마이크로어레이 분석을 통한 스트렙토마이세스 실리칼라에서의 이차대사산물 고생산 관련 후보유전자들의 선별 및 기능분석

Title
마이크로어레이 분석을 통한 스트렙토마이세스 실리칼라에서의 이차대사산물 고생산 관련 후보유전자들의 선별 및 기능분석
Authors
김선혜
Keywords
마이크로어레이분석을통한스트렙토마이세스실리칼라에서의이차대사산물고생산관련후보유전자들의선별및기능분석
Issue Date
2011
Publisher
인하대학교
Abstract
Previously, we successfully utilized Streptomyces interspecies DNA microarray analysis to identify novel regulatory genes associated with secondary metabolite overproduction in S.peucetius Doxorubicin(DXR) overproducing industrial strain(14). Especially wblA, a whiB-like putative transcription factor gene, and SCO1712, a TetR-family transcriptional regulatory gene, were identified as strong antibiotic biosynthetic down-regulators in both S. coelicolor as well as S. peucetius species. Moreover, these two strong antibiotic down-regulators were deleted in the S. coelicolor wild-type chromosome, resulting S. coelicolor△wblA△SCO1712 double knock-out (KO) mutant which showed significantly higher actinorhodin (Act) productions (16). To further increase our understanding about the genetic nature of Streptomyces antibiotic overproduction, a microarray analysis was conducted again between S. coelicolor wild-type and a S. coelicolor△wblA△SCO1712 double KO mutant. As a result, the double KO strain was found to express the Act biosynthetic cluster genes stronger than the wild-type. Also, various gene expression signatures were obtained by deletion of wblA and SCO1712. Twenty eight genes showed a five-fold or greater change in expression at two or more time points and 14 genes showed non-change in expression pattern at most of time points. First, in case of putative wblA/SCO1712 dependent genes, expression pattern was confirmed in wild-type and double KO mutant using RT-PCR. And these genes were individually over-expression into S. coelicolor to verify the biological functions of these putative targets, we selected some putative effective antibiotics-relating regulators in M145 wild-type. Second, SCO5426 relating regulation of carbon flux was found among wblA/SCO1712 independent genes(5). So SCO5426 was disrupted in S. coelicolor△wblA△SCO1712 double KO mutant additionally and Act productivity was increased than double KO mutant. This additional transcriptomics-driven approach in
Description
Abstract Ⅰ Contents Ⅱ List of Tables IV Captions for Figures V 1. Introduction 1 2. Materials and Methods 3 2.1 Bacterial strains, plasmids and condition 3 2.2 Antibiotic production 3 2.3 Isolation of total RNA and gene expression analysis by microarray 4 2.4 Real-time RT-PCR 5 2.5 Functional expression of wblA and SCO1712 dependent genes from S. coelicolor 5 2.6 Gene disruption of wblA and SCO1712 independent SCO5426 in S. coelicolor△wblA△SCO1712 double knock-out (KO) mutant 7 3. Results and Discussion 9 3.1 Comparative Transcriptome Analysis between S. coelicolor M145 and M145△wblA△SCO1712 9 3.2 Identification and characterization of wblA & SCO1712 dependent target genes in S. coelicolor M145 14 3.3 Genetic manipulation of wblA & SCO1712 independent gene for Rational Strain Improvement 18 4. Acknowledgements 23 5. References 24
URI
http://dspace.inha.ac.kr/handle/10505/22819
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College of Natural Science(자연과학대학) > Ocean Sciences (해양과학) > Theses(해양과학 석박사 학위논문)
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