[보고서]방선균의 이차대사산물 생합성 유도 및 제어 기작 연구

최선욱

방선균의 이차대사산물 생합성 유도 및 제어 기작 연구
Induction and Regulation of Secondary Metabolites Biosynthesis from Actinomycetes 원문보기

보고서 정보
주관연구기관	경남대학교 산학협력단
연구책임자	최선욱
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2011-04
과제시작연도	2010
주관부처	교육과학기술부
사업 관리 기관	한국연구재단
등록번호	TRKO201200002252
과제고유번호	1345124528
사업명	일반연구자지원
DB 구축일자	2013-05-20
키워드	이차대사산물.생합성.제어 유전자.유도.메커니즘.Secondary metabolites.Biosynthesis.Regulator.Induction.Streptomyces natalensis.Mechanism.

초록 ▼

현재 세계적으로 많은 관심을 받고 있는 방선균의 이차대사산물 생합성 유도 및 조절에 대한 연구 발전에 크게 기여 할 것이며 본 과제를 통해 구축될 제어 시스템은 유용 생리활성물질의 생산량 증대뿐만 아니라 새로운 신규 생리활성물질의 유도방법을 새롭게 제시하는 매우 유용한 기반기술이 될 것이다. 또 본 과제를 통해 확립된 기반기술을 활용하여 산업적으로 유용한 생리활성물질의 생산성 향상 및 신규 생리활성물질 획득에 활용한다면 현재 국내의 제한된 연구투자 상황 때문에 침체된 제약 및 신약 개발사업을 활성화하여 국내 제약산업 뿐만 아니라 국

Abstract ▼

This project will contribute the technology development appling the productivity improvement of bioactive materials and induction of new secondary metabolites, through the study of regulators that are concerned in biosynthesis control of secondary metabolites of actinomycetes, as well as their interrelationship. If the technology obtained by this project is used to industrial strain to improve the productivity of bioactive materials and to induce new secondary metabolites, it may contribute greatly in economic development as well as domestic pharmaceutical industry.

목차 Contents

일반연구자지원사업 최종보고서(최종본) ... 1
목차 ... 3
Ⅰ. 연구 계획 요약문 ... 4
1. 국문 요약문 ... 4
Ⅱ. 연구 결과 요약문 ... 5
1. 국문 요약문 ... 5
2. 영문 요약문 ... 6
Ⅲ. 연구내용 및 결과 ... 7
1. 연구개발과제의 개요 ... 7
2. 국내외 기술개발 현황 ... 10
3. 연구수행 내용 및 결과 ... 13
4. 목표 달성도 및 관련 분야에의 기여도 ... 50
5. 연구결과의 활용계획 ... 51
6. 연구과정에서 수집한 해외과학기술정보 ... 51
7. 주관연구책임자(공동연구원) 대표적 연구 실적 ... 53
8. 참고 문헌 ... 54
9. 연구 성과 ... 56
10. 기타사항 ... 56

표/그림 (55)

표 Structures of γ-butyrolactone autoregulators isolated from Streptomyces species.
표 Regulation mechanism by autoregulator and receptor protein.
표 Highly phosphorylated guanine nucleotide
표 Model of RNAP changed by ppGpp in global regulation.
표 Hypothetical scheme for the regulation of secondary metabolites production
표 Alignment of RelA (A), autoregulator receptor (B), and MetK (C), and design of PCR primers for cloning.
표 PCR for cloning of autoregulator receptor, metK, and relA from S. natalensis.
표 Homology search of PCR products
표 HindIII and EcoRI partial digestion test.
표 0.35U EcoRI partial digestion and elution of 50 kb DNA fragment.
표 Primer sequence for 2D pool screening
표 PCR products for 500 bp metK and 400 bp relA.
표 First template pooling PCR.
표 Secondary template pooling PCR for metK. H: Horizontal PCR, V: Vertical PCR.
표 Secondary template pooling PCR for relA. H: Horizontal PCR, V: Vertical PCR.
표 Acquirement of 5 clones for relA and 18 clones for metK by 2D pool screening using PCR.
표 Restriction map and gene organization of the 8 kb DNA fragment cloned from S. natalensis.
표 Homology search of orfs in the 8 kb DNA fragment
표 Southern hybridization for subcloning of metK (A) and relA (B).
표 (A) Gene organization in the fragment cloned from S. natalensis. Abbreviations : K: KpnI P: PstI S: SacI Sm: SmaI, Sp: SphI X: XbaI (B) Frame analysis of the region containging seven orfs. The numbers in parentheses indicate the order of restriction enzyme sites.
표 (A) Gene organization of the 4-kb PvuI-EcoRI DNA fragment cloned from S. natalensis genomic DNA. orf3 is truncated. (B) Frame analysis of the 4-kb PvuI-EcoRI region.(C) Alignment of overall amino acid sequences of MetKs.
표 Vector construction for high expression (A) and knock-out (B) of regulator gene in actinomycetes.
표 Effects of medium on transconjugation efficiency.
표 Effects of MgCl2 concentration in MS medium on the transconjugation efficiency.
표 Effects of temperature on the viability of S. natalensis spores. Spores (103/mL) in 2×YT medium were incubated for 10 min at the temperature indicated. The data are the average of three independent experiments, and expressed as a percent of the colony count obtained in the absence of heat treatment (control).
표 Effects of heat treatment of spores on transconjugation efficiency
표 Effects of the number of E. coli donor cells on transconjugation efficiency.
표 Southern blot analysis of exconjugants containing pSET152 and design of primer for attB site sequencing.
표 Phylogenetic tree among actinomycetes (A) and alignment of attB site and pseudo-attB site (pseN) sequences of S. natalensis (B).
표 Schematic representation of the strategy used for the disruption of receptor gene.
표 Southern hybridization analysis of KpnI-digested chromosomal DNA.
표 Schematic representation of the strategy used for the disruption of orf2.
표 Southern hybridization analysis of wild-type and orf2-disruptant strain.
표 Construction of vectors for relA and metK expression in streptomycetes.
표 Southern hybridization analysis of chromosomal DNA digested with BamHI from the S. lividans (A) and the S. natalensis (B). Lane 1; Wild-type strain, lane 2; pSET152ET-integrated strain, lane 3; relA expression vector, lane 4; relA expression vector-integrated strain. The probe used was a 0.5-kb relA fragment.
표 Southern hybridization analysis of EcoRI-digested genomic DNAs from the S. lividans (A) and the S. natalensis (B). (A) Lane 1, S. lividans TK24 wild-type strain lane2, pSET152ET-integrated strain lane 3, metK high expression vector-integrated strain lane 4, metK high expression vector. (B) Lane 1, S. natalensis wild-type strain lane2, pSET152ET-integrated strain lane 3-4, metK expression vector-integrated strain lane 5, metK high expression vector. The probe used was 0.5-kb PCR fragment amplified from the metK gene.
표 Gene cluster for natamycin biosynthesis.
표 Southern hybridization analysis of chromosomal DNA and vector digested with PstI (A) and BamHI (B). (A) Lane 1, S. natalensis wild-type strain; lane 2, pSET152ET integrated strain; ane 3, pSET152ET-pimM vector; lane 4, pSET152-pimM integrated strain. The 0.6-kb fragment of pimM was used as the probe. (B) Lane 1, S. natalensis wild-type strain; lane 2, pSET152ET integrated strain; lane 3, pSET152ET-pimR vector; lane 4, pSET152-pimR integrated strain. The 1-kb fragment of pimM was used as the probe.
표 Natamycin production in liquid culture of a wild-type strain (solid circles), a receptor gene-disruptant (solid squares), and a receptor gene-complemented strain (solid triangles).
표 Effect of receptor-disruption on morphological differentiation. 1, wild-type strain; 2, Receptor-disruptant strain; 3, Receptor-complemented strain.
표 Natamycin production in liquid culture of a wild-type strain (solid circles), a receptor gene-disruptant (solid squares), and a receptor gene-complemented strain (solid triangles).
표 Effect of orf2-disruption on morphological differentiation. 1, wild-type
표 Effect of receptor-disruption on morphological differentiation. 1, wild-type strain; 2, Receptor-disruptant strain; 3, Receptor-complemented strain.
표 Transcriptional comparison of the genes related to biosynthesis of natamycin from the ORF2-disruptant and S. natalensis wild-type strain cultivated for 36 h. The mRNA levels of the ORF2-disruptant strainand S. natalensis wild-type strain were determined by real-time qRT-PCR. The lysA gene was used as the reference gene. The error bars indicate the standard deviations from the means.
표 Comparison of natamycin production from the pSET152ET-integrated strain as the control (solid circles), pSET152ET-pimM -integrated strain (solid squares), and pSET152ET-pimR -integrated strain (solid triangles). Three independent pSET152ET-integrated s t r a i n s , pSET152ET-pimM-integrated strains, and pSET152ET-pimR-integrated strains showed identical patterns, and representative data from the exconjugants are shown.
표 Transcriptional comparison of pimM and pimR from the pSET152ET-integrated strain as control (C), pSET152ET-pimM-integrated strain (M), and pSET152ET-pimR-integrated strain (R), cultivated for 24 h by real-time qRT-PCR. Bar graphs of dark gray and light gray indicate the transcript amounts of pimM and pimR, respectively. The lysA gene was used as the reference. Data represent the averages of three independent experiments, and the error bars indicate the standard deviations from the means.
표 Structure of the PI-factor.
표 Gel-shift assay. MeOH, 100% Methyl alcohol; EtOH, 100% Ethyl acetate; WT, Wild-type S. natalensis; △sngA, orf2-disruptant S. natalensis; △ sngR, orf1-disruptant ; SA, S. avermitilis.
표 Effect of the introduction of high expression vector on the production of actinorhodin (blue color) in S. lividans TK24.
표 Effect of metK high expression on natamycin production in S. natalensis. Symbols: solid circles, S. natalensis wild-type strain solid square, pSET152ET-integrated strain solid triangle, metK high expression vector-integrated strain. Bars represent SD of three experiments (n=3).
표 Transcriptional comparison of metK, pimM, and pimR from the pSET152ET-integrated S. natalensis as control and pSET152ET-metK -integrated S. natalensis by real-time qRT-PCR. The lysA gene was used as the reference gene. The error bars indicate the standard deviations from the means.
표 Transcriptional comparison of regulatory genes from the pSET152ET-integrated S. lividans TK24 as control and pSET152ET-metK-integrated S. lividans TK24 cultivated for 36 h and 42h by real-time qRT-PCR. The lysA gene was used as the reference gene. The error bars indicate the standard deviations from the means.
표 Effect of relA high expression on actinorhodin production in S. lividans. Solid circles, squares, and triangles, represent wild-type strain, pSET152ET-integrated strain, and relA high e x p r e s s i o n vector-integrated strain, respectively.
표 Effect of relA high expression on morphological differentiation of S. natalensis. 1, wild-type strain; 2. pSET152ET-integrated strain; 3, relA high expression vector-integrated strain.
표 Effect of relA overexpression on natamycin production in S. natalensis. Solid circles, squares, and triangles, represent wild-type strain, pSET152ET-integrated strain, and relA high e x p r e s s i o n v ec to r- inte gr a te d strain, respectively.

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연합인증

방선균의 이차대사산물 생합성 유도 및 제어 기작 연구
Induction and Regulation of Secondary Metabolites Biosynthesis from Actinomycetes 원문보기