Novel Microorganism Saccharopolyspora sp. AN150100 Producing Spinosyn α4 and Uses Thereof

Abstract

The present invention relates to a novel microorganism of the genus Saccharopolysporasp. represented by SEQ ID NO. 1, KR0007 (AN150100) KCTC14897BP; an insecticidal composition comprising said microorganism or its culture medium, supernatant, or product isolated therefrom as an active ingredient; a compound represented by Formula 1 isolated from Saccharopolysporasp. AN150100 KCTC14897BP; and an insecticidal composition comprising said compound or a salt thereof as an active ingredient. The novel microorganism Saccharopolysporasp. AN150100 according to the present invention exhibits excellent insecticidal activity against diamondback moth larvae and can effectively replace existing chemical insecticides and commercial biological insecticides due to resistance issues. The microbial culture solution maintains stable activity even under high dilution conditions, making it highly effective in agricultural fields. Furthermore, since it is based on a novel naturally isolated strain, it offers the potential for independent industrial application distinct from existing spinosyn-producing strains. Additionally, it was confirmed that Spinosyn α4, which exhibits high insecticidal activity against diamondback moth larvae, can be produced with high efficiency.

Description

Technology Field

[0001] The present invention relates to a novel microorganism of the genus Saccharopolisphora having a 16S rDNA base sequence represented by SEQ ID NO. 1 ( Saccharopolyspora sp.) AN150100 KCTC14897BP; an insecticidal composition comprising the above microorganism or its culture medium, supernatant, or product isolated therefrom as an active ingredient; a compound represented by Formula 1 isolated from the above microorganism; and an insecticidal composition comprising the above compound or a salt thereof as an active ingredient. Background Technology

[0003] Chinese cabbage and cabbage are important vegetable crops worldwide, and are mass-produced in major agricultural regions such as Asia, Europe, and South America. These cruciferous crops are attacked by various pests, among which the diamondback moth ( Plutella xylostella It is known as the most serious pest. In particular, as high temperatures and warming environments are created due to climate change, the overwintering areas of diamondback moths are expanding, and the rapid spread of their populations is being reported as the number of generations per year increases, accelerating the decline in agricultural productivity.

[0004] Although organophosphates, carbamates, and synthetic pyrethroids have long been used to control diamondback moths, the emergence and spread of resistance to these insecticides has become a serious global issue. Furthermore, widespread resistance is also being reported for bio-insecticides, which were introduced as low-toxicity and eco-friendly alternatives. Non-patent document 1 summarizes cases of resistance regarding bio-insecticides widely used globally and points out that most bio-insecticides currently in use also eventually result in the selection of resistant individuals. For example, Bacillus thuringiensis It was stated that resistance to (Bt) toxins alone has been reported in at least 27 species of pests, and that the related resistance mechanisms are highly diverse, similar to resistance to chemical insecticides. In particular, the spinosyn class is treated as a representative biological insecticide. Spinosad and Spinetoram are Saccharopolyspora spinosa As a secondary metabolite derived from, it has been widely used worldwide for the control of pyrethroid-resistant lepidopteran larvae. However, not long after Spinosyn formulations were commercialized, Heliothis virescens , Musca domestica , Plutella xylostella , Frankliniella occidentalis , Spodoptera exigua Cases of resistance at both laboratory and field levels have been reported in various pest species, among which diamondback moths and western flower thrips exhibited particularly high levels of resistance. In Hawaii, spinosad was introduced to diamondback moth populations that already showed strong resistance to pyrethroids and other chemical insecticides; however, spinosad resistance was observed across the island within about two years, and there were reports of a resurgence of resistant individuals even during the process of reintroducing the drug after temporarily suspending its use for resistance management. According to an analysis by Siegwart et al., such cases demonstrate that even with biological insecticides, long-term and intensive reliance on a single formulation can lead to the emergence of high levels of resistance in a manner essentially no different from that of chemical insecticides.

[0005] Therefore, in the control of diamondback moths, it is essential to introduce new mechanisms of action or novel substances with the risk of resistance development not only for existing chemical insecticides but also for biological insecticides currently in use, such as spinosad or spinetoram, and the development of new microorganisms and their secondary metabolites to support this is required.

[0007] Against this background, the inventors have made research efforts to secure novel microorganisms and their metabolites that act specifically on diamondback moth larvae while possessing structural and biosynthetic characteristics different from existing formulations. As a result, diamondback moth ( Plutella xylostella A novel microorganism of the genus Saccharopolisphora represented by Sequence No. 1 exhibiting insecticidal activity against ) larvae ( Saccharopolyspora The present invention was completed by isolating sp.) AN150100 KCTC14897BP, isolating and purifying Spinosyn α4 among the secondary metabolites produced by the microorganism, and confirming that it exhibits excellent insecticidal activity against diamondback moth larvae. Prior art literature

[0009] Front. Plant Sci., 19 June 2015Sec. Plant Pathogen InteractionsVolume 6 The problem to be solved

[0010] The object of the present invention is a novel microorganism of the genus Saccharopolisphora having a 16S rDNA sequence represented by SEQ ID NO. 1 ( Saccharopolyspora sp.) KR0007(AN150100) KCTC14897BP is provided.

[0011] Another objective of the present invention is to provide an insecticidal composition comprising the microorganism or its culture medium, supernatant, or product separated therefrom as an active ingredient.

[0012] Another objective of the present invention is to provide a compound represented by the following chemical formula 1 isolated from the microorganism.

[0013] [Chemical Formula 1]

[0014]

[0015] Another objective of the present invention is to provide an insecticidal composition comprising the compound or a salt thereof as an active ingredient.

[0017] The problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0019] The present invention relates to a novel microorganism of the genus Saccharopolisphora having a 16S rDNA base sequence represented by SEQ ID NO. 1 ( Saccharopolyspora sp.) KR0007(AN150100) provides KCTC14897BP.

[0020] The above microorganisms may have been isolated from soil.

[0021] The above microorganisms may grow on Bennett's, R2A, LB, PDA, ISP2, ISP4, and NA media.

[0022] The above microorganisms may be capable of growing under conditions of 20–37°C, pH 6–10, and 7% NaCl concentration.

[0023] The genome size of the above microorganism may be 9,372,325 bp and the GC content may be 67.9%.

[0024] The microorganism may contain 8,613 coding sequences (CDS), 62 tRNAs, and 15 rRNAs.

[0025] The above microorganism is the diamondback moth ( Plutella xylostella It may exhibit insecticidal activity against larvae.

[0026] In addition, the present invention provides an insecticidal composition comprising the microorganism or its culture medium, supernatant, or product separated therefrom as an active ingredient.

[0027] The above product may contain Spinosyn α4.

[0028] The above composition may maintain insecticidal activity even at 20 times the volume of the culture medium.

[0029] The above composition may be used for controlling diamondback moth larvae in Chinese cabbage or cabbage.

[0030] In addition, the present invention provides a compound represented by the following chemical formula 1 isolated from the microorganism.

[0031] [Chemical Formula 1]

[0032]

[0033] The above compound may be extracted by an ethyl acetate solvent.

[0034] The above compound has the molecular formula C 41 H 62 O 11 It may be Spinosyn α4 having a molecular weight m / z 730.42921.

[0035] In addition, the present invention provides an insecticidal composition comprising the above compound or a salt thereof as an active ingredient.

[0036] The above composition is for diamondback moths ( Plutella xylostella It may exhibit insecticidal activity against larvae. Effects of the invention

[0038] A novel microorganism having the 16S rDNA base sequence represented by SEQ ID NO. 1 according to the present invention Saccharopolyspora sp. AN150100 exhibits excellent insecticidal activity against diamondback moth larvae and can effectively replace existing chemical insecticides and commercial biological insecticides due to resistance issues. The microbial culture maintains stable activity even under high dilution conditions, making it highly effective in agricultural settings; furthermore, as it is based on a novel naturally isolated strain, it offers the potential for independent industrial application distinct from existing spinosin-producing strains.

[0039] In addition, it was confirmed that Spinosyn α4, which exhibits high insecticidal activity against diamondback moth larvae, can be produced with high efficiency. Brief explanation of the drawing

[0041] Fig. 1 is Saccharopolyspora This shows the results of the 16S rRNA phylogenetic analysis of sp. AN150100. Figure 2 is Saccharopolyspora This shows the morphological characteristics of sp. AN150100. Fig. 3 is Saccharopolyspora This is a circular genome map showing the results of the whole-genome analysis of sp. AN150100. Fig. 4 is Saccharopolyspora The insecticidal activity of sp. AN150100 culture solution or dilution against diamondback moth larvae was evaluated. Fig. 5 is Saccharopolyspora Active compound C1 was isolated from the culture medium of sp. AN150100. Figure 6 is the measured value for determining the structure of active compound C1 1 This shows the H NMR spectrum. Figure 7 shows the HPLC chromatogram and high-resolution mass spectrometry (HR-ESIMS) spectrum analysis results of the active compound C1. Fig. 8 is Saccharopolyspora A total of 36 species, including sp. AN150100 Saccharopolyspora This shows the results of whole-genome-based phylogenetic analysis of the related strains. Fig. 9 is Saccharopolyspora This shows the results of ROARY-based pangenome analysis for four similar strains including sp. AN150100, showing the distribution of core and shell genes. Fig. 10 is Saccharopolyspora The Spinosyn biosynthetic gene cluster (BGC) identified in sp. AN150100 is a reference strain ( S. sp inosa, S. pogona This shows the results of a similarity analysis compared with the cluster of ). Figure 11 shows the functional arrangement of the major polyketide synthase (PKS) module and glycosylation-methylation related genes constituting the Spinosyn α4 biosynthetic pathway in the form of a schema. Figure 12 shows chromatograms measured under the same HPLC conditions to confirm the purity of Spinosyn α4(C1), spinosad, and spinetoram. Figure 13 shows the results of a comparison of the larval killing activities of Spinosyn α4(C1), spinosad, and spinetoram against diamondback moth larvae. Specific details for implementing the invention

[0042] Preferred embodiments according to the present invention will be described in detail below with reference to the attached drawings.

[0043] The advantages and features of the present invention and the method for achieving them will become clear by referring to the embodiments described in detail below together with the accompanying drawings.

[0044] However, the present invention is not limited by the embodiments disclosed below but may be implemented in various different forms, and these embodiments are provided merely to make the disclosure of the present invention complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0045] In addition, in describing the present invention, if it is determined that related known technologies, etc., may obscure the essence of the present invention, a detailed explanation thereof will be omitted.

[0047] The present invention will be described in detail below.

[0048] One aspect of the present invention for achieving the above objective is a novel microorganism of the genus Saccharopolisphora having a 16S rDNA sequence represented by SEQ ID NO. 1 ( Saccharopolyspora sp.) KR0007(AN150100) provides KCTC14897BP.

[0049] The terminology of the present invention, "Saccharopolisphora genus ( Saccharopolyspora "Sp. " strains refer to Gram-positive bacteria belonging to the order Actinomycetales, specifically aerobic actinomycetes found primarily in natural environments such as soil and plant residues. These Saccharopspora strains possess an actinomycete morphology characterized by branched hyphae, produce genomes with high GC content, and generate diverse secondary metabolites, particularly polyketide synthase-based metabolites. Representative examples include Saccharopolyspora spinosa It is known that spinosyns are produced. Strains of the genus Saccharopolisphora are known as a major group of strains that produce antibiotics, insecticides, and other bioactive substances, and their diversity in nature is also relatively high.

[0050] In the present invention, the genus Saccharopolisphora ( Saccharopolyspora The strain sp.) KR0007(AN150100) or AN150100 is a naturally occurring actinomycete isolated from Korean soil samples, and it has been confirmed to belong to the genus Saccharopolisphora through 16S rRNA sequencing and phylogenetic analysis. It is a novel strain that has sequence, genetic, and physiological characteristics that distinguish it from existing known strains.

[0051] The inventors of the present invention Streptomyces It was named sp. KR0007(AN150100), deposited with the Gene Bank of the Korea Research Institute of Bioscience and Biotechnology on March 14, 2022, and assigned accession number KCTC 14897BP. In this specification, the above strain is referred to as “ Streptomyces It is commonly referred to as “sp. AN150100” or “AN150100”.

[0052] In the present invention, the Saccharopolispora strain or Saccharopolyspora The term sp. can be interpreted to include AN150100 or variant strains, mutant strains, and functionally equivalent strains derived from this strain. Additionally, in the present invention, the strain may include the cells, dried cells, or crushed material of a strain of the genus Saccharopolispora. In this case, the dried cells may be dried cells such as spray-dried cells, freeze-dried cells, vacuum-dried cells, or drum-dried cells, and the crushed material may be a product obtained by crushing the cell wall of the strain itself by chemical or physical force.

[0053] According to one embodiment of the present invention, a novel microorganism having a 16S rDNA sequence represented by SEQ ID NO. 1 is obtained from a soil sample from the region of Sunchang-gun, Jeollabuk-do, Korea. Saccharopolyspora sp. KR0007(AN150100) KCTC14897BP was isolated. Soil samples were serially diluted and plated on humic acid-vitamin agar medium, then incubated at 28°C for 7 days. Afterward, a single colony was selected and subcultured to establish a pure culture of the strain.

[0054] In addition, according to one embodiment of the present invention, the 16S rRNA gene (27F / 1492R primer set) of the isolated strain was amplified to obtain a nucleotide sequence, which was then compared with the sequences in the EZBioCloud and NCBI databases, and as a result, the strain Saccharopolyspora hattusasensis , S. spinosa , S. pogona It was analyzed to have high homology with the back. Based on the above sequence, a phylogenetic tree was constructed using the Neighbor-Joining algorithm of the MEGA7 program, and as a result, AN150100 is Saccharopolyspora It was confirmed that it is a novel strain that forms an independent cluster within the genus (Fig. 1).

[0055] In addition, according to one embodiment of the present invention, the morphological characteristics of the AN150100 strain were evaluated through colony morphology on R2A agar medium and observation of cell structure using a scanning electron microscope (SEM), and it was confirmed that white circular colonies were formed on R2A medium and that a branched hyphae structure characteristic of actinomycetes was exhibited (Fig. 2).

[0056] In addition, in one embodiment of the present invention, the growth conditions of the AN150100 strain were evaluated, and it was confirmed that the strain could grow in various media such as Bennett's, R2A, LB, PDA, ISP2, ISP4, and NA media, and could grow in a temperature range of 20 to 37°C, under conditions of pH 6 to 10, and at a concentration of 0 to 7% NaCl (Table 2).

[0057] Accordingly, the above AN150100 strain may be considered to be a strain with environmental resistance suitable for industrial cultivation and mass production.

[0058] In one embodiment of the present invention, whole-genome sequencing was performed to determine the genomic structure and gene composition of the AN150100 strain. As a result, the total genome size was analyzed to be 9,372,325 bp, and the GC content of the total nucleotide sequence was 67.9%, characterized by a high GC ratio. Genome annotation results included a total of 8,613 coding sequences (CDS), and identified 62 tRNA genes and 15 rRNA genes (including 5S, 16S, and 23S) (Fig. 3). This result is consistent with the typical genome structure of actinomycetes.

[0059] In addition, in one embodiment of the present invention, the isolated novel microorganism strain AN150100 is used against diamondback moths ( Plutella xylostella It was confirmed that it exhibits insecticidal activity against larvae. The lethality of larvae was measured by treating leaf discs with the culture solution, and high insecticidal activity was observed even under dilution conditions ranging from 1 to 20 times. In addition, the number of surviving larvae was significantly reduced in whole-plant experiments using Chinese cabbage and cabbage (Fig. 4).

[0061] Another aspect of the present invention for achieving the above objective is the genus Saccharopolisphora ( Saccharopolyspora The present invention provides an insecticidal composition comprising sp.) KR0007(AN150100) KCTC14897BP or its culture medium, supernatant, or product separated therefrom as an active ingredient.

[0062] The above term, genus Saccharopolisphora ( Saccharopolyspora sp.) is as described above.

[0063] The term "culture solution" of the present invention is Saccharopolyspora It refers to a product obtained by culturing sp. AN150100 in a culture medium. Specifically, it may refer to the entire culture including the entire medium (cells + supernatant) after culturing the microorganism in the medium; more specifically, the culture medium contains all live or dead cells (mycelia, cells), all metabolites (primary and secondary metabolites) released by the cells into the medium, and other soluble components.

[0064] The above culture may refer to the entire culture, culture supernatant, lysate, fractions thereof, etc. of the above-mentioned strain of the genus Saccharopolisphora. In this case, the culture supernatant may be obtained by centrifuging the culture of the strain, the lysate may be obtained by physically or ultrasonically treating the strain, and the fractions may be obtained by applying the culture, culture supernatant, lysate, etc. to methods such as centrifugation or chromatography.

[0065] The culture medium and other culture conditions used for culturing the strain of the present invention may be any medium used for culturing microorganisms of the genus Saccharopolisphora without any particular limitations, and specifically, the strain of the present invention may be cultured under aerobic or anaerobic conditions while controlling the temperature, pH, etc. in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compound, amino acid and / or vitamin.

[0066] The above carbon sources may include, but are not limited to, carbohydrates such as glucose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvate, lactic acid, citric acid, etc.; and amino acids such as glutamate, methionine, lysine, etc. Additionally, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue, and corn steeping liquid may be used, and carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted into reducing sugars) may be used, and other appropriate amounts of carbon sources may be used without limitation. These carbon sources may be used individually or in combination of two or more types.

[0067] The above nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; and organic nitrogen sources such as amino acids, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0068] The above ingredients may include potassium monophosphate, potassium diphosphate, or corresponding sodium-containing salts. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used.

[0069] In addition, the above medium may contain amino acids, vitamins, and / or suitable precursors. Specifically, L-amino acids, etc. may be added to the culture medium of the strain. Specifically, glycine, glutamate, and / or cysteine, etc. may be added, and if necessary, L-amino acids such as lysine may be further added, but are not necessarily limited thereto.

[0070] The above medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.

[0071] In addition, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in an appropriate manner during the culture of the strain. In addition, bubble formation can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester during the culture, and oxygen or oxygen-containing gas can be injected into the culture to maintain an aerobic state of the culture, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection to maintain an anaerobic and microaerobic state.

[0072] The above fraction refers to the result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture containing various constituent components.

[0073] The fractionation method for obtaining the above fraction is not particularly limited and may be carried out according to methods commonly used in the relevant technical field as long as a fraction having the intended effect of the present invention can be obtained. Non-limiting examples of the above fractionation method include the genus Saccharopolisphora ( Saccharopolyspora sp.) AN150100 A method for obtaining a fraction from an extract obtained by adding a solvent to the supernatant of a culture and treating the extract with a predetermined fractionation solvent can be cited.

[0074] The type of fractionation solvent is not particularly limited, and any solvent known in the relevant technical field may be used as long as a fraction having the desired effect of the present invention can be obtained. Non-limiting examples of the fractionation solvent include organic solvents such as water, alcohol, ethyl acetate, acetone, or chloroform. These may be used alone or in a mixture of two or more. Specifically, the fractionation solvent may be selected from the group consisting of water, hexane, chloroform, ethyl acetate, butanol, or a mixture thereof, but is not limited thereto.

[0075] In the present invention, the product may mean the entirety of metabolites produced by the microorganism during the culture process that exhibit insecticidal activity against diamondback moth larvae, and specifically, Spinosyn α4 may be included, but other insecticidal active substances produced by the microorganism are not excluded.

[0076] In one embodiment of the present invention, the composition can maintain effective insecticidal activity even when the microbial culture solution is diluted to a high magnification. For example, an insecticidal effect was confirmed even when the culture solution was diluted 20 times (Fig. 4).

[0077] In addition, in one embodiment of the present invention, the composition of the present invention exhibited insecticidal activity against Chinese cabbage or cabbage under both leaf disc treatment and whole plant treatment conditions (Fig. 4).

[0078] This suggests that it can be applied to the control of diamondback moth larvae present in cruciferous crops such as Chinese cabbage or cabbage.

[0080] Another aspect of the present invention for achieving the above objective is the genus Saccharopolisphora ( Saccharopolyspora Provides a compound represented by the following chemical formula 1, isolated from sp.) KR0007(AN150100) KCTC14897BP.

[0081] [Chemical Formula 1]

[0082]

[0084] The above term, the genus Saccharopolisphora, is as described above.

[0085] The above compound is characterized as being an ethyl acetate extract.

[0086] The above extract is of the genus Saccharopolisphora ( Saccharopolyspora It includes the extract itself and all formulations of extracts that can be formed using the extract, such as an extract obtained by extracting a culture medium of the AN150100 strain, a diluted or concentrated extract of the extract, a dried product obtained by drying the extract, a modified or purified product of the extract, or a mixture thereof.

[0087] The method for preparing the above extract is not particularly limited and can be extracted according to methods commonly used in the relevant technical field. Non-limiting examples of the above extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, and these may be performed individually or in combination of two or more methods. In addition, to obtain a high-purity extract, the extract may be extracted one or more times using the same method.

[0088] The type of extraction solvent used for the preparation of the above extract is not particularly limited, and any solvent known in the art may be used as long as an extract having the desired effect of the present invention can be obtained. For example, one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, or chloroform may be used. More specifically, ethyl acetate may be used as a solvent.

[0089] In addition, the above compound has the molecular formula C 41 H 62 O 11 It is characterized by being Spinosyn α4 having a molecular weight m / z 730.42921.

[0090] In one embodiment of the present invention, the microorganism was cultured in a liquid medium, and then centrifuged at 8,000 rpm for 15 minutes to separate the mycelium and the supernatant. The mycelium was extracted with an equal volume of acetone, and the supernatant was separated into solvent layers by solvent partitioning in the order of ethyl acetate (EtOAc) and n-butanol (n-BuOH). As a result of evaluating the insecticidal activity of each solvent extract by dissolving them in methanol, only the ethyl acetate (EtOAc) extract showed significant insecticidal activity (Fig. 5). The active EtOAc extract was loaded into a C18 Sep-Pak cartridge and eluted under a 50–100% methanol (MeOH) gradient to obtain fractions (Fig. 5).

[0091] In addition, in one embodiment of the present invention, it was confirmed that among the fractions obtained by elution under methanol (MeOH) gradient conditions, the two fractions E-S90 and E-S100 exhibited the highest insecticidal activity against diamondback moth larvae, and after combining the E-S90 and E-S100 fractions, a single peak (C1) was isolated. The purified compound C1 was 87.4 mg in total and was detected at a retention time (Rt) of 20.31 min on the HPLC chromatogram (Fig. 7).

[0093] Another aspect of the present invention for achieving the above objective provides an insecticidal composition comprising the compound or a salt thereof as an active ingredient.

[0094] The above compound may refer to Spinosyn α4 represented by the following chemical formula 1.

[0095] [Chemical Formula 1]

[0096]

[0097] In the present invention, Spinosyn α4, the compound represented by Chemical Formula 1, can be isolated from natural materials by known methods or synthesized by known chemical synthesis methods. Additionally, the compound represented by Chemical Formula 1 can be obtained and used from the market. Specifically, the compound represented by Chemical Formula 1 is Saccharopolyspora It may be separated from sp. AN150100, but is not limited thereto.

[0098] In the present invention, the salt of the compound represented by Chemical Formula 1 is one commonly used in the art, and there are no limitations as long as the salt has an insecticidal effect. Specifically, the inorganic base may be an alkali metal such as sodium, potassium, and lithium, an alkaline earth metal such as calcium and magnesium, ammonia, etc., the organic base may be pyridine, colidine, triethylamine, and triethanolamine, etc., and the organic acid may be formic acid, acetic acid, tartaric acid, malic acid, citric acid, oxalic acid, succinic acid, benzoic acid, picric acid, methanesulfonic acid, and p-toluenesulfonic acid.

[0099] When the compound of the present invention is used as an active ingredient in an insecticidal composition, it may be used as itself or in the form of a salt without the addition of any other ingredients. Additionally, the compound of the present invention may be mixed with a solid carrier, a liquid carrier, a gas carrier, or an attractant, or absorbed into a basic substance, such as a porous ceramic plate or a nonwoven fabric, and then a surfactant and, if necessary, other auxiliary agents may be added to form it into various forms, such as an oil spray, an emulsifiable concentrate, a wet powder, a liquid, a granule, a dust, an aerosol, a fumigant, a vaporizable formulation, a smoking formulation, a toxic attractant, a tick-repellent sheet, or a resin formulation. Each of the above formulations may typically contain 0.01 to 99 weight % of one or more of the compound of the present invention as an active ingredient.

[0100] Solid carriers that can be used in formulations may include fine powders or granules of clay materials such as kaolin clay, diatomite, synthetic hydrated silicon dioxide, bentonite, fubasami clay, and acid clay; various talc, ceramics, and other inorganic materials, e.g., sericite, quartz, sulfur, activated carbon, calcium carbonate, and hydrated silica; and chemical fertilizers, e.g., ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, and ammonium chloride.

[0101] The liquid carrier may include water; alcohols, e.g., methanol and ethanol; ketones, e.g., acetone and methyl ethyl ketone; aromatic hydrocarbons, e.g., benzene, toluene, xylene, ethylbenzene and methylnaphthalene; aliphatic hydrocarbons, e.g., hexane, cyclohexane, kerosene and light oil; esters, e.g., ethyl acetate and butyl acetate; nitriles, e.g., acetonitrile and isobutyrnitrile; ethers, e.g., diisopropyl ether and dioxane; acid amides, e.g., N,N-dimethylformamide and N,N-dimethylacetamide; halogenated hydrocarbons, e.g., dichloromethane, trichloroethane and carbon tetrachloride; dimethyl sulfoxide; and vegetable oils, e.g., soybean oil and cottonseed oil.

[0102] The gas carrier or propellant may include Freon gas, butane gas, LPG, dimethyl ether, and carbon dioxide.

[0103] Basic substances for use in toxic dispersants may include dispersant substances, e.g., grain powder, vegetable oil, sugar, and crystalline cellulose; antioxidants, e.g., dibutylhydroxytoluene and nordihydroguaiaretic acid; preservatives, e.g., dehydroacetic acid; non-edible anti-sweetening substances, e.g., chili powder; and dispersible flavors, e.g., cheese flavor and onion flavor.

[0104] Surfactants may include alkyl sulfates, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and their polyoxyethylene derivatives, polyethylene glycol ethers, polyhydric alcohol esters, and sugar alcohol derivatives.

[0105] Auxiliaries such as adhesives or dispersants may include casein, gelatin; polysaccharides, e.g., starch, gum arabic, cellulose derivatives, and alginic acid; lignin derivatives, bentonite, sugar, and synthetic water-soluble polymers, e.g., polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylic acid.

[0106] Stabilizers may include PAT (isopropyl acid phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (a mixture of 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol), vegetable oil, mineral oil, surfactants, fatty acids and their esters.

[0107] The above composition of the present invention is for diamondback moths ( Plutella xylostella It may exhibit insecticidal activity against larvae.

[0108] [Chemical Formula 1]

[0109]

[0111] The above term, the genus Saccharopolisphora, is as described above.

[0112] The above compound is characterized as being an ethyl acetate extract.

[0113] The above extract is of the genus Saccharopolisphora ( Saccharopolyspora It includes the extract itself and all formulations of extracts that can be formed using the extract, such as an extract obtained by extracting a culture medium of the AN150100 strain, a diluted or concentrated extract of the extract, a dried product obtained by drying the extract, a modified or purified product of the extract, or a mixture thereof.

[0114] The method for preparing the above extract is not particularly limited and can be extracted according to methods commonly used in the relevant technical field. Non-limiting examples of the above extraction method include hot water extraction, ultrasonic extraction, filtration, and reflux extraction, and these may be performed individually or in combination of two or more methods. In addition, to obtain a high-purity extract, the extract may be extracted one or more times using the same method.

[0115] The type of extraction solvent used for the preparation of the above extract is not particularly limited, and any solvent known in the art may be used as long as an extract having the desired effect of the present invention can be obtained. For example, one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, ethyl acetate, acetone, or chloroform may be used. More specifically, ethyl acetate may be used as a solvent.

[0116] In addition, the above compound has the molecular formula C 41 H 62 O 11 It is characterized by being Spinosyn α4 having a molecular weight m / z 730.42921.

[0117] In one embodiment of the present invention, the microorganism was cultured in a liquid medium, and then centrifuged at 8,000 rpm for 15 minutes to separate the mycelium and the supernatant. The mycelium was extracted with an equal volume of acetone, and the supernatant was separated into solvent layers by solvent partitioning in the order of ethyl acetate (EtOAc) and n-butanol (n-BuOH). As a result of evaluating the insecticidal activity of each solvent extract by dissolving them in methanol, only the ethyl acetate (EtOAc) extract showed significant insecticidal activity (Fig. 5). The active EtOAc extract was loaded into a C18 Sep-Pak cartridge and eluted under a 50–100% methanol (MeOH) gradient to obtain fractions (Fig. 5).

[0118] In addition, in one embodiment of the present invention, it was confirmed that among the fractions obtained by elution under methanol (MeOH) gradient conditions, the two fractions E-S90 and E-S100 exhibited the highest insecticidal activity against diamondback moth larvae, and after combining the E-S90 and E-S100 fractions, a single peak (C1) was isolated. The purified compound C1 was 87.4 mg in total and was detected at a retention time (Rt) of 20.31 min on the HPLC chromatogram (Fig. 7).

[0120] Another aspect of the present invention for achieving the above objective provides an insecticidal composition comprising the compound or a salt thereof as an active ingredient.

[0121] The above compound may refer to Spinosyn α4 represented by the following chemical formula 1.

[0122] [Chemical Formula 1]

[0123]

[0124] In the present invention, Spinosyn α4, the compound represented by Chemical Formula 1, can be isolated from natural materials by known methods or synthesized by known chemical synthesis methods. Additionally, the compound represented by Chemical Formula 1 can be obtained and used from the market. Specifically, the compound represented by Chemical Formula 1 is Saccharopolyspora It may be separated from sp. AN150100, but is not limited thereto.

[0125] In the present invention, the salt of the compound represented by Chemical Formula 1 is one commonly used in the art, and there are no limitations as long as the salt has an insecticidal effect. Specifically, the inorganic base may be an alkali metal such as sodium, potassium, and lithium, an alkaline earth metal such as calcium and magnesium, ammonia, etc., the organic base may be pyridine, colidine, triethylamine, and triethanolamine, etc., and the organic acid may be formic acid, acetic acid, tartaric acid, malic acid, citric acid, oxalic acid, succinic acid, benzoic acid, picric acid, methanesulfonic acid, and p-toluenesulfonic acid.

[0126] When the compound of the present invention is used as an active ingredient in an insecticidal composition, it may be used as itself or in the form of a salt without the addition of any other ingredients. Additionally, the compound of the present invention may be mixed with a solid carrier, a liquid carrier, a gas carrier, or an attractant, or absorbed into a basic substance, such as a porous ceramic plate or a nonwoven fabric, and then a surfactant and, if necessary, other auxiliary agents may be added to form it into various forms, such as an oil spray, an emulsifiable concentrate, a wet powder, a liquid, a granule, a dust, an aerosol, a fumigant, a vaporizable formulation, a smoking formulation, a toxic attractant, a tick-repellent sheet, or a resin formulation. Each of the above formulations may typically contain 0.01 to 99 weight % of one or more of the compound of the present invention as an active ingredient.

[0127] Solid carriers that can be used in formulations may include fine powders or granules of clay materials such as kaolin clay, diatomite, synthetic hydrated silicon dioxide, bentonite, fubasami clay, and acid clay; various talc, ceramics, and other inorganic materials, e.g., sericite, quartz, sulfur, activated carbon, calcium carbonate, and hydrated silica; and chemical fertilizers, e.g., ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, and ammonium chloride.

[0128] The liquid carrier may include water; alcohols, e.g., methanol and ethanol; ketones, e.g., acetone and methyl ethyl ketone; aromatic hydrocarbons, e.g., benzene, toluene, xylene, ethylbenzene and methylnaphthalene; aliphatic hydrocarbons, e.g., hexane, cyclohexane, kerosene and light oil; esters, e.g., ethyl acetate and butyl acetate; nitriles, e.g., acetonitrile and isobutyrnitrile; ethers, e.g., diisopropyl ether and dioxane; acid amides, e.g., N,N-dimethylformamide and N,N-dimethylacetamide; halogenated hydrocarbons, e.g., dichloromethane, trichloroethane and carbon tetrachloride; dimethyl sulfoxide; and vegetable oils, e.g., soybean oil and cottonseed oil.

[0129] The gas carrier or propellant may include Freon gas, butane gas, LPG, dimethyl ether, and carbon dioxide.

[0130] Basic substances for use in toxic dispersants may include dispersant substances, e.g., grain powder, vegetable oil, sugar, and crystalline cellulose; antioxidants, e.g., dibutylhydroxytoluene and nordihydroguaiaretic acid; preservatives, e.g., dehydroacetic acid; non-edible anti-sweetening substances, e.g., chili powder; and dispersible flavors, e.g., cheese flavor and onion flavor.

[0131] Surfactants may include alkyl sulfates, alkyl sulfonates, alkyl aryl sulfonates, alkyl aryl ethers and their polyoxyethylene derivatives, polyethylene glycol ethers, polyhydric alcohol esters, and sugar alcohol derivatives.

[0132] Auxiliaries such as adhesives or dispersants may include casein, gelatin; polysaccharides, e.g., starch, gum arabic, cellulose derivatives, and alginic acid; lignin derivatives, bentonite, sugar, and synthetic water-soluble polymers, e.g., polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylic acid.

[0133] Stabilizers may include PAT (isopropyl acid phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (a mixture of 2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol), vegetable oil, mineral oil, surfactants, fatty acids and their esters.

[0134] The above composition of the present invention is for diamondback moths ( Plutella xylostella It may exhibit insecticidal activity against larvae.

[0136] 실험예 1. 미생물의 분류학적 특성 분석

[0137] To determine the taxonomic position of the microorganism, genomic DNA was isolated using a microbial DNA extraction kit according to the manufacturer's protocol. Using the isolated DNA as a template, the 16S rRNA gene was PCR amplified using primer sets 27F and 1492R, and homology search (Blast) was performed on the amplified sequences using EZbiocloud (https: / / www.ezbiocloud.net / ).

[0138] Saccharopolyspora The 16S rRNA sequences of 34 reference strains for the taxonomic analysis of the sp. AN150100 strain were collected from the National Center for Biotechnology Information (NCBI). Based on the collected sequences, a phylogenetic tree was constructed using the Neighbor-Joining (NJ) algorithm with MEGA 7.0 software, and the branching stability of the phylogenetic tree was evaluated through bootstrap analysis with 1,000 replicates.

[0140] 실험예 2. 배추좀나방( Plutella xylostella ) 유충에 대한 살충 활성 평가

[0141] 2-1. 잎 디스크 침지법에 의한 살충 활성 평가

[0142] Microorganisms' diamondback moth ( Plutella xylostella To evaluate the insecticidal activity against larvae, a leaf disc immersion method was performed. Cabbage leaf discs with a diameter of 3 cm were prepared, immersed in the supernatant of AN150100 culture solution for 30 seconds, and then air-dried. Ten diamondback moth larvae were inoculated onto each treated leaf disc, and the procedure was repeated three times under the same conditions.

[0143] The lethality and growth inhibition rates of the larvae were recorded at 24-hour intervals after treatment, and the final evaluation was performed on the third day after treatment. The negative control group was tested under the same conditions using distilled water.

[0145] 2-2. Whole-plant Bioassay

[0146] At the whole-plant level, adult diamondback moths were inoculated to induce larval development, and to confirm insecticidal activity against the larvae, cabbage and Chinese cabbage were cultivated for 21 days under greenhouse conditions (30 / 25 ± 5℃, light / dark cycle 14 / 10 h). After inoculating the cultivated plants with adult diamondback moths, 20 mL of AN150100 culture solution (5x dilution) was sprayed onto each cabbage seedling (2 pots). The sprayed plants were placed in plastic boxes (30 cm × 25 cm × 30 cm) for management.

[0147] One hundred adult diamondback moths were inoculated per box, and a total of four pots, including two control pots, were tested under identical conditions. Two days after the initial inoculation, the supernatant of the AN150100 culture solution was re-sprayed under the same conditions. Insecticidal activity was evaluated by counting the number of larvae in each group after the second treatment. All experiments were repeated independently three times.

[0149] Experimental Example 3. Statistical Analysis

[0150] The data obtained from the experiment were expressed as mean ± standard deviation (SD), and all experiments were performed independently in three repetitions. The collected data were statistically analyzed using IBM SPSS Statistics 27.0 software, and graphing and visualization were performed using Microsoft Excel.

[0152] Example 1. Saccharopolyspora AN150100 separation

[0153] 1-1. Microbial screening

[0154] Soil samples were collected from Gurim-myeon, Sunchang-gun, Jeollabuk-do to isolate the strain. First, the collected soil samples were serially diluted with distilled water, and a portion of the diluted solution was plated onto a humic acid-vitamin agar medium (1 g humic acid, 0.5 g Na2HPO4, 1.71 g KCl, 0.05 g MgSO4·7H2O, 0.01 g FeSO4·7H2O, 1 g CaCl2, Vitamin B group (0.5 mg each of thiamine-HCl, riboflavin, niacin, pyridoxine, calcium-pantothenic acid, inositol, and p-aminobenzoic acid, and 0.25 mg biotin), 15 g agar, 1 L water, pH 7.2). The plated media were incubated at 28°C for 7 days, and single colonies formed after incubation were selected and repeatedly subcultured until pure cultures were obtained, isolating a total of 500 streptomycetes. The obtained strains were stored at -80°C as an aqueous suspension containing 20% ​​(v / v) glycerol for subsequent experiments.

[0155] To select the above 500 isolated strains, each strain was cultured in liquid M3 medium (1% Soytone, 1% Glucose, 2% Soluble-Starch, 0.3% CaCO3, 0.02% FeSO4, 1 L water, pH 7.2) under identical conditions of 28°C and 160 rpm for 7 days. The cultured microbial solution was centrifuged at 8,000 rpm for 15 minutes to separate the supernatant.

[0156] The microorganisms isolated as described above were taxonomically classified based on 16S rRNA initial analysis and morphological observation by the method described in Experimental Example 1, and were distributed into a total of 44 genera. The distribution ratio Streptomyces (68%), Kitasatospora (4%), Rhodococcus (4%), Nocardia (4%), Kitasatospora (3%) etc., Saccharopolyspora A total of two strains of the genus were isolated.

[0158] 1-2. Insecticidal activity screening

[0159] Using the supernatant obtained by culturing a total of 500 strains of the above 44 genera under the same conditions, diamondback moth ( Plutella xylostella Screening for insecticidal activity against larvae was performed.

[0160] The specific experimental method is as described in Experimental Example 2 above.

[0161] As a result, the strain that was the only one among 500 strains to exhibit insecticidal activity against diamondback moth larvae was Saccharopolyspora sp. AN150100 was only one type.

[0163] Example 2. Saccharopolyspora sp. AN150100 characteristic analysis

[0164] 2-1. Taxonomic Analysis

[0165] In Example 1, the only one confirmed to have insecticidal activity against diamondback moth larvae Saccharopolyspora To analyze the taxonomic position of the sp. AN150100 strain, the 16S rRNA sequences of 34 reference strains were collected and analyzed from the National Center for Biotechnology Information (NCBI).

[0166] Saccharopolyspora As a result of analyzing the 16S rRNA gene sequence (1399 nt) of sp. AN150100, as shown in Fig. 1, Saccharopolyspora hattusasensis CR3506 T , Saccharopolyspora spinosa NRRL18395 T , Saccharopolyspora pogona NRRL 30141 T Showing homology of 99.4%, 98.9%, and 98.0%, respectively, the genus Saccharopolisphora ( Saccharopolyspora It was named sp.) AN150100, and this strain was deposited at the National Institute of Biotechnology and Bioengineering (NIBB) Biological Resource Center and assigned accession number KCTC14897BP on March 14, 2022.

[0168] 2-2. Morphological Analysis

[0169] The above Saccharopolyspora The morphological characteristics of the AN150100 strain classified into the genus were examined.

[0170] Morphological characteristics were observed using a scanning electron microscope (Philips SEM 515), and for this purpose, cells cultured in R2A medium at 28°C for 5 days were used.

[0171] As a result, as shown in Figures 2a and 2b, Saccharopolyspora sp. AN150100 exhibited a white, circular colony shape (Fig. 2a), and the cells observed under a scanning electron microscope showed a rod-shaped structure (Fig. 2b).

[0173] 2-3. Analysis of Physiological and Biochemical Characteristics

[0174] Saccharopolyspora The antibiotic susceptibility, carbon source availability, and enzyme activity of the sp. AN150100 strain were analyzed according to the manufacturer's instructions for the API ZYM and API 20NE kits (bioMirieus). Additionally, to determine the growth range, the feasibility of growth was evaluated by varying the temperature (4–42°C) and pH (5–10, in 1 pH increments) in R2A medium. Salt tolerance was tested by adding various concentrations of NaCl (1–10%, w / v) to R2A medium.

[0175] As a result, as shown in Table 1 below, AN150100 showed sensitivity to tetracycline, amikacin, kanamycin, vancomycin, chloramphenicol, teicoplanin, streptomycin, gentamicin, rifampicin, lincomycin, and erythromycin.

[0176] Antibiotics (weight / disc) Clear zone (diameter, mm) Nalidixic acid (30ug) - Tetracycline (30ug) 3.6 Amikacin (30ug) 3.4 Ampicilin / Sulbactam (20ug) - Kanamycin (30ug) 4 Vancomycin (30ug) 4.2 Chloramphenicol (30ug) 3.5 Teicoplanin (30ug) 3.7 Streptomycin (25ug) 3 Gentamicin (30ug) 2 Spectinomycin (25ug) - Rifampicin (30ug) 1.5 Lincomycin (15ug) 1.1 Erythromycin (30ug) 3.2

[0178] In addition, as shown in Table 2, SaccharopolysporaThe sp. AN150100 strain can grow on various media, including Bennett's medium, R2A medium, LB medium, PDA medium, ISP2 medium, ISP4 medium, and NA medium. It was capable of growth within a temperature range of 20–37℃ and a pH range of 6–10, and exhibited salt tolerance up to a maximum NaCl concentration of 7%. API ZYM and carbon source utilization tests revealed a total of 11 enzyme activities, and the strain assimilated most tested carbon sources, with the exception of capric acid and phenylacetic acid.

[0179] Characteristics AN150100 Colony color White Shape Rod Growth temperature (range, optimum) (℃) 20 - 37 (28) NaCl tolerance (range, optimum) (%) 0 - 7 (0) Growh pH (range, optimum) 6 - 10 (6 - 7) Growth media Bennett's, R2A, LB, PDA, ISP2, ISP4, NA API ZYM Lipase (C14) + Trypsin + α-chymotrypasin + Acid phosphatase + α-galactosidase + -galactosidase + -glucuronidase + α-glucosidase + -glucosidase + α-mannosidase + α-fucosidase + 20NE UREase + -glucosidase + Protease + D-glucose + L-arabinose + D-mannose + D-mannitol + N-acetyl-glucosamine + D-maltose + Potassium gluconate + Adipic acid + Malic acid + Trisodium citrate +

[0181] Example 3. Saccharopolyspora Genomic analysis of sp. AN150100

[0182] 3-1. Genome Sequencing

[0183] Saccharopolyspora For whole-genome analysis of the sp. AN150100 strain, genomic DNA isolated from the strain was analyzed using the PacBio RSII sequencing platform. Genomic DNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific) and a Qubit fluorometer (Qubit Fluorometer) according to the manufacturer's protocol. Libraries were prepared in 10 μL volumes containing 3–10 kb DNA using the PacBio DNA Template Prep Kit 1.0. The prepared SMRTbell templates were annealed using the PacBio DNA / Polymerase Binding Kit P6.

[0184] Sequencing was performed on a total of eight SMRT cells using the PacBio DNA Sequencing Kit (v4.0) and C4 chemistry, and each SMRT cell was captured for 240 minutes using the PacBio RSII platform (Pacific Biosciences). All sequencing was performed at Macrogen, and the PacBio Sample Net-Shared Protocol (http: / / pacificbiosciences.com) was used for data analysis. The acquired sequencing data were de novo assembled using HGAP 3.0.

[0186] 3-2. Comparative Genome Analysis

[0187] Saccharopolyspora The phylogenetic analysis of sp. AN150100 was obtained from NCBI Saccharopolyspora Based on the genus genome sequence, the study was performed using the Type Strain Genome Server (TYGS, https: / / tygs.dsmz.de).

[0188] For genome annotation, pangenome analysis was performed using GFF files generated with PROKKA v1.14.6 as input data for ROARY v3.13.0. Core and accessory genes of the target genome were identified using the ROARY output results.

[0190] As a result, as shown in Fig. 3, SaccharopolysporaThe total genome size of sp. AN150100 was confirmed to be 9,372,325 bp, and the GC content was 67.9%. The whole genome contains a total of 8,613 protein-coding genes (CDS), 62 tRNAs, and 15 rRNAs, and consists of a single chromosome and two plasmids. Genomic components such as CDS, RNA genes, GC content, CRISPR-Cas, and antimicrobial resistance genes (CARD) were derived through whole-genome annotation and visualized as a circular genome map.

[0192] Example 4. Saccharopolyspora of sp. AN150100 culture medium Plutella xylostella Evaluation of insecticidal activity

[0193] Saccharopolyspora The insecticidal activity of the sp. AN150100 culture solution was evaluated through leaf disc analysis and whole plant analysis.

[0194] The specific experimental method is as described in Experimental Example 2 above.

[0195] In the case of leaf disc analysis, cabbage leaf discs Saccharopolyspora The samples were immersed for 30 seconds in the undiluted solution or 1 / 5, 1 / 10, and 1 / 20 diluted solutions of sp. AN150100 culture medium, and then air-dried. The insecticidal effect of each treatment group was measured at 24, 48, and 72 hours after treatment.

[0196] As a result, as shown in Figures 4a and 4b, Saccharopolyspora Leaf discs treated with sp.AN150100 culture solution remained undamaged even at a 10-fold dilution (1 / 10), while a small number of larvae survived in the 20-fold dilution (1 / 20) treatment group (Fig. 4a). The undiluted solution and the 5-fold dilution treatment groups maintained a 100% insecticidal effect throughout the entire experimental period. The insecticidal rate of the 1 / 10 dilution treatment group decreased to 90% 24 hours after treatment, but returned to 100% insecticidal effect after 48 hours. The insecticidal rate was maintained at over 80% even at a 1 / 20 dilution (Fig. 4b).

[0198] The insecticidal effect at the whole plant level was evaluated on Chinese Cabbage and Cabbage using a 5-fold diluted culture solution.

[0199] As a result, as shown in Figures 4c and 4d, the insecticidal activity against diamondback moth larvae (Figure 4c) and the insecticidal activity value (Figure 4d) were confirmed. In Chinese cabbage, the lethality rate of diamondback moth larvae was 16%, and in cabbage, the number of surviving diamondback moth larvae decreased by 15%, Saccharopolyspora It was confirmed that the culture solution of sp.AN150100 exhibited significant insecticidal activity even at the plant level.

[0201] Example 5. Saccharopolyspora Isolation and identification of insecticidal active compounds of sp. AN150100

[0202] 5-1. Separation of Active Inducing Compounds

[0203] Saccharopolyspora After confirming insecticidal activity in the sp. AN150100 culture medium, the culture medium was solvent-divided to obtain the active ingredient.

[0204] Specifically, Saccharopolyspora 20 L of culture medium of sp. AN150100 was centrifuged to separate the mycelium and the supernatant. The mycelium was extracted with an equal volume of acetone, while the supernatant was extracted by sequential liquid-liquid partitioning with ethyl acetate (EtOAc) and n-butanol (n-BuOH). Each extract was concentrated using a rotary evaporator at 40°C, dissolved in methanol, and prepared for the insecticidal activity test. The insecticidal activity test was performed in the same manner as previously described.

[0205] As a result of the fractionation, insecticidal activity was confirmed only in the ethyl acetate (EtOAc) fraction, and no significant activity was observed in the butanol (n-BuOH) or water-soluble fractions.

[0206] Subsequently, EtOAc extracts were serially eluted using a C18 Sep-Pak cartridge under 50–100% MeOH conditions, and each eluted fraction was concentrated to evaluate insecticidal activity. As a result, the highest activity was confirmed in the two fractions E-S90 and E-S100. The two active fractions were purified using Prep-HPLC (flow rate 6 mL / min) under mobile phase conditions containing 55% acetonitrile (ACN) and 0.02% TFA, during which the C1 peak was obtained. The C1 fraction was used for the insecticidal activity test after solvent removal.

[0207] As a result, as shown in Figure 5, a single compound, C1, was purified with a total yield of 87.4 mg.

[0209] 5-2. Determination of the Structure of the Active Compound

[0210] 5-2-1. HPLC Analysis

[0211] High-performance liquid chromatography (HPLC) analysis was performed using a YL9100 HPLC system (four YL9100 pumps, a YL9160 PDA detector, and a YL9150 autosampler), and an Atlantis T3 column (4.6 × 250 mm, 5 μm; Waters) was used. The injection volume was 20 μL, and the mobile phase consisted of 5–100% acetonitrile (A) and a 0.02% TFA-containing aqueous solution (B). The flow rate was set to 0.8 mL / min and the detection wavelength to 254 nm.

[0212] As a result, as shown in Figure 7a, the single compound C1 separated by preparative HPLC appeared as a light gray powder, with a retention time of 20.31 min, and the UV absorption maximum (λmax) in PDA detection was confirmed to be 200 nm and 250 nm.

[0214] The structure of C1 is 1 H and 13It was determined through 3C NMR, second-order correlation analysis (COSY), heterojunction correlation (HMBC), and high-resolution mass spectrometry (HR-ESIMS).

[0216] 5-2-2. NMR Analysis

[0217] 1 1H NMR (700 MHz) and 13 The 175 MHz NMR spectrum was measured using a Bruker AVANCE HD 700 instrument under CD3OD solvent and TMS internal standard conditions.

[0218] 1 H NMR and 13 C1 NMR spectrum analysis confirmed that C1 possesses a macrolide system structurally consistent with the spinosyn scaffold. 1 The major signals observed in the H NMR spectrum included characteristic methan and methylene resonances, which were consistent with the macrocyclic backbone of spinosyn A. 1 Two olefinic proton signals identified in the H NMR spectrum suggested that C1 contains an unsaturated side chain.

[0219] Correlation spectroscopy (COSY) analysis confirmed spin coupling between H-21 and adjacent olefin protons, and these protons were additionally coupled to methylene and methyl groups. These chemical shift and binding patterns supported the presence of a butenyl group (-CH=CH-CH-CH3) at the C-21 position instead of an ethyl group in spinosyn A.

[0220] Downfield movement of H-25 also supported the modification of the side chain structure compared to spinosyn A.

[0221] In addition, COSY and HMBC spectral analysis confirmed that a sugar is attached to the hydroxyl group of C-17. This sugar was identified as damicetose, a monohydroxylated hexose. No additional methoxy group signal was observed, which clearly distinguishes C1 from methylated spinosine derivatives. In particular, no signals characteristic of methylamine or methoxy groups were observed in the NMR signals, confirming that C1 possesses a simpler and more compact glycoside and side chain structure (Table, Fig. 6).

[0222] Position Experimental data in CD 3 OD δ C δ H (multi) HMBC correlations 1 172.0 - 2 33.9 3.09 (m), 2.44 (dd) 1, 3, 4, 14 3 48.0 2.98 (m) 14 4 41.3 3.41 (m) 5 5 128.3 5.85 (dt) 7, 12 6 129.3 5.95 (dt) 5, 7, 8, 11 7 41.1 2.18 (m) 8 36.0 2.01 (m), 1.40 (m) 9, 10, 11 9 76.5 4.35 (q) 7, 11 10 37.1 2.36 (quin), 1.39 (m) 7, 8, 9, 11 11 46.1 0.97 (m) 7, 10 12 49.6 2.88 (ddt) 14 13 148.8 7.08 (s) 3, 11, 12, 15 14 143.7 - 15 203.3 - 16 47.1 3.43 (m) 15, 17 17 81.0 3.67 (m) 15 18 33.7 1.64 (m), 1.59 9m) 16, 17, 19 19 20.9 1.78 (m), 1.26 (m) 17, 18, 20 20 31.4 1.58 (m) 18, 19, 21 21 76.0 5.04 (m) 1, 19, 20 22 128.0 5.34 (ddt) 21, 24 23 134.3 5.67 (dt) 21, 24, 25 24 24.7 2.02 (m) 22, 25 25 12.3 0.97 (t) 24 26 15.2 1.20 (d) 15, 16 1' 95.6 4.88 (s) 2', 3', 5' 2' 77.2 3.59 (dd) 1', 3' 3' 81.1 3.46 (dd) 4' 4' 82.1 3.07 (t) 3', 5' 5' 67.7 3.56 (m) 1', 3', 4' 6' 16.7 1.24 (d) 5' 2'-OMe 57.7 3.48 (s) 2' 3'-OMe 56.3 3.47 (s) 3' 4'-OMe 59.7 3.53 (s) 4' 1" 102.6 4.66 (dd) 17, 2", 3", 5" 2" 29.6 2.12 (br dq), 1.62 (m) 1", 3", 4" 3" 19.2 2.16 (m), 1.89 (qd) 1", 2", 4", 5" 4" 65.5 3.11 (m) 5" 5" 69.7 3.87 (m) 1", 4" 6" 17.0 1.38 (d) 4", 5",

[0224] 5-2-3. 질량 분석(HR-ESIMS)

[0225] High-resolution electrospray ionization mass spectrometry (HR-ESIMS) was measured using the Q-TOF mass spectrometer (SYNAPT G2) at the Korea Basic Science Institute (KBSI, Ochang Center).

[0227] As a result of the above, as shown in Fig. 7, C1 exhibited a molecular ion peak at m / z 730.42921, and based on this, molecular formula C 41 H 62 O 11 It was decided as.

[0229] 실시예 6. Saccharopolyspora sp. AN150100의 유전체 비교 분석

[0230] Saccharopolyspora, Allosaccharopolyspora, Halosaccharopolyspora The genome sequences of a total of 36 strains belonging to the genus were obtained from NCBI, and based on this, the Type Strain Genome Server (TYGS, https: / / tygs.dsmz.de) was used.

[0231] The size of the obtained genome ranged from approximately 4 to 10 Mbp, and about 70% of the entire genome consisted of GC (Table 4).

[0232] Strains Accession number Genome size GC percent Gene Protein Allosaccharopolyspora coralli E2A NZ CP045929 4.8 68.5 4,509 4,328 Halosaccharopolyspora lacisalsi DSM 45975 NZ JACGWZ000000000 5.4 69 5,336 5,258 Saccharopolyspora sp. AN150100 9.4 67.9 8,690 8,613 Saccharopolyspora antimicrobica DSM 45119 RBXX01000000 8.3 70.5 7,749 7,522 Saccharopolyspora aridisoli 16K404 SMKV01000000 6 69.5 5,389 5,147 Saccharopolyspora cebuensis JCM 18116 BAABII010000000 6.2 73 5,953 5,881 Saccharopolyspora dendranthemae DSM 46699 VIWX01000000 6.4 69.5 5,858 5,782 Saccharopolyspora elongata 7K502 SMKW01000000 10.3 69.5 9,326 8,751 Saccharopolyspora endophytica KCTC 19397 JAGPXE010000000 7.2 70 6,666 6,528 Saccharopolyspora erythraea DSM 40517 PDBV01000000 8.2 71 7,409 7,069 Saccharopolyspora flava DSM 44771 FOZX01000000 6.3 71 5,676 5,601 Saccharopolyspora gloriosae DSM 45582 JACHIV010000000 6.8 71 6,053 5,984 Saccharopolyspora gregorii JCM 9687 BAAAYK010000000 7.7 72.5 7,322 7,223 Saccharopolyspora halophila JCM 16221 BAAARA010000000 5.5 70 5,113 5,042 Saccharopolyspora hattusasensis KCTC 29104 8.8 67.8 8,356 8,281 Saccharopolyspora hirsuta VKM And 666 VWPH01000000 7.5 71.5 6,851 6,658 Saccharopolyspora hordei DSM 44065 JACCFJ010000000 5.8 72.5 5,376 5,307 Saccharopolyspora indica KCTC 29208 JAQGLB010000000 8.6 71 8,037 7,924 Saccharopolyspora ipomoeae TS4A08 JASAOF010000000 6.6 70.5 6,031 5,953 Saccharopolyspora karakumensis 5K548 SMLA01000000 6.7 69.5 6,234 5,983 Saccharopolyspora kobensis ATCC 20501 FNVB01000000 7.7 71 7,221 7,149 Saccharopolyspora mangrovi S2 29 NZ JAWLNX000000000 7.2 69.5 6,846 6,692 Saccharopolyspora oryzae WRP15 2 JAQGLA010000000 8.1 70 7,646 7,524 Saccharopolyspora phatthalungensis DSM 45584 JACHIW01000000 8.5 67.5 7,673 7,606 Saccharopolyspora pogona NRRL30141 NZ CP031142 9.6 68 9,318 8,788 Saccharopolyspora rectivirgula DSM 43747 AYJW01000000 4 69 3,843 3,692 Saccharopolyspora rhizosphaerae H219 RSAA01000000 5.8 70.5 5,331 5,142 Saccharopolyspora rosea JCM 19121 BAABLT010000000 6.5 72 6,146 6,075 Saccharopolyspora shandongensis CGMCC 4 3530 FNOK01000000 10.2 70 9,312 9,236 Saccharopolyspora soli K220 JALBWV010000000 9.4 68.5 8,846 8,623 Saccharopolyspora spinosa DSM 44228 PJNB01000000 9 68 8,146 7,401 Saccharopolyspora spinosporotrichia JCM 10303 BAAAGS010000000 8.1 71 7,332 7,235 Saccharopolyspora subtropica CGMCC 4 7206 BMMT01000000 5.4 71 5,084 5,021 Saccharopolyspora taberi JCM 9383 BAAAUX010000000 7 71 6,776 6,698 Saccharopolyspora terrae 16K309 SMKS01000000 6.7 69.5 6,243 6,004 Saccharopolyspora thermophila JCM 10664 BAAAHC010000000 5.3 71 5,061 4,998

[0234] As a result of phylogenetic analysis, as shown in Figure 8, Saccharopolyspora sp. AN150100 is Saccharopolyspora pogona NRRL 30141, Saccharopolyspora spinosa NRRL 19395, Saccharopolyspora hattusasensis It formed the same cluster as CR3506, and this classification pattern was consistent with the results of 16S rRNA-based phylogenetic tree analysis.

[0236] As shown in Figure 9, the results of the ROARY-based pangenome analysis identified a total of 20,383 gene clusters in the four most similar strains. Of these, 2,982 were classified as core genes and 17,401 as shell genes. Core genes were found to be conserved as structural and functional components present in the genomes of all four strains.

[0237] The BGCs predicted in the AntiSMASH analysis are classified into several categories, such as non-ribosomal peptide synthases (NRPS) and polyketide synthases type I (PKS I), among which the butenyl-spinosyn biosynthetic gene cluster is Saccharopolyspora It was identified in the genomes of three strains, including sp. AN150100. The biosynthetic cluster was approximately 60.5 kb long and contained a total of 5 open reading frames (ORFs).

[0238] As a result of the BLAST-based comparative analysis, as shown in Fig. 10, the cluster of AN150100 is S. spinosa Spinosin D biosynthetic gene cluster in DSM 44228, S. pogona It was found to have high similarity to the BGC of NRRL 30141. In particular, the butyl spinosine biosynthetic cluster was found to be AN150100 and S. pogona It was commonly identified in NRRL 30141, and the BGC of the two strains S. spinosa It was analyzed to have a pattern very similar to the spinosin D cluster of DSM 44228.

[0240] 실시예 7. 게놈 기반 Spinosyn α4 생합성 유전자 클러스터 분석

[0241] Secondary metabolite biosynthesis gene cluster analysis is Saccharopolyspora AntiSMASH 7.0 (bacterial version) was performed on 36 genomes including sp. AN150100. Network analysis was performed on the finally identified Biosynthetic Gene Clusters (BGCs) using BiG-SCAPE v1.1.5 (Python 3.6.15), and the similarity networks between BGCs were visualized using Cytoscape v3.10.2 (https: / / cytoscape.org).

[0242] Saccharopolyspora As shown in Figure 11, the results of genome sequencing analysis of sp. AN150100 showed that antiSMASH predicted a total of 43 secondary metabolites (BGCs), and all identified biosynthetic gene clusters were found to be located on the chromosome. Genes annotated as proteins related to spinosine α4 biosynthesis in AN150100 were located in the interval from 5,569,661 bp to 5,647,811 bp, and the cluster was classified as the 27th secondary metabolite (BGC 27).

[0243] Biosynthetic genes were distributed over a range of approximately 148,662 bp, and the function of each gene was confirmed through NCBI BLAST analysis (Table 5).

[0244] Gene Locus tag Location Length (nt) Function Similarity spnA ctg_1 5038 5,617,247 - 5,630,320 13074 type I polyketide synthase 91.66 spnB ctg_1 5037 5,610,795 - 5,617,250 6456 type I polyketide synthase 91.18 spnC ctg_1 5036 5,601,244 - 5,610,750 9507 type I polyketide synthase 94.67 spnD ctg_1 5035 5,586,407 - 5,601,196 14790 type I polyketide synthase 92.19 spnE ctg_1 5034 5,569,661 - 5,586,355 16695 type I polyketide synthase 92.39 spnF ctg_1 5039 5,630,436 - 5,631,221 786 methyltransferase 96.17 spnG ctg_1 5040 5,631,716 - 5,632,888 1173 glycosyltransferase 95.67 spnH ctg_1 5041 5,632,928 - 5,633,680 753 TylF / MycF family methyltransferase 99.2 spnI ctg_1 5042 5,633,686 - 5,634,873 1188 class I SAM-dependent methyltransferase 100 spnJ ctg_1 5043 5,635,062 - 5,636,630 1569 FAD-binding oxidoreductase 94.6 spnK ctg_1 5044 5,636,644 - 5,637,837 1194 class I SAM-dependent methyltransferase 96.7 spnL ctg_1 5045 5,637,834 - 5,638,733 900 SAM-dependent methyltransferase 95.3 spnM ctg_1 5046 5,638,733 - 5,639,863 1131 Lipase 96.0 spnN ctg_1 5047 5,640,023 - 5,641,033 1011 Gfo / Idh / MocA family protein 94.9 spnO ctg_1 5048 5,641,030 - 5,642,490 1461 dTDP-4-dehydro-6-deoxy-alpha-D-glucopyranose 2,3-dehydratase 96.9 spnP ctg_1 5049 5,642,752 - 5,644,071 1320 activator-dependent family glycosyltransferase 94.7 spnQ ctg_1 5050 5,644,560 - 5,645,864 1305 dTDP-4-dehydro-2,6-dideoxy-D-glucose 3-dehydratase 97.2 spnR ctg_1 5051 5,645,902 - 5,647,038 1137 DegT / DnrJ / EryC1 / StrS family aminotransferase 95.2 spnS ctg_1 5052 5,647,071 - 5,647,811 741 class I SAM-dependent DNA methyltransferase 95.9

[0246] In addition, the basic structure of spinosine α4 is generated by type I polyketide synthase (PKS I), which is encoded by a cluster of five major genes: spn A, spn B, spn C, spn D, and spn E. This gene group occupies a total of 60.7 kb and includes 13 extension modules. Specifically, spn A consists of three biosynthetic initiation modules (M1, M2, M3) in which a butenyl group is added to the terminal, and spn J, spn L, spn M, and spn F were analyzed as genes involved in the formation of intramolecular CC bonds. spn G played a role in inducing rhamnose attachment, and it was confirmed that hydroxyl group methylation of rhamnose is carried out by spn H, spn I, and spn K.

[0247] In addition, the gene clusters included genes related to CC bond formation (spn F, J, L, M) and genes related to rhamnose attachment and methylation (spn G, H, I, K).

[0248] Additionally, the spn N, spn O, spn P, spn Q, spn R, and spn S genes were also detected in the genome, but it was confirmed that these genes may not be applicable to some butenyl spinosine biosynthesis.

[0250] 실시예 8. C1, 스피노사드 및 스피네토람의 산란기피성 및 살유충 활성 비교

[0251] First, to verify the accurate concentration and purity of C1, HPLC-based quantitative analysis was performed, and through chromatogram analysis obtained under the same HPLC conditions shown in Figure 12, the purity of C1 was confirmed to be 90%, and the purity of spinosad and spinetoram was 90% and 80%, respectively.

[0252] Subsequently, to evaluate the oviposition-repellent and larval-killing activities of the active compound C1, a comparative experiment was conducted by treating cabbage grown in a greenhouse for 21 days with C1, spinosad, and spinetoram.

[0253] As a result, as shown in Figure 13, the larval lethality of C1 at a concentration of 0.025 μg / mL was 26.7%, which was lower than that of spinetoram (63.3%) but similar to that of spinosad. On the other hand, at a concentration of 0.05 μg / mL, C1 exhibited insecticidal activity similar to that of spinetoram.

[0255] According to one embodiment of the present invention so far Saccharopolyspora Although specific examples regarding the insecticidal activity of sp. AN150100 and spinosin α4 derived from the microorganism above against diamondback moths have been described, it is obvious that various modifications are possible within the scope of the present invention.

[0256] Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined by the claims set forth below as well as equivalents thereof.

[0257] That is, the aforementioned embodiments should be understood as exemplary in all respects and not limiting, and the scope of the invention is defined by the claims set forth below rather than by the detailed description, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the invention.

[0259] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC14897BP Date of Deposit: 2022-03-14

Claims

Claim 1 A novel microorganism of the genus Saccharopolisphora having the 16S rDNA sequence denoted by SEQ ID NO. 1, deposited under accession ID KCTC14897BP ( Saccharopolyspora sp.) KR0007(AN150100) strain. Claim 2 A strain according to claim 1, characterized in that the strain is isolated from soil. Claim 3 The strain according to claim 1, characterized in that the strain grows in Bennett's agar medium (Bennett's), Reasoner's 2A agar (R2A), Luria-Bertani medium (LB), Potato Dextrose Agar (PDA), International Streptomyces Project Medium No. 2 (Yeast Extract-Malt Extract Agar) (ISP2), International Streptomyces Project Medium No. 4 (Inorganic Salts-Starch Agar) (ISP4), and Nutrient Agar (NA) medium. Claim 4 The strain according to claim 1, characterized in that the strain is capable of growing under conditions of 20–37℃, pH 6–10, and a 7% NaCl concentration. Claim 5 A strain according to claim 1, characterized in that the genome size of the strain is 9,372,325 bp and the GC content is 67.9%. Claim 6 The strain according to claim 1, characterized in that the strain comprises 8,613 coding sequences (CDS), 62 tRNAs, and 15 rRNAs. Claim 7 In paragraph 1, the strain is a diamondback moth ( Plutella xylostella A strain characterized by exhibiting insecticidal activity against larvae. Claim 8 Saccharopolisphora deposited under deposit number KCTC14897BP ( Saccharopolyspora An insecticidal composition for controlling diamondback moth (Plutella xylostella) larvae, comprising the strain sp.) KR0007(AN150100) or its culture medium, supernatant, or product isolated therefrom as an active ingredient. Claim 9 A composition according to claim 8, characterized in that the product comprises Spinosyn α4. Claim 10 A composition according to claim 8, characterized in that the above composition maintains insecticidal activity against diamondback moth larvae even at 20 times the volume of the culture medium. Claim 11 In claim 8, the composition is characterized by being used for controlling diamondback moth larvae in Chinese cabbage or cabbage. Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete

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