KR102313936B1 - Novel Streptomyces having various biological activity and use thereof - Google Patents
Novel Streptomyces having various biological activity and use thereof Download PDFInfo
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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Abstract
본 발명은 다양한 생리활성을 갖는 신규 방선균 및 이의 용도에 관한 것으로, 본 발명에서는 항진균 활성이 우수한 폴리엔 화합물을 생산하는 방선균을 유전체 검색기술로 선별한 후, NGS 기반으로 유전체를 해독하여 해당 생리활성물질의 특성을 분자수준에서 규명하고, 이들 유용 방선균 (혹은 방선균 유래 생리활성물질)을 생물농약으로 실용화하기 위한 배양공정 최적화를 수행하였다. 이에, 본 발명의 Streptomyces sp. AN090726(Inha501) 균주는 농작물 병원성 진균을 효과적으로 제어하므로 생물농약으로 유용하게 활용될 가능성이 있다.The present invention relates to novel actinomycetes having various physiological activities and uses thereof. In the present invention, actinomycetes that produce polyene compounds having excellent antifungal activity are selected by genome search technology, and then the genome is decoded based on NGS to decipher the physiological activity The characteristics of the substance were identified at the molecular level, and the culture process was optimized to put these useful actinomycetes (or actinomycetes-derived physiologically active substances) into practical use as biological pesticides. Accordingly, Streptomyces sp of the present invention. AN090726 (Inha501) strain effectively controls crop pathogenic fungi, so it has the potential to be useful as a biological pesticide.
Description
본 발명은 다양한 생리활성을 갖는 신규 방선균 및 이의 용도에 관한 것이다.The present invention relates to novel actinomycetes having various physiological activities and uses thereof.
미생물 기반 친환경 생물농약의 필요성은 더 이상의 설명이 필요 없을 정도로 절실하다. 현재 생물농약으로는, 바실러스 (Bacillus) 기반의 살충제를 포함한 다양한 미생물 종이 사용되고 있으나, 농업용 작물에 대한 항진균 생물농약으로는 방선균, 특히 스트렙토마이세스 (Streptomyces) 종이 다수를 차지하고 있다.The need for microbial-based eco-friendly biological pesticides is so urgent that no further explanation is needed. Currently the biopesticide is Bacillus (Bacillus), but based on a variety of microorganisms used paper, including pesticides, the anti-fungal biopesticide for agricultural crops is accounted for actinomycetes, particularly Streptomyces (Streptomyces) paper number.
국내 생물농약 (일명 천연식물보호제) 등록현황 (2012년 12월말 현재)에 따르면, 방선균 기반의 생물 농약으로는 (주)케이아이비씨의 방선균 2종 (스트렙토마이세스 고시키엔시스 더블유와이이324 액제, 스트렙토마이세스 콜롬비엔시스 더블유와이이20 액제)이 등록되어 사용되고 있다. According to the registration status of domestic biological pesticides (aka natural plant protection agents) (as of the end of December 2012), actinomycetes-based biological pesticides include two types of actinomycetes of KIBC (Streptomyces goshikiensis W.Y324 liquid formulation). , Streptomyces colombiensis W.Y.Y. 20 liquid) has been registered and used.
국외에서는 더욱 활발하게 방선균 기반 생물농약이 사용되고 있으며, 특히 미국 노보자임의 Streptomyces lydicus WYEC108 (상품명 Actinovate)와 핀란드 Verdera의 Streptomyces K61 (상품명 Mycostop)은 방선균을 주성분으로 하고 있다. 중국의 경우에도 폴리엔계 켄디시신 및 나타마이신을 생산하는 방선균 스트렙토마이세스 (Streptomyces)를 생물농약으로 활용하여 작물의 진균 감염 및 진균 공생 해충을 친환경적으로 제어하고 있다.Overseas, actinomycete-based biological pesticides are being used more actively, especially Streptomyces of Novozyme in the United States. lydicus WYEC108 (trade name Actinovate) and Streptomyces K61 (trade name Mycostop) from Verdera, Finland (trade name: Mycostop) contain actinomycetes as main ingredients. In the case of China utilizing polyene canned body and receive Actinomycetes Streptomyces (Streptomyces) to produce azithromycin as biopesticide and has eco-friendly control of the fungal infection and fungal symbiotic pest of the crop.
본 발명의 목적은 식물병원성 진균에 대한 항진균 활성을 갖는 스트렙토마이세스 속(Streptomyces sp.) AN090726(Inha501) 균주(KCTC13999BP) 및 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 식물병원성 진균 방제용 조성물을 제공하는데 있다. An object of the present invention is Streptomyces sp. AN090726 (Inha501) strain (KCTC13999BP) having antifungal activity against phytopathogenic fungi and a composition for controlling phytopathogenic fungi comprising the strain or a culture solution thereof as an active ingredient is to provide
상기 목적을 달성하기 위하여, 본 발명은 KCTC13999BP로 기탁되고, 식물병원성 진균에 대한 항진균 활성을 갖는 스트렙토마이세스 속(Streptomyces sp.) AN090726(Inha501) 균주를 제공한다. In order to achieve the above object, the present invention provides a strain of Streptomyces sp. AN090726 (Inha501) deposited as KCTC13999BP and having antifungal activity against phytopathogenic fungi.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 식물병원성 진균 방제용 조성물을 제공한다.In addition, the present invention provides a composition for controlling phytopathogenic fungi comprising the strain or a culture solution thereof as an active ingredient.
본 발명은 다양한 생리활성을 갖는 신규 방선균 및 이의 용도에 관한 것으로, 본 발명에서는 항진균 활성이 우수한 폴리엔 화합물을 생산하는 방선균을 유전체 검색기술로 선별한 후, NGS 기반으로 유전체를 해독하여 해당 생리활성물질의 특성을 분자수준에서 규명하고, 이들 유용 방선균 (혹은 방선균 유래 생리활성물질)을 생물농약으로 실용화하기 위한 배양공정 최적화를 수행하였다. 이에, 본 발명의 Streptomyces sp. AN090726(Inha501) 균주는 농작물 병원성 진균을 효과적으로 제어하므로 생물농약으로 유용하게 활용될 가능성이 있다.The present invention relates to novel actinomycetes having various physiological activities and uses thereof. In the present invention, actinomycetes that produce polyene compounds with excellent antifungal activity are selected by genome search technology, and then the genome is decoded based on NGS to decipher the physiological activity The properties of the substance were identified at the molecular level, and the culture process was optimized to put these useful actinomycetes (or actinomycetes-derived physiologically active substances) into practical use as biological pesticides. Accordingly, Streptomyces sp of the present invention. AN090726 (Inha501) strain effectively controls crop pathogenic fungi, so it has the potential to be useful as a biological pesticide.
도 1은 방선균 배양액 샘플들의 항진균 활성을 확인한 결과이다.
도 2는 여러 가지 식물병원성 진균에 대한 Streptomyces sp. AN090726(Inha501) 균주의 항진균 활성 결과를 나타낸다.1 is a result confirming the antifungal activity of the actinomycetes culture medium samples.
2 is Streptomyces sp. for various phytopathogenic fungi. Shows the results of antifungal activity of strain AN090726 (Inha501).
본 발명은 KCTC13999BP로 기탁되고, 식물병원성 진균에 대한 항진균 활성을 갖는 스트렙토마이세스 속(Streptomyces sp.) AN090726(Inha501) 균주를 제공한다. The present invention provides a Streptomyces sp. AN090726 (Inha501) strain deposited as KCTC13999BP and having antifungal activity against phytopathogenic fungi.
바람직하게는, 상기 균주는 칸디다 알비칸스(Candida albicans), 아스퍼질러스 나이거(Aspergillus niger), 푸자리움 옥시스포럼(Fusarium oxysporum), 푸자리움 솔라니(Fusarium solani), 푸자리움 그라미네아룸(Fusarium graminearum), 푸자리움 베르티실리오이드(Fusarium verticillioides), 푸자리움 세미텍툼(Fusarium semitectum), 알테르나리아 알테르나타(Alternaria alternate), 보트리티스 시네레아(Botrytis cinerea), 파이토프토라 칵토룸(Phytophthora cactorum), 라이족토니아 세레알리스(Rhizoctonia cerealis), 콜레토트리쿰 글로에오스포리오이데스(Colletotrichum gloeosporioides) 및 커부라리아 루나타(Curvularia lunata)로 이루어진 군에서 선택된 어느 하나 이상의 진균에 항진균 활성을 갖을 수 있으나, 이에 제한되는 것은 아니다.Preferably, the strain is Candida albicans ( Candida albicans ), Aspergillus niger ( Aspergillus niger ), Fusarium oxysporum ( Fusarium ) oxysporum ), Fusarium solani ), Fusarium graminearum ( Fusarium ) graminearum ), Fusarium verticillioides , Fusarium semitectum ( Fusarium) semitectum ), Alternaria Alternaria alternate , Botrytis cinerea , Phytophthora Phytophthora cactorum ), Rhizoctonia cerealis ), Colletotrichum gloeosporioides ) and Curvularia lunata ) To have antifungal activity against any one or more fungi selected from the group consisting of However, it is not limited thereto.
바람직하게는, 상기 균주는 서열번호 1로 표시되는 16S rDNA 염기서열을 포함할 수 있으나, 이에 제한되는 것은 아니다.Preferably, the strain may include the 16S rDNA base sequence represented by SEQ ID NO: 1, but is not limited thereto.
바람직하게는, 상기 균주는 펜타 폴리엔을 가진 선형 폴리케타이드 화합물을 생산할 수 있으나, 이에 제한되는 것은 아니다.Preferably, the strain can produce a linear polyketide compound with penta polyene, but is not limited thereto.
또한, 본 발명은 상기 균주 또는 이의 배양액을 유효성분으로 포함하는 식물병원성 진균 방제용 조성물을 제공한다.In addition, the present invention provides a composition for controlling phytopathogenic fungi comprising the strain or a culture solution thereof as an active ingredient.
본 발명에 있어, 식물병원성 진균 방제용 조성물은 본 발명에 의한 균주를 유효량 함유하고 농화학적으로 허용 되는 담체를 포함한다. 본 발명에 의한 조성물은 당업계에 공지된 방식의 미생물 농약 제제화 방식에 따라, 예컨대 배양된 균체의 배양액, 동결건조 균체와 전분, 조단백질 및 암석분 등을 포함하도록 분말, 펠렛 또는 과립, 마이크로캡슐 등으로 제형화하여 그대로 사용할 수 있다. 상기 조성물은 표면 활성화제, 비활성 담체, 보존제, 습윤제, 공급 촉진제, 유인제, 캡슐화제, 결합제, 유화제, 염료, U.V. 보호제, 완충제 등 목적 병원균 및 작물에 따라 적용하기 용이한 다른 성분을 부가함으로 수득할 수 있다. 본 발명의 조성물은 적용하기 전에 적당한 양의 물 또는 다른 희석제로 희석을 하여 직접 적용하기에 적합한 형태 또는 농축액 또는 1차 조성물일 수 있다. 항진균성 농도는 특히 농축 또는 직접 사용하거나 하는 것은 특정 제제의 성질, 적용 식물의 종류, 재배 지역의 토양 및 기후 등에 따라 다양할 수 있음은 당업계에 명백히 이해될 것이다.In the present invention, the composition for controlling phytopathogenic fungi contains an effective amount of the strain according to the present invention and an agrochemically acceptable carrier. The composition according to the present invention may be prepared according to a method of formulating a microbial pesticide known in the art, such as powder, pellet or granule, microcapsule, etc. It can be formulated and used as it is. The composition comprises a surface active agent, an inert carrier, a preservative, a wetting agent, a supply enhancer, an attractant, an encapsulant, a binder, an emulsifier, a dye, a U.V. It can be obtained by adding other components that are easy to apply depending on the target pathogen and crop, such as a protective agent and buffer. The composition of the present invention may be in a form suitable for direct application or a concentrate or primary composition by dilution with an appropriate amount of water or other diluent prior to application. It will be apparent to those skilled in the art that the antifungal concentration may vary depending on the nature of the particular formulation, the type of plant applied, the soil and climate of the cultivation area, and the like, particularly whether concentrated or used directly.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시에는 오로지 본 발명을 예시하기 위한 것으로, 본 발명이 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.
<< 실험예Experimental example >>
하기의 실험예들은 본 발명에 따른 각각의 실시예에 공통적으로 적용되는 실험예를 제공하기 위한 것이다.The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.
1. 샘플 수집 및 분리1. Sample Collection and Isolation
항진균 활성을 갖는 균주를 선별하기 위해 한국생명공학연구원의 김창진 박사님 연구실로부터 2419개의 방선균 배양액을 이용하여 진균에 대한 저해환 실험을 실시하였다. 그 중 2009년 보성의 유기농 녹차밭에서 동정한 Streptomyces sp. AN090726(Inha501) 균주를 분양받았다. 생물학적 특징으로는 Fusarium oxysporum (KCTC 46453), Candida albicans (ATCC 14053)에 대한 항진균 활성을 갖는 균주로 확인하였다.In order to select strains with antifungal activity, an inhibition test against fungi was conducted using 2419 actinomycetes culture solutions from Dr. Changjin Kim's laboratory at the Korea Research Institute of Bioscience and Biotechnology. Among them, Streptomyces sp. identified in organic green tea fields in Boseong in 2009. AN090726 (Inha501) strain was sold. Biological features include Fusarium. oxysporum (KCTC 46453), Candida It was confirmed as a strain having antifungal activity against albicans (ATCC 14053).
2. 성장 조건 및 배지2. Growth Conditions and Medium
Streptomyces sp. AN090726(Inha501) 균주는 M3 agar (soluble starch 20 g/L, agar 20 g/L, soytone 10 g/L, glucose 10 g/L, CaCO3 3 g/L, FeSO4·7H2O 0.2 g/L) 에서 30 ℃, 5일간 고체 정치배양을 한다. 또한 이 균주는 TSB (tryptone 17 g/L, soybean 3 g/L, NaCl 5 g/L, K2HPO4 2.5 g/L, glucose 2.5 g/L)에서 30℃, 220rpm, 3일간 진탕 배양을 통해 전배양을 실시하고 GSS soybean flour 25 g/L, glucose 20 g/L, soluble starch 10 g/L, yeast extract 4 g/L, NaCl 2 g/L, CaCO3 2 g/L, Beef extract 1 g/L, K2HPO4 0.25 g/L)에서 30℃, 220rpm, 5일간 진탕 배양을 통해 생산배양을 실시한다. 식물병원성 13종[Fusarium oxysporum (KCTC 46453, KACC 40051, KACC 42795), Fusarium solani (KACC 44891), Fusarium graminearum (KACC 47495), Fusarium verticillioides (KCTC 16672). Fusarium semitectum (KCTC 6065), Alternaria alternate (KACC 40019), Botrytis cinerea (KACC 40574), Phytophthora cactorum (KACC 40166), Rhizoctonia cerealis (KACC 40153), Colletotrichum gloeosporioides (KACC 4003), Curvularia lunata (KACC 40861)]은 PDA (potato starch 4 g/L, glucose 2 g/L, agar 15 g/L)에서 30 ℃, 3~7일간 고체 정치배양을 한다. Candida albicans (ATCC 14053) 균주는 YM agar (yeast extract 3 g/L, malt extract 3 g/L, peptone 5 g/L, glucose 10 g/L, agar 20 g/L)에서 30℃, 220rpm, 10시간 동안 진탕 배양을 한다. Streptomyces sp. AN090726 (Inha501) strain is M3 agar (soluble starch 20 g/L, agar 20 g/L, soytone 10 g/L, glucose 10 g/L, CaCO 3 3 g/L, FeSO 4 7H 2 O 0.2 g/ L) at 30 ℃, solid-state culture for 5 days. In addition, this strain was cultured in TSB (tryptone 17 g/L, soybean 3 g/L, NaCl 5 g/L, K 2 HPO 4 2.5 g/L, glucose 2.5 g/L) at 30°C, 220 rpm, shaking culture for 3 days. Pre-culture was carried out through GSS soybean flour 25 g/L, glucose 20 g/L, soluble starch 10 g/L, yeast extract 4 g/L, NaCl 2 g/L, CaCO 3 2 g/L, Beef extract 1 g/L, K 2 HPO 4 0.25 g/L) at 30° C., 220 rpm, and cultured with shaking for 5 days. 13 phytopathogenic species [ Fusarium oxysporum (KCTC 46453, KACC 40051, KACC 42795), Fusarium solani (KACC 44891), Fusarium graminearum (KACC 47495), Fusarium verticillioides (KCTC 16672). Fusarium semitectum (KCTC 6065), Alternaria alternate (KACC 40019) , Botrytis cinerea (KACC 40574) , Phytophthora factorum (KACC 40166) , Rhizoctonia cerealis (KACC 40153) , Colletotrichum gloeosporioides (KACC 4003) , Curvularia lunata (KACC 40861)] is solid-state culture in PDA (potato starch 4 g/L, glucose 2 g/L, agar 15 g/L) at 30 °C for 3 to 7 days. Candida albicans (ATCC 14053) strain was incubated in YM agar (yeast extract 3 g/L, malt extract 3 g/L, peptone 5 g/L, glucose 10 g/L, agar 20 g/L) at 30°C, 220rpm, 10 hours. during the shaking culture.
3. 추출 및 3. Extraction and HPLCHPLC 분석 analysis
생산 배양액과 n-butanol을 1:1의 비율로 상온에서 220rpm, 4시간 동안 섞어주면서 추출을 실시한다. 혼합물을 10000×g, 15분간 원심분리를 한 후 n-butanol층(상층부)만 솜으로 걸러 모은다. 모아진 추출물들은 evaporator를 이용하여 가루형태가 되도록 농축을 시키고 메탄올(Methanol)로 녹여 HPLC 분석 및 항진균 활성확인 실험을 진행한다.Extraction is performed while mixing the production culture medium and n- butanol at a ratio of 1:1 at 220 rpm at room temperature for 4 hours. After centrifuging the mixture at 10000×g for 15 minutes, collect only the n- butanol layer (upper layer) with cotton. The collected extracts are concentrated to a powder form using an evaporator and dissolved in methanol to perform HPLC analysis and antifungal activity confirmation experiments.
HPLC 분석 조건은 다음과 같다. reversed-phase C-18 column (5 μm particles, 4.6 × 150 mm; Agilent)이 장착된 Shimadzu SPD M10A (Shimadzu, Japan)가 사용되었다. 컬럼은 50% solvent A (0.05 M ammonium acetate, pH 6.5) 및 50% solvent B (methanol)로 평형화시킨 후, 다음의 농도 구배(gradient)로 진행하였다. 1.0 ml/min 유속으로, 50% B (0 min), 75% B (3 min), 100% B (30 min), 50% B (33 min) 및 50% B (40 min). HPLC analysis conditions are as follows. Shimadzu SPD M10A (Shimadzu, Japan) equipped with a reversed-phase C-18 column (5 μm particles, 4.6 × 150 mm; Agilent) was used. The column was equilibrated with 50% solvent A (0.05 M ammonium acetate, pH 6.5) and 50% solvent B (methanol), followed by the following concentration gradient. At a flow rate of 1.0 ml/min, 50% B (0 min), 75% B (3 min), 100% B (30 min), 50% B (33 min) and 50% B (40 min).
4. 플레이트 대면 분석을 통한 항진균 분석4. Antifungal Assay via Plate-Face Assay
생산 배양액 10 ul를 paper disc에 로딩(loading) 한 뒤 충분히 말린다. 저해활성을 보고자 하는 곰팡이가 포함된 PDA agar plate을 준비하기 위해 고체 정치배양을 한 곰팡이의 포자를 2 cm × 2 cm 크기 정도로 긁어 2% 아가로스젤(agarose gel)과 섞어준 뒤 PDA agar plate 위를 덮도록 부어주고 30분간 굳게 둔다. 곰팡이가 포함된 PDA agar plate 위에 충분히 말린 paper disc을 적절한 위치에 올린 후 30 ℃, 3~7일간 고체 정치배양을 하면서 저해환을 관찰하고 이 크기를 기록한다.After loading 10 ul of the production culture on a paper disc, dry it sufficiently. To prepare a PDA agar plate containing the mold for which inhibitory activity is to be reported, scrape the spores of the solid stationary cultured mold to a size of 2 cm × 2 cm, mix it with 2% agarose gel, and place it on the PDA agar plate. Pour over to cover and leave to harden for 30 minutes. Place a sufficiently dried paper disc on a PDA agar plate containing mold in an appropriate position, and observe the inhibition ring while culturing the solid at 30 °C for 3 to 7 days, and record the size.
5. 전체 게놈 서열분석 및 클러스터 예측5. Whole Genome Sequencing and Cluster Prediction
Streptomyces sp. AN090726(Inha501) 균주의 전체 염기서열은 PacBio RSII, Illumina platform and De novo assembly를 이용하여 한국 macrogen에 외주를 맡겨 분석하였다. 전체 염기서열 속 이차대사산물의 생합성 유전자 클러스터(biosynthetic gene cluster)는 antiSMASH bacterial version 5.0((https://antismash.secondarymetabolites.org/#!/start)을 이용하여 분석하였다. Streptomyces sp. The entire nucleotide sequence of the AN090726 (Inha501) strain was analyzed by outsourcing to Korean macrogen using PacBio RSII, Illumina platform and De novo assembly. The biosynthetic gene cluster of secondary metabolites in the entire base sequence was analyzed using antiSMASH bacterial version 5.0 (https://antismash.secondarymetabolites.org/#!/start).
6. 온실 조건하에서 식물 비료로서의 적용 가능성 평가 6. Evaluation of applicability as plant fertilizer under greenhouse conditions
수원의 한국식물환경연구소에 용역의뢰 후 확인실험을 진행하였다(pot test).After commissioning the service to the Korea Plant Environment Research Institute in Suwon, a confirmation experiment was conducted (pot test).
가. 대상병해: 시들음병(Fusarium oxysporium, KACC40051) go. Target disease: Fusarium oxysporium , KACC40051)
나. 시험작물: 고추(PR마니따), 토마토(호용), 딸기(설향)me. Test crops: Red pepper (PR Manita), Tomato (Hoyong), Strawberry (Seolhyang)
※ 대상병해 발생상황※ Target disease outbreak status
농촌진흥청 국립농업과학원 농업생물부부터 대상 병원균인 시들음병균 (Fusarium oxysporium)을 분양받아 PDA(Potato Dextrose Agar) 배지에 배양하여 접종원을 준비하였으며, 모종에 접종하여(접종일자: 11월 14일) 시들음병 발생을 유도하였다. Rural Development Administration, National Academy of Agricultural Sciences, Department of Agriculture and Biology, the target pathogen, Fusarium oxysporium , was purchased and cultured in PDA (Potato Dextrose Agar) medium to prepare an inoculum, and seedlings were inoculated (inoculation date: November 14). development was induced.
다. 처리내용 : 토양관주처리 - 배양액 100ml 또는 상등액 100ml (희석배수 1배), 처리 폿트 수 : 각각 4개 all. Treatment contents: Soil irrigation treatment - 100ml of culture medium or 100ml of supernatant (1 times dilution), Number of treatment pots: 4 each
라. 경종개요 : 재배양식 (시설재배, 폿트재배), 폿트크기 : 지름 6cm La. Seedling overview: Cultivation culture (facility cultivation, pot cultivation), pot size: 6cm in diameter
마. 조사방법 및 판정기준 : 약효시험 (이병주수, 약제처리 7일 후 작물별 병발생 정도), 약해시험 (외관상 약해유무, 약제처리 3, 5, 7일 후 외관확인)mind. Investigation method and criteria: Drug efficacy test (number of infected weeks, the degree of disease occurrence by crop after 7 days of drug treatment), drug toxicity test (appearance of weakness, appearance check after 3, 5, 7 days of drug treatment)
<실시예 1> 항진균 활성을 갖는 Streptomyces sp. AN090726(Inha501) 방선균 선별<Example 1> Streptomyces sp. AN090726 (Inha501) actinomycete screening
KRIBB의 김창진 박사님 실험실로부터 방선균 배양액을 분주 받아 항진균 활성을 확인할 때 자주 사용되는 C. albicans와 식물병원성 진균인 F. oxysporum을 이용해 항진균 활성 비교실험을 진행하였다.An antifungal activity comparison experiment was conducted using C. albicans and F. oxysporum , a phytopathogenic fungus, which are frequently used to check antifungal activity by dispensing an actinomycetes culture medium from Dr. Changjin Kim's laboratory at KRIBB.
2419개의 샘플들 중 149개의 샘플에서 억제 영역(inhibition zone)을 보이며 항진균 활성을 확인하였다(도 1).Of 2419 samples, 149 samples showed an inhibition zone and confirmed antifungal activity (FIG. 1).
149개의 샘플들을 HPLC 분석을 통해 그 중 51개의 샘플에서 항진균 활성이 나타나는 폴리엔 화합물(polyene compound)이 생성됨을 확인하였다.Through HPLC analysis of 149 samples, it was confirmed that a polyene compound exhibiting antifungal activity was produced in 51 of them.
16s rRNA sequence, RNA polymerase B 등 특정 유전자의 서열 비교를 통해 신규 균주라고 판단되는 Streptomyces sp. AN090726(Inha501) 균주를 선별하였다. Streptomyces sp., which is determined to be a novel strain through sequence comparison of specific genes such as 16s rRNA sequence and RNA polymerase B. AN090726 (Inha501) strain was selected.
<실시예 2> Streptomyces sp. AN090726(Inha501) 균주 전체 게놈 서열분석과 항진균 활성물질의 특성 분석<Example 2> Streptomyces sp. Whole genome sequencing of AN090726 (Inha501) strain and characterization of antifungal active substances
전체 게놈 서열분석을 통해 Streptomyces 계열의 신규 균주임을 확인하였다(표 1). Streptomyces through whole genome sequencing It was confirmed that it is a new strain of the series (Table 1).
antiSMASH 프로그램을 이용하여 폴리엔 화합물(polyene compound)을 만들어내는 유전자 클러스터(gene cluster)를 예측한 결과, 펜타 폴리엔을 가지고 있는 선형의 폴리케타이드 화합물로 예상되었다.As a result of predicting a gene cluster producing a polyene compound using the antiSMASH program, it was expected to be a linear polyketide compound having pentapolyene.
총 35개의 영역에서 이차대사산물을 만드는 클러스터(cluster)를 찾아냈고 22번째 영역이 선형 폴리케타이드 화합물의 클러스터로 예측되었다. 이는 기존에 알려진 ECO-02301 클러스터와 유사성을 보였다.A cluster producing secondary metabolites was found in a total of 35 regions, and the 22nd region was predicted to be a cluster of linear polyketide compounds. This showed similarity to the previously known ECO-02301 cluster.
폴리엔 화합물의 분리 및 정제를 통해 항진균 활성을 확인하였고, High resolution Mass analysis를 통해 얻은 분자량을 통해 아직 보고되지 않은 폴리엔(polyene) 분자량임을 확인하였다. 예상되는 폴리엔 화합물의 정보는 다음과 같다. Chemical Formula: C67H101NO18, Exact Mass: 1207.7019, Molecular Weight: 1208.5147The antifungal activity was confirmed through separation and purification of the polyene compound, and the molecular weight obtained through high resolution mass analysis was confirmed to be a polyene molecular weight that has not yet been reported. Information on the expected polyene compound is as follows. Chemical Formula: C 67 H 101 NO 18 , Exact Mass: 1207.7019, Molecular Weight: 1208.5147
<< 실시예Example 3> 항진균 활성 확인 3> Check antifungal activity
아래의 여러 가지 식물병원성 진균에 대한 항진균 활성을 확인한 결과, 확인한 모든 진균에서 활성을 확인하였다(도 2). As a result of confirming the antifungal activity against various phytopathogenic fungi below, activity was confirmed in all the confirmed fungi (FIG. 2).
Fusarium oxysporum (KCTC 46453, KACC 40051, KACC 42795), Fusarium solani (KACC 44891), Fusarium graminearum (KACC 47495), Fusarium verticillioides (KCTC 16672). Fusarium semitectum (KCTC 6065), Alternaria alternate (KACC 40019), Botrytis cinerea (KACC 40574), Phytophthora cactorum (KACC 40166), Rhizoctonia cerealis (KACC 40153), Colletotrichum gloeosporioides (KACC 4003), Curvularia lunata (KACC 40861). Fusarium oxysporum (KCTC 46453, KACC 40051, KACC 42795), Fusarium solani (KACC 44891), Fusarium graminearum (KACC 47495), Fusarium verticillioides (KCTC 16672). Fusarium semitectum (KCTC 6065), Alternaria alternate (KACC 40019) , Botrytis cinerea (KACC 40574) , Phytophthora factorum (KACC 40166) , Rhizoctonia cerealis (KACC 40153) , Colletotrichum gloeosporioides (KACC 4003) , Curvularia lunata (KACC 40861).
<< 실시예Example 4> 미생물제제로서의 가능성 확인하기 위해 화분 수준에서 항진균 활성확인 4> Confirmation of antifungal activity at pollen level to confirm its potential as a microbial agent
한국식물환경연구소(수원)에 용역을 의뢰하고 진행하였다. 고추, 토마토, 딸기에 대한 항진균 활성을 확인하였다. 약효시험의 결과, 고추와 딸기에는 항진균 활성이 확인되었다. 약해시험의 결과, Streptomyces sp. AN090726(Inha501)의 배양액이 식물에 해를 끼치지 않음을 확인하였다.The service was commissioned and carried out by the Korea Plant Environment Research Institute (Suwon). Antifungal activity against pepper, tomato, and strawberry was confirmed. As a result of the drug efficacy test, antifungal activity was confirmed in red pepper and strawberry. As a result of the toxicity test, Streptomyces sp. It was confirmed that the culture medium of AN090726 (Inha501) did not harm plants.
이상으로, 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그것들의 등가물에 의해서 정의된다고 할 것이다.In the above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Novel Streptomyces having various biological activity and use thereof <130> ADP-2019-0437 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1514 <212> RNA <213> Streptomyces sp. AN09726(Inha501) <400> 1 aggtgatcca gccgcacctt ccggtacggc taccttgtta cgacttcgtc ccaatcgcca 60 gtcccacctt cgacgattcc ctcccacaag gggttgggcc accggcttcg ggtgttaccg 120 actttcgtga cgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcagcactg 180 ctgatctgcg attactagag actccgactt catggggtcg agttgcagac cccaatccga 240 actgagaccg gctttttgag attcgctcca cctcacggca tcgcaactca ttgtaccggc 300 cattgtagca cgtgtgcagc ccaagacata aggggcatga tgacttgacg tcgtccccac 360 cttcctccga gttgaccccg gcagtctccc gtgagtcccc atcaccccga agggcatgct 420 ggcaacacag gacaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga 480 gctgacgaca gccatgcacc acctgtacac cgaccacaag ggggcaccca tctctggatg 540 tttccgatgt atgtcaagcc ttggtaaggt tcttcgcgtt gcgtcgaatt aagccacatg 600 ctccgcctct tgtgcgggcc cccgtcaatt cctttgagtt ttagccttgc ggccgtactc 660 cccaggcggg gaacttaatg cgttagctgc ggcacggaca acgtggaatg tcgcccacac 720 ctagttccca acgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacg 780 ctttcgctcc tcagcgtcag tatcggccca gagatccgcc ttcgccaccg gtgttcctcc 840 tgatatctgc gcatttcacc gctacaccag gaattccgat ctcccctacc gaactctagc 900 ctgcccgtat cgaatgcaga cccggggtta agccccgggc tttcacatcc gacgcgacaa 960 gccgcctacg agctctttac gcccaataat tccggacaac gctcgcaccc tacgtattac 1020 cgcggctgct ggcacgtagt tagccggtgc ttcttctgca ggtaccgtca cttgcgcttc 1080 ttccctgctg aaagaggttt acaacccgaa ggccgtcatc cctcacgcgg cgtcgctgca 1140 tcaggctttc gcccattgtg caatattccc cactgctgcc tcccgtagga gtctgggccg 1200 tgtctcagtc ccagtgtggc cggtcgccct ctcaggccgg ctacccgtcg tcgccttggt 1260 aggccatcac cccaccaaca agctgatagg ccgcgggctc atcctgcacc gccggagctt 1320 tccacccgaa agcatgcgct cccaggtaat atccggtatt agaccccgtt tccagggctt 1380 gtcccagagt gcagggcaga ttgcccacgt gttactcacc cgttcgccac taatccaccc 1440 cgaagggctt catcgttcga cttgcatgtg ttaagcacgc cgccagcgtt cgtcctgagc 1500 caggatcaaa ctct 1514 <110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Novel Streptomyces having various biological activity and use it <130> ADP-2019-0437 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1514 <212> RNA <213> Streptomyces sp. AN09726 (Inha501) <400> 1 aggtgatcca gccgcacctt ccggtacggc taccttgtta cgacttcgtc ccaatcgcca 60 gtcccacctt cgacgattcc ctcccacaag gggttgggcc accggcttcg ggtgttaccg 120 actttcgtga cgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcagcactg 180 ctgatctgcg attactagag actccgactt catggggtcg agttgcagac cccaatccga 240 actgagaccg gctttttgag attcgctcca cctcacggca tcgcaactca ttgtaccggc 300 cattgtagca cgtgtgcagc ccaagacata aggggcatga tgacttgacg tcgtccccac 360 cttcctccga gttgaccccg gcagtctccc gtgagtcccc atcaccccga agggcatgct 420 ggcaacacag gacaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga 480 gctgacgaca gccatgcacc acctgtacac cgaccacaag ggggcaccca tctctggatg 540 tttccgatgt atgtcaagcc ttggtaaggt tcttcgcgtt gcgtcgaatt aagccacatg 600 ctccgcctct tgtgcgggcc cccgtcaatt cctttgagtt ttagccttgc ggccgtactc 660 cccaggcggg gaacttaatg cgttagctgc ggcacggaca acgtggaatg tcgcccacac 720 ctagttccca acgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacg 780 ctttcgctcc tcagcgtcag tatcggccca gagatccgcc ttcgccaccg gtgttcctcc 840 tgatatctgc gcatttcacc gctacaccag gaattccgat ctcccctacc gaactctagc 900 ctgcccgtat cgaatgcaga cccggggtta agccccgggc tttcacatcc gacgcgacaa 960 gccgcctacg agctctttac gcccaataat tccggacaac gctcgcaccc tacgtattac 1020 cgcggctgct ggcacgtagt tagccggtgc ttcttctgca ggtaccgtca cttgcgcttc 1080 ttccctgctg aaagaggttt acaacccgaa ggccgtcatc cctcacgcgg cgtcgctgca 1140 tcaggctttc gcccattgtg caatattccc cactgctgcc tcccgtagga gtctgggccg 1200 tgtctcagtc ccagtgtggc cggtcgccct ctcaggccgg ctacccgtcg tcgccttggt 1260 aggccatcac cccaccaaca agctgatagg ccgcgggctc atcctgcacc gccggagctt 1320 tccacccgaa agcatgcgct cccaggtaat atccggtatt agaccccgtt tccagggctt 1380 gtcccagagt gcagggcaga ttgccccacgt gttactcacc cgttcgccac taatccaccc 1440 cgaagggctt catcgttcga cttgcatgtg ttaagcacgc cgccagcgtt cgtcctgagc 1500 caggatcaaa ctct 1514
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