유전체 분석을 활용한 식물 유래 세균 Streptomyces sp. N50의 이차대사산물 연구
Investigation of secondary metabolites from plant-derived bacterium, Streptomyces sp. N50 through genomic analysis
- 주제(키워드) 도움말 Streptomyces , Genome mining , Naphthomycin , Phosphonate , Siderophore
- 발행기관 강릉원주대학교
- 지도교수 도움말 박진수, 김형섭
- 심사위원 박진수, 김형섭, 권재영
- 사용가능일 20190201
- 저작권일 20200301
- 발행년도 2018
- 학위수여년월 2019. 2
- 학위명 석사
- 학과 및 전공 도움말 일반대학원 학연협동과정
- 실제URI http://www.dcollection.net/handler/kangnung/000000010427
- UCI I804:42001-000000010427
- 본문언어 한국어
초록/요약 도움말
As the antibiotic-resistant pathogens have been constantly founded, a discovery of new antibiotics come to an important element in recent natural product chemistry. Fortunately, an advance of genomics along with NGS technology has provided a new approach to that tasks. In this study, I exploited secondary metabolites from the strain Streptomyces sp. N50 based on diverse biosynthetic gene clusters acquired from its genome sequence. Strain N50 was isolated from Selaginella tamariscina sample collected in Mt. Nochu, Gangwon-do. The 16S rRNA showed that N50 belongs to the genus Streptomyces. The genome of N50 was fully sequenced and there were 32 secondary metabolites predictable gene clusters involved in 11 different types from 2 contigs. The results of genomic analysis were used to predict and confirm the production of naphthomycin, uncertain phosphonate, scabichelin and desferrioxamine B in N50. The presence of phosphonate and desferrioxamine B were demonstrated by 31P NMR and CAS assay, respectively, but they were not isolated as a single compounds. Four new naphthomycins (1–4) were isolated, and planar structures of 1–3 were definitely identified. Although the structure of compound 4 was not clearly confirmed due to less amount of substance, but it seems new 15-deoxy derivative of naphthomycin C according to spectroscopic data. Unlike previous reported naphthomycins, these four compounds were dehydroxylated at C15, which accords closely with its biosynthetic context containing ER domain. Among the isolated naphthomycins, compound 1 were found to possess toxicity for the producer N50, whereas 2 and 3 were not active. Compound 3 is considered as an intermediate between 1 and 2 based on the detoxificative effect of mycothiol structure. Therefore, this suggest that mycothiol binds to compound 1 to inhibit the self-toxicity, and this complex are changed into 2 through post modification for reuse of glucosamine-inositol unit of mycothiol. These results suggest that genome inspired investigation is effective approach to avoid a re-isolation to discover new secondary metabolites in natural products chemistry.
more목차 도움말
목 차
List of Figures 1
List of Tables 4
Ⅰ. Abstract 5
Ⅱ. 서론 7
Ⅲ. 재료 및 방법 11
1. 실험재료 및 시약 11
2. 분석기기 12
3 유전체 분석 13
3.1 DNA extraction 13
3.2 Whole genome sequencing 14
3.3 16S rRNA analysis 14
3.4 ANI (Average Nucleotide Identity) analysis 15
4. 성분 연구 16
4.1 Phosphonate 화합물 탐색 16
4.1.1생장조건 비교 16
4.1.2 추출물/분획물 제조 및 phosphonate 화합물 생산 분석 18
4.2 Siderophore 화합물 탐색 20
4.2.1 추출물 및 분획물 제조 20
4.2.2 Blue CAS assay 21
4.3 Naphthomycin 화합물 탐색 22
4.3.1 성분 분석 22
4.3.2 추출 및 소분획불 확보 22
4.3.3 화합물의 분리 정제 (compound 1–3) 23
4.3.4 화합물의 분리 정제 (compound 4) 24
Ⅳ. 결과 25
1. N50의 유전체와 생합성 유전자 정보 25
2. 16S rRNA 분석 31
3. ANI (Average Nucleotide Identity) 분석 34
4. 실험 대상 BGCs 선정 35
5. Phosphonate 화합물 생산 확인 41
6 Siderophore 화합물 검출반응 42
7. EA, 물 분획으로부터 화합물의 분리 44
8. 분리한 신규 화합물의 구조 동정 48
8.1 Compound 1 49
8.2 Compound 2 60
8.3 Compound 3 68
8.4 Compound 4 76
Ⅴ. 결론 및 고찰 79
Ⅵ. 참고문헌 83
