide contained in mycostilin (hereinafter referred to as "mycostilin heptapeptide"). In the Bacillus subtilis genome, the four ORFs encoding fenF, mycA, mycB, and mycC constitute a single operon (herein referred to as the "mycostilin operon").

[0032] As used herein, "mycosubtilin synthase subunit A (mycA)" refers to an enzyme that catalyzes a series of reactions that produce an asparagine thioester bound to a β-amino fatty acid in the biosynthesis of mycostilin. Like ituA and the like, mycA contains an AL domain, an ACP domain, a KS domain, an AMT domain, a C domain, a PCP domain, and an A domain. The full-length amino acid sequence of wild-type mycA from Bacillus subtilis is shown in SEQ ID NO:9.

[0033] As used herein, the term "acyl ligase domain (AL domain)" refers to a domain contained in the N-terminus of iturin family lipopeptide synthase A that can function in incorporating a fatty acid, which serves as a β-amino fatty acid substrate, into an iturin family lipopeptide. For example, the AL domain of ituA corresponds to the region consisting of positions 1 to 584 in the amino acid sequence shown in SEQ ID NO: 1, and the AL domain of mycA corresponds to the region consisting of positions 1 to 585 in the amino acid sequence shown in SEQ ID NO: 9. The amino acids in the AL domains of ituA and mycA are highly conserved, with amino acid similarity of 96% or more (566 a.a. / 586 a.a.) and amino acid identity of 84% or more (496 a.a. / 586 a.a.).

[0034] As used herein, "bacillomycin synthetase subunit A (bmyA)" refers to an enzyme that catalyzes a series of reactions that produce an asparagine thioester bound to a β-amino fatty acid in the biosynthesis of bacillomycin. bmyA may be an iturin-family lipopeptide synthetase A corresponding to any bacillomycin, such as bacillomycin D synthase A (KEGG T02050: BANAU 1943) corresponding to bacillomycin D. The full-length amino acid sequence of wild-type bacillomycin D synthase A from Bacillus subtilis is shown in SEQ ID NO: 22.

[0035] As used herein, the term "amino acid" encompasses both standard and non-standard amino acids, and may be either an L-amino acid or a D-amino acid. In the context of polypeptides, excluding cyclic lipopeptides, unless otherwise specified, amino acids refer to the 20 L-amino acids that normally constitute proteins (specifically, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, cysteine, glutamine, glycine, proline, tyrosine, alanine, aspartic acid, asparagine, glutamic acid, and serine). When distinguishing between L- and D-amino acids, the amino acid is designated herein by adding "L-" and "D-" to the beginning of the three-letter amino acid designation, respectively.

[0036] As used herein, the term "identity" of amino acids refers to the percentage (%) of identical amino acid residues between two amino acid sequences relative to the total amino acid residues in one amino acid sequence when the two amino acid sequences are aligned, with gaps introduced as necessary, to maximize the degree of amino acid identity between the two. Amino acid identity can be calculated using protein search systems and software (e.g., GENETYX), including BLAST and FASTA.

[0037] Bacillus bacteria produce a variety of iturin family lipopeptides depending on the species, subspecies, and strain (Dunlap CA, Bowman MJ, and Rooney AP, Frontiers in Microbiology, 2019, Vol. 10, Article 1794.).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Examples of species of Bacillus bacteria that produce mohavencin include Bacillus swezeyi, Bacillus halotolerans, Bacillus genomospecies #1, and Bacillus tequilensis.

[0042] 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.

[0043] 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.

[0044] As used herein, the term "target plant" refers to a plant to which iturin produced by the production method of the present invention described below is applied. This plant includes plants that have developed specific plant diseases due to infection with bacteria, fungi, and / or viruses, or plants that are at risk of infection with bacteria, fungi, and / or viruses.

[0045] 1-3.Configuration In the acyl ligase domain of the mutant iturin-family lipopeptide synthetase A of the present invention or an active fragment thereof, the amino acid residue corresponding to position 208 in the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than Phe, Arg, Trp, or Tyr. As used herein, "the amino acid residue corresponding to position 208 in the amino acid sequence shown in SEQ ID NO: 1" refers to the amino acid residue corresponding to position 208 in the amino acid sequence shown in SEQ ID NO: 1 in the amino acid sequence of any iturin-family lipopeptide synthetase A. This amino acid residue can be identified as the amino acid residue in the amino acid sequence of any iturin-family lipopeptide synthetase A that corresponds to position 208 in the amino acid sequence shown in SEQ ID NO: 1, for example, by aligning the amino acid sequence of any iturin-family lipopeptide synthetase A with the amino acid sequence shown in SEQ ID NO: 1 and introducing gaps as necessary to maximize the degree of amino acid identity between the two. In the acyl ligase domain of the mutant iturin-family lipopeptide synthetase A of the present invention, the amino acid residue corresponding to position 208 in the amino acid sequence shown in SEQ ID NO: 1 is specifically an Ala residue, a Cys residue, an Asp residue, a Glu residue, a Gly residue, a His residue, an Ile residue, a Lys residue, a Leu residue, a Met residue, an Asn residue, a Pro residue, a Gln residue, a Ser residue, a Thr residue, or a Val residue.

[0046] In one embodiment, in the acyl ligase domain of a mutant iturin-family lipopeptide synthetase A of the present invention, the amino acid residue corresponding to position 208 in the amino acid sequence set forth in SEQ ID NO: 1 is an amino acid residue other than Arg, Trp, Tyr, Phe, Ala, Cys, Asp, Glu, Gly, His, Asn, Pro, Ser, or Thr, and may be, for example, an Ile, Lys, Leu, Met, Gln, or Val residue.

[0047] In one embodiment, the mutant iturin-family lipopeptide synthetase A of the present invention is a mutant ituA, and in its acyl ligase domain, the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than a Phe residue, an Arg residue, a Trp residue, or a Tyr residue.

[0048] In one embodiment, the mutant iturin-family lipopeptide synthetase A of the present invention is a mutant mycA, and in its acyl ligase domain, the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than a Phe residue, an Arg residue, a Trp residue, a Leu residue, or a Tyr residue.

[0049] The mutant iturin family lipopeptide synthase A of the present invention can be derived from a wild-type iturin family lipopeptide synthase A of any biological species. The biological species from which the iturin family lipopeptide synthase A is derived is not limited as long as it is a biological species that possesses an iturin family lipopeptide synthase A. Examples of microorganisms that possess an iturin family lipopeptide synthase A include bacteria such as gram-positive bacteria. Examples of Gram-positive bacteria include the genus Bacillus, Paenibacillus, Geobacillus, Oceanobacillus, and Brevibacillus, and preferably bacteria of the genus Bacillus, such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Bacillus atrophaeus. The Bacillus bacterium may be the above-mentioned iturin-producing Bacillus bacterium, mycostilin-producing Bacillus bacterium, bacillomycin-producing Bacillus bacterium, or mohavencin-producing Bacillus bacterium. Specific examples of wild-type iturin family lipopeptide synthase A include wild-type ituA of Bacillus subtilis (for example, ituA consisting of the amino acid sequence shown in SEQ ID NO: 1) and its orthologs.

[0050] In the mutant iturin-family lipopeptide synthetase A of the present invention, the amino acid sequence other than the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1 is not limited. For example, the amino acid sequence may be identical to that of the original wild-type iturin-family lipopeptide synthetase A except for the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1, or may have additional mutations (e.g., amino acid addition, deletion, and / or substitution). For example, the mutant iturin-family lipopeptide synthetase A of the present invention may consist of an amino acid sequence that has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 1. Note that the percentages of identity exemplified above are calculated excluding the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1. Alternatively, the mutant iturin-family lipopeptide synthetase A of the present invention may consist of an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1. In Bacillus subtilis, the amino acid identity between wild-type ituA and wild-type mycA is 79%, and wild-type mycA is included in amino acid sequences that share 75% or more identity with the amino acid sequence shown in SEQ ID NO: 1.

[0051] In one embodiment, the acyl ligase domain of the mutant iturin-family lipopeptide synthetase A of the present invention consists of the acyl ligase domain of mycostylein synthase subunit A (mycA), or consists of an amino acid sequence having 85% or more identity with the amino acid sequence set forth in SEQ ID NO: 2. As described above, the amino acid identity between the AL domains of iturin-family lipopeptide synthetase A and mycA is 84% ​​or more (496 a.a. / 586 a.a.).

[0052] Also provided is an active fragment of the mutant iturin-family lipopeptide synthetase A of the present invention. The "active fragment" of the mutant iturin-family lipopeptide synthetase A of the present invention refers to a polypeptide fragment that comprises a partial region of the mutant iturin-family lipopeptide synthetase A of the present invention, including the amino acid residue corresponding to position 208 of the amino acid sequence set forth in SEQ ID NO: 1, and that retains at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or a similar or greater amount of the activity of the full-length protein. The amino acid length of the active fragment is not particularly limited as long as it retains the activity of the protein; for example, it may be a region of at least 2000, 2500, 3000, 3500, 3600, 3700, 3800, 3900, 3950, 3960, 3970, or 3980 consecutive amino acids. Specific examples of active fragments include a fragment comprising the AL domain to the A domain of the mutant iturin family lipopeptide synthetase A of the present invention, or a fragment comprising the AL domain to the PCP domain located at the most C-terminal end, and more preferably a fragment comprising the AL domain to the C domain located at the most C-terminal end.

[0053] In one embodiment, the mutant iturin-family lipopeptide synthetase A of the present invention can be selected from the group consisting of the following (i) to (iv): (i) A mutant ituA, wherein in its acyl ligase domain, the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than Phe, Arg, Trp, or Tyr (for example, an amino acid residue other than Phe, Leu, Arg, Trp, or Tyr), and the mutant ituA consists of or comprises an amino acid sequence that, excluding the amino acid residue corresponding to position 208, has 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or is 100% identical thereto. (ii) A mutant mycA, wherein in its acyl ligase domain, the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than a Phe residue, an Arg residue, a Trp residue, a Leu residue, or a Tyr residue, and the mutant mycA consists of or comprises an amino acid sequence that, excluding the amino acid residue corresponding to position 208, has 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with, or is 100% identical to, the amino acid sequence shown in SEQ ID NO: 9. (iii) A mutant bacillomycin synthase subunit A (mutant bmyA, e.g., mutant bacillomycin D synthase A), in which, in its acyl ligase domain, the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than Phe residue, Arg residue, Trp residue, or Tyr residue (e.g., an amino acid residue other than Phe residue, Leu residue, Arg residue, Trp residue, or Tyr residue), and the mutant bmyA consists of or contains an amino acid sequence that, excluding the amino acid residue corresponding to position 208, has 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with, or is 100% identical to, the amino acid sequence shown in SEQ ID NO: 22. (iv) A mutant mohavencin synthase subunit A, wherein in its acyl ligase domain, the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1 is an amino acid residue other than Phe, Arg, Trp, or Tyr (for example, an amino acid residue other than Phe, Leu, Arg, Trp, or Tyr), and the mutant mohavencin synthase subunit A consists of or comprises an amino acid sequence that, excluding the amino acid residue corresponding to position 208, has 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with, or is 100% identical to, the amino acid sequence shown in SEQ ID NO: 23.

[0054] In a further embodiment, the mutant iturin-family lipopeptide synthetase A of the present invention can be selected from the group consisting of the following (v) to (vii): (v) A mutant ituA, the acyl ligase domain of which consists of the acyl ligase domain of mycA, or consists of or contains an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 2. (vi) A mutant bmyA, the acyl ligase domain of which consists of the acyl ligase domain of mycA, or consists of or contains an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 2. (vii) A mutant mohavencin synthase subunit A, the acyl ligase domain of which consists of the acyl ligase domain of mycostylen synthase subunit A (mycA), or consists of or contains an amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2.

[0055] 1-4.Effects The mutant iturin family lipopeptide synthetase A or active fragment thereof of the present invention enables the production of iturin family lipopeptides that contain a higher proportion of fatty acid chains having 17 or more carbon atoms than wild-type iturin family lipopeptide synthetase A or active fragment thereof. For example, iturin family lipopeptides can be produced in which 20% or more of the total weight of the iturin family lipopeptides contains fatty acid chains having 17 or more carbon atoms.

[0056] For example, by constructing an iturin A synthase complex with four enzyme units, namely mutant ituA, which is the mutant iturin family lipopeptide synthase A of the present invention, iturin A synthase D (ituD), iturin A synthase B (ituB), and iturin A synthase C (ituC), it can be produced that contains a higher proportion of fatty acid chains with 17 or more carbon atoms than an iturin A synthase complex containing wild-type ituA (e.g., iturin A in which 20% or more of the total weight of iturin A contains fatty acid chains with 17 or more carbon atoms).

[0057] 2. Nucleic acid encoding mutant iturin family lipopeptide synthetase A or an active fragment thereof 2-1. Overview A second aspect of the present invention is a nucleic acid encoding a mutant iturin-family lipopeptide synthetase A or an active fragment thereof.

[0058] 2-2.Configuration The "nucleic acid encoding a mutant iturin-family lipopeptide synthetase A or an active fragment thereof" may be any nucleic acid encoding any of the mutant iturin-family lipopeptide synthetase A or active fragments thereof described in the first aspect. The nucleotide sequence of such a nucleic acid is not limited. For example, a codon-optimized nucleotide sequence or a nucleotide sequence having an initiation codon (ATG) added to the 5'-end thereof is also exemplified.

[0059] 3. Mutant iturin family lipopeptide synthase A expression vector Overview A third aspect of the present invention is a gene expression vector (herein referred to as a "mutant iturin family lipopeptide synthase A expression vector") that contains, in an expressible state, a nucleic acid encoding the mutant iturin family lipopeptide synthase A or an active fragment thereof according to the second aspect. By introducing the gene expression vector of this aspect into any cell, such as a bacterial cell or a eukaryotic cell, it is possible to produce an iturin family lipopeptide that contains a high proportion of fatty acid chains having 17 or more carbon atoms.

[0060] 3-2.Configuration The gene expression vector of this aspect comprises the nucleic acid and promoter according to aspect 2, and is capable of expressing a mutant iturin-family lipopeptide synthetase A or an active fragment thereof in a cell. In addition to the nucleic acid and promoter, which are the components, the gene expression vector may also comprise components such as a drug resistance gene, an intron, an enhancer, a terminator, a replication origin, and / or a poly(A) signal, as necessary.

[0061] As used herein, the term "gene expression vector" refers to a vector that contains a gene or a gene fragment (hereinafter referred to as "gene, etc.") in an expressible state and that includes an expression unit that can control the expression of the gene, etc. The gene expression vector may be a plasmid vector or a viral vector. The gene expression vector may be a vector that contains only the mutant iturin family lipopeptide synthetase A gene as the introduced gene, or a vector that also contains the acyl carrier protein-S-malonyltransferase gene, the iturin family lipopeptide synthetase B gene, and the iturin family lipopeptide synthetase C gene, which will be described later. These genes may form an operon on the gene expression vector.

[0062] As used herein, "in an expressible state" refers to the placement of a gene to be expressed downstream of a promoter under the control of the promoter. Known vectors include plasmid vectors and viral vectors, and any of these vectors can be used. Generally, a plasmid vector that is easy to manipulate for genetic recombination is sufficient.

[0063] The plasmid vector may be a commercially available expression vector for mammalian cells, such as the pSI vector from PROmega, or a shuttle vector that can replicate between mammalian cells and bacteria such as E. coli.

[0064] As used herein, a "promoter" refers to a gene expression regulatory region capable of controlling the expression of a gene or the like located downstream (on the 3' end) in a cell into which a gene expression vector has been introduced. Promoters are classified into constitutively active promoters, inducible promoters, and the like based on the timing of expression. Constitutively active promoters are capable of constitutively expressing a target gene or the like in cells. Inducible promoters are capable of inducing the expression of a target gene or the like in cells at any time.

[0065] In the gene expression vector of this embodiment, the promoter is a promoter that can induce the expression of a nucleic acid encoding a mutant iturin-family lipopeptide synthetase A or an active fragment thereof in cells.

[0066] A "drug resistance gene" is a gene that encodes a drug resistance protein that confers resistance to a drug, such as an antibiotic, added to a culture medium, etc. Examples include genes that encode β-lactamase, which confers resistance to ampicillin, aminoglycoside 3'-phosphotransferase, which confers resistance to kanamycin, tetracycline efflux transporter, which confers resistance to tetracycline, and CAT (chloramphenicol acetyltransferase), which confers resistance to chloramphenicol.

[0067] As used herein, the term "enhancer" is not particularly limited as long as it can enhance the expression efficiency of a gene or a fragment thereof in a vector.

[0068] As used herein, a "terminator" refers to a sequence that can terminate transcription of a gene or the like expressed by the activity of the promoter. The type of terminator is not particularly limited. Preferably, the terminator is derived from the same organism as the promoter. Particularly preferred is a terminator that is paired with the promoter on the genome in a single gene expression control system.

[0069] 4. Non-natural iturin family lipopeptide synthase complex Overview A fourth aspect of the present invention is a non-naturally occurring iturin family lipopeptide synthase complex.

[0070] 4-2.Configuration In the non-natural iturin family lipopeptide synthetase complex of this embodiment, the amino acid residue corresponding to position 208 in the amino acid sequence set forth in SEQ ID NO: 1 in the acyl ligase domain contained in iturin family lipopeptide synthetase A or an active fragment thereof is an amino acid residue other than Phe, Arg, Trp, or Tyr. The non-natural iturin family lipopeptide synthetase complex of this embodiment essentially contains three constituent units as enzyme units other than iturin family lipopeptide synthetase A or an active fragment thereof: acyl carrier protein-S-malonyltransferase or an active fragment thereof, iturin family lipopeptide synthetase B or an active fragment thereof, and iturin family lipopeptide synthetase C or an active fragment thereof. Each enzyme unit may be either wild-type or mutant. Furthermore, the "active fragment" of each enzyme unit other than iturin family lipopeptide synthetase A or an active fragment thereof is configured as described above for the active fragment of iturin family lipopeptide synthetase A.

[0071] As used herein, the term "acyl carrier protein S-malonyltransferase" refers to a polypeptide that catalyzes the reaction of binding malonyl-CoA with a 4-phosphopantetheine cofactor to produce malonyl thioester in the biosynthesis of iturin family lipopeptides. Specific examples of acyl carrier protein S-malonyltransferases include iturin A synthase D (e.g., ituD consisting of the amino acid sequence set forth in SEQ ID NO: 10) that catalyzes iturin A biosynthesis, fenF (e.g., fenF consisting of the amino acid sequence set forth in SEQ ID NO: 11) that catalyzes mycostilin biosynthesis, bmyD (e.g., bmyD consisting of the amino acid sequence set forth in SEQ ID NO: 19) that catalyzes bacillomycin biosynthesis, and mohavencin synthase subunit D (e.g., the amino acid sequence set forth in SEQ ID NO: 26) that catalyzes mohavencin biosynthesis. Both enzymes are classified as K00645 ([acyl-carrier-protein]S-malonyltransferase) in KEGG ORTHOLOGY.

[0072] As used herein, "iturin family lipopeptide synthase B" refers to a polypeptide that catalyzes the incorporation of amino acid residues at positions 2 to 5 of a heptapeptide in the biosynthesis of an iturin family lipopeptide. Specific examples of iturin family lipopeptide synthase B include iturin A synthase B (e.g., ituB consisting of the amino acid sequence shown in SEQ ID NO: 12), which catalyzes the incorporation of amino acid residues at positions 2 to 5 of iturin A heptapeptide; mycostilin synthase subunit B (e.g., mycB consisting of the amino acid sequence shown in SEQ ID NO: 13), which catalyzes the incorporation of amino acid residues at positions 2 to 5 of mycostilin heptapeptide; bacillomycin D synthase B (e.g., bmyB consisting of the amino acid sequence shown in SEQ ID NO: 20), which catalyzes the incorporation of amino acid residues at positions 2 to 5 of a heptapeptide contained in bacillomycin (hereinafter referred to as "bacillomycin heptapeptide" herein); and mohavencin synthase subunit B (e.g., the amino acid sequence shown in SEQ ID NO: 24), which catalyzes the incorporation of amino acid residues at positions 2 to 5 of a heptapeptide contained in mohavencin (hereinafter referred to as "mohavencin heptapeptide" herein).

[0073] As used herein, "iturin family lipopeptide synthase C" refers to a polypeptide that catalyzes the incorporation of amino acid residues at positions 6 to 7 of a heptapeptide in the biosynthesis of an iturin family lipopeptide. Specific examples of iturin family lipopeptide synthase C include iturin A synthase C (e.g., ituC consisting of the amino acid sequence shown in SEQ ID NO: 14), which catalyzes the incorporation of amino acid residues at positions 6 to 7 of an iturin A heptapeptide; mycostilin synthase subunit C (e.g., mycC consisting of the amino acid sequence shown in SEQ ID NO: 15), which catalyzes the incorporation of amino acid residues at positions 6 to 7 of a mycostilin heptapeptide; bacillomycin D synthase C (e.g., bmyC consisting of the amino acid sequence shown in SEQ ID NO: 21), which catalyzes the incorporation of amino acid residues at positions 6 to 7 of a bacillomycin heptapeptide; and mohavencin synthase subunit C (e.g., the amino acid sequence shown in SEQ ID NO: 25), which catalyzes the incorporation of amino acid residues at positions 6 to 7 of a mohavencin heptapeptide.

[0074] The biological species from which the acyl carrier protein-S-malonyltransferase or an active fragment thereof, the iturin family lipopeptide synthase B or an active fragment thereof, and the iturin family lipopeptide synthase C or an active fragment thereof are derived is not limited, and may be, for example, a Gram-positive bacterium. Examples of Gram-positive bacteria include bacteria of the genus Bacillus, Paenibacillus, Geobacillus, Oceanobacillus, or Brevibacillus, and preferably bacteria of the genus Bacillus (e.g., iturin-producing Bacillus bacteria, mycostilin-producing Bacillus bacteria, bacillomycin-producing Bacillus bacteria, or mohavencin-producing Bacillus bacteria), such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Bacillus atrophaeus.

[0075] The non-natural iturin family lipopeptide synthetase complex of this embodiment includes the following mutant iturin family lipopeptide synthetase complexes and chimeric iturin family lipopeptide synthetase complexes. Each embodiment will be described in detail below.

[0076] 4-2-1. Mutant iturin family lipopeptide synthase complex The mutant iturin family lipopeptide synthetase complex of the present invention comprises iturin family lipopeptide synthetase A or an active fragment thereof, acyl carrier protein-S-malonyltransferase or an active fragment thereof, iturin family lipopeptide synthetase B or an active fragment thereof, and iturin family lipopeptide synthetase C or an active fragment thereof, and among these, iturin family lipopeptide synthetase A or an active fragment thereof is mutant iturin family lipopeptide synthetase A or an active fragment thereof. The configuration of mutant iturin family lipopeptide synthetase A or an active fragment thereof is similar to that described in the first aspect, and therefore further explanation is omitted here. The mutant iturin family lipopeptide synthetase complex of the present invention may be a non-chimeric iturin family lipopeptide synthetase complex or a chimeric iturin family lipopeptide synthetase complex. When the mutant iturin family lipopeptide synthetase complex of the present invention is a non-chimeric iturin family lipopeptide synthetase complex, the complex may be an iturin synthetase complex, a mycostylen synthetase complex, a bacillomycin synthetase complex, or a mohavencin synthetase complex comprising any of the mutant iturin family lipopeptide synthetase A or active fragments thereof described in the first aspect.

[0077] 4-2-2. Chimeric iturin family lipopeptide synthase complex The chimeric iturin family lipopeptide synthetase complex of the present invention comprises iturin family lipopeptide synthetase A or an active fragment thereof, acyl carrier protein-S-malonyltransferase or an active fragment thereof, iturin family lipopeptide synthetase B or an active fragment thereof, and iturin family lipopeptide synthetase C or an active fragment thereof, wherein at least one of the four constituent units is derived from an iturin family lipopeptide synthetase complex corresponding to an iturin family lipopeptide that is different from the other constituent elements.

[0078] Each enzyme unit constituting the chimeric iturin family lipopeptide synthetase complex may be either wild-type or mutant, and may be, for example, a chimeric mutant formed by combining two or more enzyme units corresponding to different iturin family lipopeptides (e.g., a fusion protein formed by linking the N-terminal region of MycB with the C-terminal region of ItuB). Iturin family lipopeptide synthetase A may also be wild-type or mutant, but if it is mutant, it may be a mutant iturin family lipopeptide synthetase A or an active fragment thereof, as described above in "4-2-1. Mutant iturin family lipopeptide synthetase complex." The configuration of the mutant iturin family lipopeptide synthetase A or an active fragment thereof in this case is also similar to that described in the first embodiment, and therefore further explanation is omitted here.

[0079] In one embodiment, the chimeric iturin family lipopeptide synthetase complex of the present invention comprises wild-type or mutant mycostylein synthase subunit A (mycA) or an active fragment thereof as iturin family lipopeptide synthetase A or an active fragment thereof, and at least one of the other three enzyme units is derived from an iturin family lipopeptide synthetase complex that synthesizes an iturin family lipopeptide other than mycostylein (e.g., iturin). The mycA contained in the chimeric iturin family lipopeptide synthetase complex of this embodiment or the mycA from which the active fragment thereof is derived may be either wild-type or mutant mycA. Examples of mutant mycA herein include mutant mycAs consisting of amino acid sequences that share 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with the amino acid sequence set forth in SEQ ID NO:9. In the acyl ligase domain of the mutant mycA of this embodiment, the amino acid residue corresponding to position 208 of the amino acid sequence set forth in SEQ ID NO: 1 may be any amino acid residue other than Phe, Arg, Trp, or Tyr, and may be the same Leu residue as in wild-type mycA. An example of a chimeric iturin family lipopeptide synthase complex is one composed of mycostilin synthase subunit A (mycA); iturin A synthase D (ituD), fenF, bmyD, or mohavencin synthase subunit D; iturin A synthase B (ituB), mycostilin synthase subunit B (mycB), bacillomycin synthase subunit B (bmyB), or mohavencin synthase subunit B; and iturin A synthase C (ituC), mycostilin synthase subunit C (mycC), bacillomycin synthase subunit C (bmyC), or mohavencin synthase subunit C.

[0080] In the non-natural or chimeric iturin family lipopeptide synthetase complex of the present invention, ituD may be wild-type ituD or a mutant ituD consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 10; fenF may be wild-type fenF or a mutant fenF consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 11; and bmyD may be a wild-type bmyD or a mutant bmyD consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 19; mohavensin synthase subunit D may be a wild-type mohavensin synthase subunit D or a mutant mohavensin synthase subunit D consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 26; and ituB may be a wild-type it mycB may be wild-type mycB or a mutant mycB consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 13; bmyB may be wild-type bmyB or a mutant mycB consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 20; The mohavencin synthase subunit B may be a mutant mohavencin synthase subunit B consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the wild-type mohavencin synthase subunit B or the amino acid sequence shown in SEQ ID NO: 24, and the ituC may be a mutant mohavencin synthase subunit B consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the wild-type ituC or the amino acid sequence shown in SEQ ID NO: 14.or a mutant ituC consisting of an amino acid sequence having 99% or more identity to the amino acid sequence shown in SEQ ID NO: 15, and mycC may be a wild-type mycC or a mutant mycC consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 15, and either enzyme unit is a chimeric mutant consisting of a combination of two or more enzyme units corresponding to different iturin family lipopeptides (e.g., a fusion protein consisting of the N-terminal region of MycB linked to the C-terminal region of ItuB). The bmyC may be a wild-type bmyC or a mutant bmyC consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 21, and the mohavensin synthase subunit C may be a wild-type mohavensin synthase subunit C or a mutant mohavensin synthase subunit C consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 25.

[0081] 5. Nucleic acid encoding a non-natural iturin family lipopeptide synthase complex Overview A fifth aspect of the present invention is a nucleic acid encoding a non-naturally occurring iturin family lipopeptide synthase complex.

[0082] 5-2.Configuration The "nucleic acid encoding a non-natural iturin family lipopeptide synthetase complex" may be any nucleic acid encoding any of the non-natural iturin family lipopeptide synthetase complexes described in the fourth aspect. The nucleic acid of this aspect essentially includes a gene encoding iturin family lipopeptide synthetase A or an active fragment thereof (hereinafter referred to as an "iturin family lipopeptide synthetase A gene"), a gene encoding acyl carrier protein-S-malonyltransferase or an active fragment thereof (hereinafter referred to as an "acyl carrier protein-S-malonyltransferase gene"), a gene encoding iturin family lipopeptide synthetase B or an active fragment thereof (hereinafter referred to as an "iturin family lipopeptide synthetase B gene"), and a gene encoding iturin family lipopeptide synthetase C or an active fragment thereof (hereinafter referred to as an "iturin family lipopeptide synthetase C gene"). The nucleotide sequences of the genes encoding each unit constituting the complex are not particularly limited, and the organisms from which they are derived are also not limited. For example, they may be derived from Gram-positive bacteria. Examples of Gram-positive bacteria include bacteria of the genus Bacillus, Paenibacillus, Geobacillus, Oceanobacillus, or Brevibacillus, and preferably bacteria of the genus Bacillus (e.g., iturin-producing Bacillus bacteria, mycostilin-producing Bacillus bacteria, bacillomycin-producing Bacillus bacteria, or mohavencin-producing Bacillus bacteria), such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, or Bacillus atrophaeus. Other examples include codon-optimized nucleotide sequences and nucleotide sequences with an initiation codon (ATG) added to the 5' end. In the nucleic acid of this embodiment, the genes encoding each unit may constitute an operon, or the above four genes may not constitute an operon, but may be, for example, independent vectors.

[0083] 6. Transformed Microorganisms Overview A sixth aspect of the present invention is a transformed microorganism.

[0084] 6-2.Configuration The transformed microorganism of this embodiment comprises a gene encoding the mutant iturin-family lipopeptide synthetase A of the first embodiment or an active fragment thereof (hereinafter often referred to as a "mutant iturin-family lipopeptide synthetase A gene" in this specification) (for example, the transformed microorganism of this embodiment comprises, as an essential component, a nucleic acid encoding the mutant iturin-family lipopeptide synthetase A or an active fragment thereof described in the second embodiment), or comprises a nucleic acid encoding the non-naturally occurring iturin-family lipopeptide synthetase complex of the fifth embodiment.

[0085] In the transformed microorganism of this embodiment, the nucleic acid encoding the mutant iturin family lipopeptide synthetase A gene or the non-naturally occurring iturin family lipopeptide synthetase complex may be contained in a vector. For example, the transformed microorganism of this embodiment may be a microorganism transformed with the gene expression vector of the third embodiment or an expression vector containing a nucleic acid encoding a non-naturally occurring iturin family lipopeptide synthetase complex. Alternatively, in the transformed microorganism of this embodiment, the nucleic acid encoding the mutant iturin family lipopeptide synthetase A gene or the non-naturally occurring iturin family lipopeptide synthetase complex may be integrated into the genome.

[0086] The type of transformed microorganism is not limited herein. Any microorganism can be transformed as long as it can express a mutant iturin family lipopeptide synthase A gene or a nucleic acid encoding a non-naturally occurring iturin family lipopeptide synthase complex and can express the mutant iturin family lipopeptide synthase A or a non-naturally occurring iturin family lipopeptide synthase complex. Examples of transformed microorganisms include bacteria and eukaryotic cells. The bacteria may be either Gram-positive or Gram-negative bacteria. Examples of Gram-positive bacteria include the genera Bacillus, Paenibacillus, Geobacillus, Oceanobacillus, and Brevibacillus. Examples of Bacillus bacteria include Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus atrophaeus. The Bacillus bacterium may be the above-mentioned iturin-producing Bacillus bacterium, mycostilin-producing Bacillus bacterium, bacillomycin-producing Bacillus bacterium, or mohavencin-producing Bacillus bacterium. Examples of Paenibacillus bacteria include Paenibacillus polymyxa. Examples of Brevibacillus bacteria include Brevibacillus choshinensis. Examples of Gram-negative bacteria include Escherichia coli. Examples of eukaryotic cells include fungal cells (e.g., yeast cells).

[0087] In one embodiment, the transformed microorganism of this aspect further comprises an acyl carrier protein-S-malonyltransferase gene, an iturin family lipopeptide synthetase B gene, and an iturin family lipopeptide synthetase C gene in addition to the mutant iturin family lipopeptide synthetase A gene. The acyl carrier protein-S-malonyltransferase gene, the iturin family lipopeptide synthetase B gene, and / or the iturin family lipopeptide synthetase C gene may be endogenous genes in the transformed microorganism or genes introduced into the transformed microorganism. Furthermore, in the transformed microorganism of this aspect, the mutant iturin family lipopeptide synthetase A gene, the acyl carrier protein-S-malonyltransferase gene, the iturin family lipopeptide synthetase B gene, and the iturin family lipopeptide synthetase C gene may constitute an operon. Alternatively, the above four genes may not constitute an operon but may be introduced, for example, by independent vectors.

[0088] In one embodiment, the transformed microorganism of this aspect is derived from any of the species, subspecies, or strains of the Bacillus bacteria described above as iturin-producing bacteria having the iturin A operon, and comprises the iturin A synthase D (ituD) gene, the iturin A synthase B (ituB) gene, and the iturin A synthase C (ituC) gene as endogenous genes on its genome, and can comprise a gene encoding wild-type mycostylen synthase subunit A (mycA) or a mutant mycA (e.g., a mutant mycA consisting of an amino acid sequence having 75% or more, 80% or more, 85% or more, or 90% or more identity to the amino acid sequence shown in SEQ ID NO: 9) as an exogenous gene or transgene. In this embodiment, the gene encoding mycA may be introduced into the genome by transformation or as an extragenomic genetic element (e.g., a gene on a plasmid). The location on the genome is not particularly limited; it may be introduced into the endogenous iturin operon as one of the genes constituting the operon, or it may be introduced at any genomic location outside the iturin operon. The transformed microorganism of this embodiment may or may not have an endogenous ituA gene, but preferably the endogenous ituA gene is deleted or disrupted. For example, in the transformed microorganism of this embodiment, the endogenous ituA gene may be replaced with a gene encoding wild-type or mutant mycA, in which case the exogenous or introduced gene encoding mycA forms an operon on the genome together with the other three enzyme unit genes.

[0089] Effects When the transformed microorganism of this embodiment contains an acyl carrier protein-S-malonyltransferase gene, an ituB gene or a mycB gene, and an ituC gene in addition to the iturin family lipopeptide synthase A gene, iturin A rather than mycostilin can be produced. This is because iturin A and mycostilin differ in the amino acid residues at positions 6 and 7 of the heptapeptide, and therefore, if the iturin family lipopeptide synthase C that catalyzes the uptake reaction is ituC, iturin A can be produced. For example, when the transformed microorganism of the present invention contains, in addition to a mutant ituA gene or a wild-type or mutant mycA gene, which is one embodiment of the first aspect, a gene encoding iturin A synthase D (ituD), a gene encoding iturin A synthase B (ituB), and a gene encoding iturin A synthase C (ituC), it can produce iturin A containing a high proportion of fatty acid chains with 17 or more carbon atoms (e.g., iturin A in which 20% or more of the total weight of iturin A contains fatty acid chains with 17 or more carbon atoms).

[0090] 7. Method for producing iturin family lipopeptides Overview A seventh aspect of the present invention is a method for producing an iturin family lipopeptide containing a fatty acid chain having 17 or more carbon atoms (hereinafter referred to as the "iturin family lipopeptide production method"). The iturin family lipopeptide production method of this aspect includes a culture step as an essential step, and includes an introduction step and / or an extraction step as selection steps. According to the production method of this aspect, iturin family lipopeptides containing a fatty acid chain having 17 or more carbon atoms can be produced.

[0091] 7-2. Method Each step in the method for producing an iturin family lipopeptide of this embodiment will be specifically described below.

[0092] (Introduction process) The "introduction step" is a step of introducing into a host microorganism a nucleic acid of the second aspect, a mutant iturin family lipopeptide synthase A expression vector of the third aspect, a nucleic acid encoding mycA or an expression vector containing the same, or a nucleic acid encoding a non-naturally occurring iturin family lipopeptide synthase complex of the fifth aspect. This step is a selection step and is not essential.

[0093] In this step, the "nucleic acid encoding mycA" may be a nucleic acid encoding either wild-type mycA or mutant mycA. Examples of mutant mycA include mutant mycAs consisting of amino acid sequences that share 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 9. In this case, in the acyl ligase domain of mutant mycA, the amino acid residue corresponding to position 208 of the amino acid sequence shown in SEQ ID NO: 1 may be any amino acid residue other than Phe, Arg, Trp, or Tyr, and may be the same Leu residue as in wild-type mycA.

[0094] In this step, the method for introducing the nucleic acid of the second aspect, the mutant iturin family lipopeptide synthase A expression vector of the third aspect, the nucleic acid encoding mycA or an expression vector containing the same, or the nucleic acid encoding the non-natural iturin family lipopeptide synthase complex of the fifth aspect into a host microorganism is not particularly limited. For example, a gene transfer method (transformation 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. Specific examples include the heat shock method, lipofection, electroporation, microinjection, calcium phosphate method, DEAE-dextran method, CI / CII method, introduction via cationic lipids, introduction via cationic polymers (e.g., polyethyleneimine (PEI)), introduction via nanoparticles, introduction via viruses, and particle bombardment.

[0095] In this step, a host microorganism into which the nucleic acid of the second aspect, the mutant iturin family lipopeptide synthase A expression vector of the third aspect, the nucleic acid encoding mycA or an expression vector containing the same, or the nucleic acid encoding the non-natural iturin family lipopeptide synthase complex of the fifth aspect has been introduced can be appropriately selected in a medium containing an antibiotic, for example, based on a drug resistance gene in the vector.

[0096] (Culture process) The "culturing step" refers to a step of culturing the transformed microorganism of the sixth embodiment described above. The purpose of this step is to have the transformed microorganism produce an iturin family lipopeptide before the extraction step described below.

[0097] The method for culturing the transformed microorganism in this step is not particularly limited and can be appropriately selected depending on the type of transformed microorganism. 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.

[0098] The medium used to culture the transformed microorganism in this step may be either a liquid medium or a solid medium. The medium may contain one or more components selected from the group consisting of enzymatic protein hydrolysates such as peptone and tryptone, biological extracts such as potato dextrose, yeast extract, soybean hydrolysate, ground beans or 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 growth by the transformed microorganism (e.g., required amino acids for amino acid-requiring microorganisms, required vitamins for vitamin-requiring microorganisms) may be added. Specific media and compositions include LB medium (tryptone, yeast extract, sodium chloride), soybean flour 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, 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).

[0099] When the transformed microorganism to be cultured in this step contains a drug resistance gene in the vector, etc., this step can be carried out in the presence of antibiotics, such as ampicillin, kanamycin, tetracycline, spectinomycin, erythromycin, lincomycin, or chloramphenicol.

[0100] 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 20 to 40°C, 25 to 35°C, 28 to 32°C, or 30°C.

[0101] Furthermore, the culture time in this step is not limited as long as a sufficient amount of iturin family lipopeptide can be obtained, and may be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 10 days or more, or 2 weeks or more.

[0102] (extraction process) The "extraction step" is a step of extracting iturin family lipopeptides from the culture medium obtained in the culture step. Note that this step is a selection step and is not essential.

[0103] As used herein, the term "culture medium" refers to the culture supernatant and / or the cultured transformed microorganism, because the culture supernatant may also contain iturin family lipopeptides secreted from the transformed microorganism.

[0104] Iturin family lipopeptides can be extracted from the culture medium using known methods and purified as needed. For example, the iturin family lipopeptides can be collected from the supernatant obtained by mixing a solvent such as ethanol with the culture medium or culture supernatant and then centrifuging, sedimenting, or a combination thereof. Alternatively, the precipitate obtained from the culture medium by centrifugation, sedimentation, filtration, or a combination thereof can be collected and, if necessary, suspended to obtain a suspension, and the precipitate or suspension can then be extracted with a solvent such as ethanol. Alternatively, the transformed microorganisms can be disrupted, and a fraction containing iturin family lipopeptides can be extracted as an extract from the resulting disrupted material or disrupted solution (or from the supernatant or precipitate obtained from the disrupted solution by centrifugation, sedimentation, filtration, or a combination thereof, if necessary, or from the suspension obtained by suspending the precipitate, if necessary) using water, ethanol, an organic solvent, or a mixture thereof. The resulting extract can be further purified by filtration using a filter or the like. Furthermore, if necessary, the extract can be concentrated to increase the iturin family lipopeptide concentration. Furthermore, when a more highly purified iturin family lipopeptide is required, the extract can be separated and / or purified by HPLC or the like to obtain a fraction containing the desired iturin family lipopeptide.

[0105] Effects The method for producing an iturin family lipopeptide of this embodiment makes it possible to produce an iturin family lipopeptide having dramatically enhanced antibacterial activity, in which 20% or more (e.g., 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more) of the total weight of the iturin family lipopeptide contains fatty acid chains having 17 or more carbon atoms. This proportion is significantly higher than the composition of previously reported iturin family lipopeptide extracts. The production method of the present invention makes it possible to stably and mass-produce iturin family lipopeptides with dramatically enhanced antibacterial activity.

[0106] 8. Iturin-containing composition 8-1. Overview An eighth aspect of the present invention is an iturin-containing composition. In the iturin-containing composition of the present invention, 20% or more of the total weight of iturin is iturin containing a fatty acid chain having 17 or more carbon atoms. The iturin-containing composition of the present invention exhibits strong antibacterial activity due to its long fatty acid chain structure, and can be used for plant disease control, etc.

[0107] 8-2.Configuration 8-2-1. Components The composition of this embodiment contains iturin as an active ingredient as an essential component. It can also contain an agriculturally acceptable carrier and / or medium to the extent that it does not inhibit or suppress the antibacterial activity of iturin. The composition of the present invention can prevent or treat plant diseases through its potent antibacterial activity, based on the activity of iturin, which contains a fatty acid chain having 17 or more carbon atoms, to destroy the membrane structure of plant pathogens and to induce a defense response in plants.

[0108] In the composition of this embodiment, 20% or more of the total weight of iturin is iturin containing a fatty acid chain having 17 or more carbon atoms. The "fatty acid chain having 17 or more carbon atoms" of the iturin contained in the composition of this embodiment is not limited as long as it has 17 or more carbon atoms, and may be, for example, 17, 18, 19, or more carbon atoms. Furthermore, in iturin containing a fatty acid chain having 17 or more carbon atoms, the branched structure of the fatty acid chain or the type of isomer is not limited, and it may be, for example, a linear type (n), an isotype (i), or an anteisotype (ai).

[0109] As used herein, "20% or more of the total weight of iturin contains fatty acid chains with 17 or more carbon atoms" means that the total weight of iturin containing fatty acid chains of any number of carbon atoms, 17 or more, is 20% or more of the total weight of iturin. Therefore, even if iturin having a 17-carbon fatty acid chain does not alone account for 20% or more, it is understood that the total weight of iturin containing 17-carbon and 18-carbon fatty acid chains, or the total weight of iturin containing 17-carbon, 18-carbon, and 19-carbon fatty acid chains, is 20% or more of the total weight of iturin.

[0110] Furthermore, in the iturin-containing composition of this embodiment, the total weight of iturin containing fatty acid chains of any carbon number of 17 or more may be 20% or more of the total weight of iturin, and for example, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 99.5% or more of the total weight of iturin may contain fatty acid chains of 17 or more carbon atoms.

[0111] In one embodiment, in the iturin-containing composition of this aspect, 30% or more of the total weight of iturin is iturin containing fatty acid chains with 17 or more carbon atoms.

[0112] In a further embodiment, in the iturin-containing composition of this aspect, 50% or more of the total weight of iturin is iturin containing fatty acid chains with 17 or more carbon atoms.

[0113] In one embodiment, the iturin-containing composition of this aspect contains iturin containing a fatty acid chain having 18 or more carbon atoms. For example, the iturin-containing composition of this aspect contains iturin containing a fatty acid chain having 18 or more carbon atoms. In the iturin-containing composition of this embodiment, the weight of the iturin containing a fatty acid chain having 18 or more carbon atoms is not particularly limited. For example, the weight of the iturin containing a fatty acid chain having 18 or more carbon atoms may be 0.1% or more, 0.5% or more, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 99.5% or more of the total weight of the iturin.

[0114] In a further embodiment, in the iturin-containing composition of this aspect, 1% or more of the total weight of iturin is iturin containing a fatty acid chain having 18 carbon atoms.

[0115] The total weight of iturin relative to the weight of the iturin-containing composition of this embodiment is not particularly limited and may be, for example, 0.0001% or more, 0.001% or more, 0.01% or more, 0.1% or more, or 1% or more, and / or 99% or less, 90% or less, 50% or less, 20% or less, 10% or less, or 5% or less. Furthermore, the weight of iturin containing a fatty acid chain having 17 or more carbon atoms relative to the weight of the iturin-containing composition of this embodiment is not particularly limited, as long as it is 20% or more of the total weight of iturin, as described above. For example, it may be 0.0001% or more, 0.001% or more, 0.01% or more, 0.1% or more, or 1% or more, and / or 99% or less, 90% or less, 50% or less, 20% or less, 10% or less, or 5% or less of the weight of the iturin-containing composition. This is because even if the iturin content relative to the weight of the iturin-containing composition of this embodiment is low, high antibacterial activity can be obtained as long as the iturin containing fatty acid chains with 17 or more carbon atoms accounts for 20% or more of the total weight of iturin.

[0116] In one embodiment, the iturin-containing composition of this aspect comprises a culture medium of Bacillus bacteria, a precipitate (or a suspension obtained by suspending the precipitate) or supernatant (culture supernatant) obtained from the culture medium, or a disruption solution or a precipitate (or a suspension obtained by suspending the precipitate) or supernatant (disruption solution supernatant) obtained from the disruption solution. The culture medium of this aspect is obtained by culturing Bacillus bacteria, and the precipitate or culture supernatant can be obtained from the culture medium by centrifugation, sedimentation, or a combination thereof. The precipitate can also be a suspension obtained by suspending it in a buffer or the like. The culture medium of Bacillus bacteria, or the precipitate, suspension, or culture supernatant derived from the culture medium, can also be one that does not contain live bacteria. A culture solution or a culture solution-derived precipitate, suspension, or culture supernatant that does not contain live bacteria can be obtained, for example, by removing live bacteria from the culture solution, culture supernatant, or suspension by centrifugation, sedimentation, and / or filtration, or by killing live bacteria by UV irradiation, radiation exposure, and / or heat treatment of the culture solution, precipitate, suspension, or culture supernatant. The disruption solution of this embodiment can be prepared by any disruption method. For example, a disruption solution can be obtained using ultrasonication, freeze-thawing, mechanical disruption using glass beads, or the like, osmotic disruption using hypotonic solutions, surfactants, proteases, or the like. A precipitate or supernatant derived from the disruption solution can be obtained from the disruption solution by centrifugation, sedimentation, or a combination thereof.

[0117] In a further embodiment, the iturin-containing composition of this aspect is an extract of Bacillus bacteria. The extract of this embodiment can be obtained by extracting iturin from a culture solution obtained by culturing Bacillus bacteria. The extraction method is not limited, and iturin can be extracted using a known method and purified as needed. For example, iturin can be recovered from the supernatant obtained by mixing a Bacillus bacteria culture solution or culture supernatant with a solvent such as ethanol, followed by centrifugation, sedimentation, filtration, or a combination thereof. Alternatively, a precipitate obtained from the culture solution by centrifugation, sedimentation, filtration, or a combination thereof can be recovered and, if necessary, suspended to obtain a suspension, and the precipitate or suspension can then be extracted with a solvent such as ethanol. Alternatively, Bacillus bacteria can be disrupted, and an iturin-containing fraction can be extracted as an extract from the resulting disrupted material or disrupted solution (or from the supernatant or precipitate obtained from the disrupted solution by centrifugation, sedimentation, filtration, or a combination thereof, if necessary, or from the suspension obtained by suspending the precipitate, if necessary) using water, ethanol, an organic solvent, a mixture thereof, or the like. The obtained extract may be further purified by filtration using a filter or the like. Furthermore, if necessary, the extract may be concentrated to increase the iturin concentration. Furthermore, if iturin of higher purity is required, the extract may be separated and / or purified using HPLC or the like to obtain a fraction containing the desired iturin.

[0118] In a further embodiment, the above-mentioned culture medium, culture supernatant, culture medium-derived precipitate (a precipitate obtained from the culture medium by centrifugation, sedimentation, filtration, or a combination thereof), or a suspension of said precipitate, a disruption solution obtained by disrupting the culture medium or a culture medium-derived precipitate, a disruption solution supernatant, a disruption solution-derived precipitate (a precipitate obtained from the disruption solution by centrifugation, sedimentation, filtration, or a combination thereof), or a suspension of said precipitate, or an extract (an extract obtained by extracting the above-mentioned culture medium, disruption solution, or any of the above supernatants, precipitates, or suspensions) is derived from the transformed microorganism of the sixth aspect. For example, the method for producing an iturin family lipopeptide of the seventh aspect can also be used to obtain an iturin-containing composition that is a culture medium, culture supernatant, culture medium-derived precipitate or a suspension thereof, a disruption solution, a disruption solution supernatant, a disruption solution-derived precipitate or a suspension thereof, or an extract.

[0119] As used herein, "agriculturally acceptable carriers and / or vehicles" refer to substances that are capable of maintaining at least part of the antibacterial activity of the active ingredient iturin, that have no or very little harmful effect on soil, etc., and that are no or very little harmful to humans, etc. Specific examples of agriculturally acceptable carriers include excipients, etc. Specific examples of agriculturally acceptable solvents include water (including aqueous solutions) and buffers, etc.

[0120] 8-2-2. Dosage form The formulation of the iturin-containing composition of the present invention may be any formulation that can maintain the antibacterial activity of the active ingredient iturin. For example, the iturin-containing composition may be in the form of a liquid formulation or a solid powder.

[0121] 8-3. Application method When used as a plant disease control composition, the method for applying the iturin-containing composition of the present invention may be any method known in the art, and is not particularly limited, as long as it is a method that allows the iturin-containing composition to be applied to target plants. For example, the method described below in "9-2. Method" under "9. Plant disease control method" can also be used.

[0122] 8-4. Target plants The target plants to which the iturin-containing composition of the present invention is applied may be any plants for which iturin can exert a control effect, and the type of plant is not particularly limited, as long as the plant is susceptible to plant diseases caused by infectious pathogens such as bacteria, fungi, and / or viruses. It may be either angiosperms or gymnosperms. Furthermore, it does not matter whether the plant is herbaceous or woody.

[0123] 8-5.Effects The iturin-containing composition of the present invention exhibits stronger antibacterial activity than iturin produced by wild-type Bacillus subtilis. For example, the iturin-containing composition of the present invention exhibits stronger antibacterial activity than an iturin composition derived from wild-type Bacillus subtilis, the majority of which has fatty acid chains with 14 to 16 carbon atoms. Furthermore, the iturin-containing composition of the present invention exhibits stronger antibacterial activity than an iturin-containing composition containing iturin with fatty acid chains with 16 or fewer carbon atoms, a composition containing almost no iturin with fatty acid chains with 17 or more carbon atoms, or an iturin-containing composition in which iturin with fatty acid chains with 17 or more carbon atoms accounts for 10% or less of the total weight of iturin. Therefore, the iturin-containing composition of this embodiment can be used as an antibacterial composition.

[0124] 9. Plant disease control methods 9-1. Overview A ninth aspect of the present invention is a method for controlling plant diseases, characterized in that the plant disease of a target plant is controlled by applying the iturin-containing composition described in the eighth aspect to the target plant.

[0125] According to the plant disease control method of the present invention, for example, bacterial, fungal, and / or viral plant diseases can be controlled in target plants.

[0126] 9-2. Method The plant disease control method of the present invention includes a contacting step as an essential step.

[0127] The "contacting step" is a step of contacting the iturin-containing composition according to the eighth embodiment with a target plant.

[0128] In this embodiment, "contact" refers to contact between an iturin-containing composition and a target plant. More specifically, it refers to contact of iturin, the active ingredient of the iturin-containing composition, with the target plant, a site affected by an infectious pathogen, or a site infected or at risk of infection with a target bacterium, fungus, and / or virus. This step aims to allow the active ingredient iturin to act on the target, thereby, for example, destroying the membrane structure of the target bacterium or fungus, inactivating the virus, and / or inducing a defense response in the plant. As a result, a control effect against plant diseases can be exerted.

[0129] Contact may be either direct or indirect. In this embodiment, direct contact refers to direct contact of the iturin-containing composition with a predetermined site of the target plant. Specifically, for example, it refers to applying, spraying, sprinkling, or immersing a liquid iturin-containing composition on the plant body of the target plant. On the other hand, indirect contact in this embodiment refers to contact of the iturin-containing composition with a predetermined site of the target plant via an intermediary. For example, it refers to applying the iturin-containing composition to the soil around the roots of the target plant.

[0130] 9-3.Effects Iturin, which is an active ingredient in the iturin-containing composition of the present invention and has a fatty acid chain with 17 or more carbon atoms, destroys the membrane structure of target bacteria and fungi, inactivates viruses, and / or induces a defense response in plants, thereby helping to prevent plant diseases. [Example]

[0131] 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.

[0132] <Example 1: Preparation of various mutant strains> (the purpose) Various mutant strains are prepared using Bacillus subtilis strain 168 (hereinafter abbreviated as "strain 168") as a parent strain.

[0133] (Methods and Results) The genetic manipulations described below were carried out with reference to "Molecular Cloning" (Cold Spring Harbor Laboratory Press, 1989) as necessary. Transformation of Bacillus subtilis was carried out using the CI / CII method (Microbial Genetics Experimental Methods, Genetics Experimental Lecture Series 3, Kyoritsu Shuppan).

[0134] [1] Construction of mutant strains 1 to 5 Mutant strains 1 to 5 shown in Table 1 below were prepared by the method described below.

[0135] [Table 1]

[0136] (Creation of mutant strain 1) A DNA fragment was prepared for inserting a spectinomycin resistance cassette into the sigF gene locus of Bacillus subtilis. A DNA fragment (SEQ ID NO: 27) containing the upstream and downstream sequences of the sigF gene and the spectinomycin resistance cassette was generated by PCR using synthetic oligonucleotides. Strain 168 was transformed with this DNA fragment, and then plated onto an LB agar plate containing spectinomycin dihydrochloride pentahydrate (150 μg / mL) and cultured at 37°C to obtain transformants. From the resulting transformants, one strain was isolated in which the sigF gene was deleted from the initiation codon to the termination codon and the spectinomycin resistance cassette was inserted. This strain (mutant 1) was designated the "sigF::spec strain."

[0137] (Creation of mutant strain 2) A DNA fragment (SEQ ID NO: 28) containing the upstream and downstream sequences of the sigF gene was generated by PCR using synthetic oligonucleotides to remove the spectinomycin resistance cassette from mutant strain 1. Mutant strain 1 was transformed with this DNA fragment, and the strain was then plated on an LB agar plate. Colonies were then replicated on an LB agar plate and an LB agar plate containing spectinomycin dihydrochloride pentahydrate (100 μg / mL), respectively, and transformants exhibiting spectinomycin sensitivity were selected. From the resulting transformants, one strain was isolated in which the region from the start codon to the stop codon of the sigF gene was deleted. This strain (mutant strain 2) was designated "ΔsigF strain."

[0138] (Creation of mutant strain 3) A DNA fragment was prepared for inserting a kanamycin resistance cassette into the srfAB gene of Bacillus subtilis. A DNA fragment (SEQ ID NO: 29) carrying a homologous region for homologous recombination and a kanamycin resistance cassette for insertion into the srfAB gene locus was prepared by PCR using synthetic oligo DNA. Mutant strain 2 was transformed with this DNA fragment, and the strain was plated on an LB agar plate containing kanamycin sulfate (7.5 μg / mL). From the resulting transformants, one strain in which the kanamycin resistance cassette was inserted into the srfAB gene was isolated, and this strain (mutant strain 3) was designated "ΔsigF,srfAB::kan strain."

[0139] (Creation of mutant strain 4) The lpa-14 gene was introduced onto the chromosome of mutant 3. The lpa-14 gene encodes 4-phosphopantetheinyl transferase and can confer the ability to produce cyclic lipopeptides (J. Ferment. Bioeng., 76:6, 445-450 (1993)).

[0140] A DNA fragment was prepared for introducing the lpa-14 gene into the sfp locus on the chromosome. A DNA fragment (SEQ ID NO: 30) containing a homologous region for homologous recombination to introduce the lpa-14 gene into the sfp locus, lpa-14, and a chloramphenicol resistance cassette was prepared by PCR using synthetic oligonucleotides. Mutant strain 3 was transformed with this DNA fragment, and the strain was plated on an LB agar plate containing chloramphenicol (7.5 μg / mL). One strain in which lpa-14 and the chloramphenicol resistance cassette were inserted into the sfp locus was isolated from the resulting transformants. This strain (mutant strain 4) was designated "ΔsigF, srfAB::kan, sfp::lpa14-cat strain."

[0141] (Creation of mutant strain 5) The region downstream of the AL domain of ituA in the iturin A operon is introduced onto the chromosome of mutant strain 4. Specifically, the region from the downstream of the AL domain of ituA (hereinafter referred to as "ituAL") to the termination codon of ituC (ituA'-ituB-ituC) of the iturin A operon (ituD-ituA-ituB-ituC) derived from Bacillus subtilis RB14 strain (hereinafter referred to as "RB14 strain") is introduced into the full-length deletion site of the pps operon (ppsA-ppsB-ppsC-ppsD-ppsE) on the chromosome of mutant strain 4.

[0142] The ituA'-ituB-ituC gene was divided into four fragments and introduced into the full-length deletion site of the pps operon through four transformations. In the fourth transformation, the strain was plated on an LB agar plate containing spectinomycin dihydrochloride pentahydrate (150 μg / mL). From the resulting transformants, one strain was isolated in which the region from the initiation codon of the ppsA gene to the termination codon of the ppsE gene had been deleted, and a spectinomycin resistance cassette and the sequence consisting of ituA'-ituB-ituC (SEQ ID NO: 31) had been introduced into the deletion site. This strain (mutant 5) was designated "ΔsigF, srfAB::kan, sfp::lpa14-cat, pps::spec-ituA'-ituB-ituC strain."

[0143] [2] Creation of mutant strains 6-26 Mutant strains 6 to 26 shown in Table 2 below were prepared by the method described below.

[0144] [Table 2]

[0145] (Creation of mutant strain 6) Of the iturin A operon derived from the RB14 strain, the region from the initiation codon of ituD to the AL domain of ituA (ituAL) (ituD-ituA_ituAL) was introduced onto the chromosome of mutant 5.

[0146] Specifically, a DNA fragment (SEQ ID NO: 32) containing the homologous region for homologous recombination, the erythromycin resistance gene, the promoter sequence, and the above-mentioned ituD-ituA_ituAL was prepared by PCR using synthetic oligo DNA. Mutant strain 5 was transformed with this DNA fragment, and the strain was plated on an LB agar plate containing erythromycin (1 μg / mL). From the resulting transformants, one strain containing the erythromycin resistance gene, the promoter sequence, and ituD-ituA_ituAL was isolated, and this strain (mutant strain 6) was named "ituAL strain."

[0147] (Creation of mutant strain 7) The region of the iturin A operon from the RB14 strain, from the initiation codon of ituD to the intergenic region between ituD and ituA, and the DNA sequence (ituD-mycA_mycAL) encoding the AL domain of mycA (mycA_mycAL) of the mycostilin operon (fenF-mycA-mycB-mycC) from Bacillus subtilis ATCC 6633 were introduced onto the chromosome of mutant 5.

[0148] Specifically, a DNA fragment (SEQ ID NO: 33) containing the homologous region for homologous recombination, the erythromycin resistance gene, the promoter sequence, and the above-mentioned ituD-mycA_mycAL was prepared by PCR using synthetic oligo DNA. Mutant strain 5 was transformed with this DNA fragment, and the strain was plated on an LB agar plate containing erythromycin (1 μg / mL). From the resulting transformants, one strain containing the erythromycin resistance gene, the promoter sequence, and the above-mentioned ituD-mycA_mycAL was isolated, and this strain (mutant strain 7) was designated the "mycAL strain."

[0149] (Creation of mutant strain 8) A DNA sequence (ituD-ituA_ituAL_F208A) containing a mutation that replaces the Phe residue at position 208 of the amino acid sequence shown in SEQ ID NO: 1 with an Ala residue in the region (ituD-ituA_ituAL) from the initiation codon of iturin A operon derived from the RB14 strain to the AL domain (ituAL) of iturin A operon derived from the RB14 strain was introduced onto the chromosome of mutant strain 5.

[0150] Specifically, a DNA fragment containing the homologous region for homologous recombination, the erythromycin resistance gene, the promoter sequence, and the ituD-ituA_ituAL_F208A sequence was prepared by PCR using synthetic oligonucleotides. Mutant strain 5 was transformed with this DNA fragment, and the resulting strain was plated on an LB agar plate containing erythromycin (1 μg / mL). From the resulting transformants, one strain containing the erythromycin resistance gene, the promoter sequence, and the ituD-ituA_ituAL_F208A sequence was isolated. This strain (mutant strain 8) was designated "F208A strain."

[0151] (Creation of mutant strains 9 to 26) Using a method similar to that used to prepare mutant strain 8, mutant strains 9 to 26 were prepared in which the Phe residue at the position corresponding to position 208 was replaced with various amino acid residues (Table 2).

[0152] <Example 2: Production of iturin A using mycAL and ituAL strains> (the purpose) Iturin A is produced using the mycAL strain and the ituAL strain prepared in Example 1, and the composition of the iturin A is compared.

[0153] (Methods and Results) The mycAL strain (mutant 7) and the ituAL strain (mutant 6) were cultured under the following conditions to produce iturin A. Specifically, each mutant was inoculated into LB medium (containing 5 μg / mL kanamycin sulfate, 5 μg / mL chloramphenicol, and 0.5 μg / mL erythromycin) and cultured overnight at 37°C with shaking at 300 rpm. This culture was then inoculated into 2.5 mL of soybean flour 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, and 0.022 g / L manganese chloride tetrahydrate) and cultured at 30°C with shaking at 300 rpm for 72 hours. The iturin A in the resulting culture medium was analyzed by HPLC. The weight ratio of iturin A with each fatty acid chain length to the total weight of iturin A was calculated from the area of ​​each peak in the sum of the areas of the peaks corresponding to each fatty acid chain length (C14 to C19). Each peak in the HPLC analysis was assigned using the m / z value in LC-TOFMS measurement and the m / z value of the product ion obtained by MS / MS measurement, and the peak corresponding to iturin A of each fatty acid chain length was identified. Iturin A (Sigma-Aldrich) was used as a standard sample for quantification. The results of the fatty acid chain length analysis are shown in Table 3 below and in FIG.

[0154] [Table 3]

[0155] As shown in Table 3 and Figure 2, in the iturin A produced by the ituAL strain in which ituA contains the AL domain of ituA (ituAL), 83% had a 15-carbon fatty acid chain, while only 3.4% had a 17-carbon fatty acid chain. No iturin A with a fatty acid chain longer than 18 carbons was detected. In contrast, in the iturin A produced by the mycAL strain in which ituA contains the AL domain of mycA (mycAL), 75.2% had a 17-carbon fatty acid chain, and an additional 4.4% had an 18-carbon fatty acid chain.

[0156] These results revealed that the ratio of iturin A with fatty acid chains of 17 or more carbon atoms was significantly increased by replacing the AL domain of ituA with the AL domain of mycA.

[0157] <Example 3: Identification of important amino acid residues in the AL domain> (the purpose) We identify amino acid residues in the AL domain that may affect the fatty acid chain length of iturin A.

[0158] (Methods and Results) Mutant strains were prepared in ituA by substituting each of the amino acid residues in the AL domain that differ between ituA and mycA with the mycA type, and each mutant strain was cultured in the same manner as in Example 2 to produce iturin A. The composition of fatty acid chain lengths (C14 to C19) of the iturin A in the resulting culture medium was analyzed, and the weight ratio of iturin A having each fatty acid chain length relative to the total weight of iturin A was calculated.

[0159] Surprisingly, we found that substituting the Phe residue at position 208 in ituA with a Leu residue in mycA produced an effect equivalent to that of the mycAL strain (mutant 7). This result is shown in Table 4 below as F208L strain (mutant 16).

[0160] Example 4: Introduction of various amino acid substitutions at position 208 of ituA (the purpose) Iturin A is produced using mutant strains 8 to 26 in which the Phe residue at position 208 of the amino acid sequence shown in SEQ ID NO: 1 in ituA is replaced with various amino acid residues, and the effect on the composition of the fatty acid chain is examined.

[0161] (Methods and Results) Each of the mutant strains (mutants 8 to 26 in Table 2) such as the F208A strain prepared in Example 1 was cultured in the same manner as in Example 2 to produce iturin A. The iturin A in the resulting culture medium was analyzed by HPLC, and the weight ratio of iturin A having each fatty acid chain length to the total weight of iturin A was calculated from the area value of each peak in the sum of the area values ​​of the peaks corresponding to each fatty acid chain length (C14 to C19). The results of the fatty acid chain length analysis are shown in Table 4 below and in FIG.

[0162] [Table 4]

[0163] As shown in Table 4 and Figure 2, when the Phe residue at position 208 in the amino acid sequence shown in SEQ ID NO: 1 in iturin A was substituted with an amino acid residue other than Arg, Trp, or Tyr (Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Ser, Thr, or Val), the proportion of iturin A having a fatty acid chain with 17 or more carbon atoms increased to 20% or more.

[0164] Example 5: Evaluation of antibacterial activity (the purpose) The antibacterial activity of iturin A produced by the mycAL and ituAL strains will be evaluated.

[0165] (Methods and Results) (1) Preparation of iturin A solution The mycAL strain (mutant 7) was cultured in the same manner as in Example 2 to produce iturin A. The resulting culture medium was centrifuged, and the resulting precipitate was suspended in ethanol. The resulting suspension was then centrifuged, and the supernatant was collected. The resulting supernatant was sterilized by filtration using a 0.2 μm filter. The resulting filtrate was concentrated to dryness and then redissolved in ethanol to prepare an iturin A solution. Hereinafter, the resulting iturin A solution is referred to as "iturin A solution (mycAL)."

[0166] Next, the ituAL strain (mutant strain 6) was cultured in the same manner to prepare an iturin A solution. Hereinafter, the obtained iturin A solution is referred to as "iturin A solution (ituAL)."

[0167] Iturin A solution (mycAL) and iturin A solution (ituAL) were mixed in ratios of 75:25, 50:50, or 25:75. The resulting solutions are referred to as "iturin A solution (75:25)," "iturin A solution (50:50)," and "iturin A solution (25:75)," respectively.

[0168] (2) Analysis of fatty acid chain length of iturin A in each iturin A solution The iturin A in each iturin A solution prepared in (1) above was subjected to HPLC analysis, and the weight ratio of iturin A having each fatty acid chain length to the total weight of iturin A was calculated from the area value of each peak in the sum of the area values ​​of the peaks corresponding to each fatty acid chain length (C14 to C19). The results of the fatty acid chain length analysis are shown in Table 5 below and in FIG.

[0169] [Table 5]

[0170] (3) Evaluation of the antibacterial activity of each iturin A solution The antibacterial activity of the iturin A in each of the iturin A solutions prepared in (1) above was evaluated by the following procedure.

[0171] The test bacterium Saccharomyces cerevisiae BY4742 was inoculated into YPD medium and cultured with shaking at 30°C for 20 to 24 hours. The culture broth was then added to YPD medium to adjust the OD600 to 0.01. Each of the iturin A solutions prepared in (1) above was diluted appropriately with ethanol and added to the YPD medium inoculated with the test bacterium. After 24 hours of culture with shaking at 30°C, the OD620 of the culture broth was measured. The OD620 measurement results are shown in Table 6 below and FIG.

[0172] [Table 6]

[0173] Next, the minimum inhibitory concentration, which indicates the strength of antibacterial activity, was determined for each iturin A solution. The minimum inhibitory concentration was determined as the minimum iturin A concentration that could suppress the OD620 of the culture solution to 20% or less of the maximum value. The minimum inhibitory concentrations for each iturin A solution are shown in Table 7 below.

[0174] [Table 7]

[0175] These results demonstrate that iturin A with dramatically enhanced antibacterial activity can be produced by replacing the AL domain of ituA with the AL domain of mycA. Furthermore, it was revealed that the greater the proportion of iturin A with fatty acid chains of 17 or more carbon atoms, the greater the antibacterial activity.

[0176] Example 6: Mass spectrometry of iturin A (the purpose) The structure of iturin A produced by the F208G strain (mutant strain 12) prepared in Example 1 was analyzed by mass spectrometry.

[0177] (Methods and Results) The F208G strain (mutant 12) prepared in Example 1 was cultured in the same manner as in Example 2 to produce iturin A. Methanol was added to the resulting culture medium to prepare an 80% methanol solution, and the supernatant obtained after centrifugation was used as a sample for the following LC-TOFMS analysis.

[0178] Liquid chromatography (LC) was performed using a Shimadzu UFLC Nexera X2 column, Kinetex 2.6 μm EVO C18 100A 150 × 4.6 mm. The liquid chromatography conditions were a 10 μL sample, a column temperature of 40 °C, and a flow rate of 0.7 mL / min. 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 were 40% (0 min to 120 min), 80% (120.1 min to 150 min), and 40% (150.1 min to 180 min) for mobile phase B. Detection was performed at a UV detection wavelength of 205 nm.

[0179] Next, for mass spectrometry, an AB SCIEX TripleTOF6600 was used as the MS device, and the ESI method in positive ion mode was used as the ionization method. The results of the LC-TOFMS analysis are shown in Table 8 below.

[0180] [Table 8]

[0181] Product ion assignment was performed based on the MS / MS measurement results for the 11 peaks in Table 8. The product ion assignment results for Peaks No. 1 to No. 11 are shown in Tables 9 to 19 below.

[0182] [Table 9]

[0183] [Table 10]

[0184] [Table 11]

[0185] [Table 12]

[0186] [Table 13]

[0187] [Table 14]

[0188] [Table 15]

[0189] [Table 16]

[0190] [Table 17]

[0191] [Table 18]

[0192] [Table 19]

[0193] From the results in Tables 9 to 19, peaks No. 1 to No. 11 shown in Table 8 were identified as iturin A with different fatty acid chain lengths. The identification results are shown in Table 20 below.

[0194] [Table 20]

[0195] It was suggested that two peaks with the same m / z value in Table 20 correspond to different isomers of branched structure (linear type (n), isotype (i), or anteisotype (ai), etc.).

Claims

1. An iturin-containing composition, wherein 20% or more of the total weight of iturin is iturin containing a fatty acid chain having 17 or more carbon atoms.

2. The composition according to claim 1, wherein 30% or more of the total weight of iturin is iturin containing fatty acid chains having 17 or more carbon atoms.

3. The composition according to claim 1, wherein 50% or more of the total weight of iturin is iturin containing fatty acid chains having 17 or more carbon atoms.

4. The composition according to any one of claims 1 to 3, wherein the number of carbon atoms is 17.

5. The composition according to any one of claims 1 to 3, comprising iturin containing a fatty acid chain having 18 carbon atoms.

6. An iturin-containing composition, wherein 1% or more of the total weight of iturin is iturin containing a fatty acid chain having 18 carbon atoms.

7. The composition according to any one of claims 1 to 3 and 6, which is an extract of a bacterium of the genus Bacillus.

8. The composition according to any one of claims 1 to 3 and 6, which is a culture solution or a supernatant thereof, a precipitate derived from a culture solution or a suspension thereof, a disruption solution or a supernatant thereof, or a precipitate derived from a disruption solution or a suspension thereof, of a Bacillus bacterium.

9. A method for controlling plant diseases, comprising a contacting step of contacting a target plant with the composition according to any one of claims 1 to 3 and 6.

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