Transcriptional regulatory network analysis and its application for increasing antibiotics production in Streptomyces coelicolor : 방선균에서 전사 조절 네트워크 분석 및 항생제 생산 증가에의 활용

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2013. 2. 김병기.-
dc.description.abstractTranscriptional regulatory networks in Streptomyces coelicolor A3(2) have evolved to execute various cellular processes affecting antibiotic production. Several key transcriptional regulators involved in biosynthesis of secondary metabolites have been characterized, including ActII-ORF4, RedD, and CdaR. However, new regulators are still needed to be identified to understand the complex transcriptional regulatory networks delineating antibiotic production. Here, we identified a list of regulators controlled by AbsB acting as a higher level regulator in the antibiotic production. These potential regulators were identified to show different expression patterns in the absence of absB gene. RNase cleavage assay revealed that sco6808 transcripts, one of the potential regulators, are cleaved by AbsB. This result indicates that sco6808 is a direct target of AbsB. Therefore, our approach provides a comprehensive list of novel regulators controlled by AbsB, which serves as a basis for understanding multi-layered regulatory networks.
Manipulation of multiple transcription regulators controlling non-redundant pathways related to antibiotics production would effectively increase the antibiotics production. Here, we present an approach that uses a combination of two transcriptional regulators that control independent pathways to increase the antibiotics production. AfsS is one of key master activators of antibiotics production. Using the microarray data of afsS disruption mutants, we first selected the regulators transcriptionally independent of afsS. Among them, we focused on sco4677, an antagonistic regulator of sigma factor F, and sco4228 (phoU), a phosphate transport system regulator. Using the combination of sco4677 with afsS (BG4677S) and phoU with afsS (BG4228S), we observed the intracellular actinorhodin production increased in BG4677S by approximately 11-folds higher and BG4228S by approximately 149-folds higher than wild type.
Time-course microarray experiments were performed using ndgR deletion mutants for expanding the fragmented transcriptional regulatory network. Previously, NdgR was identified by DNA affinity capture assay (DACA) and mass spectrometric analysis as a global regulator that controls amino acids synthesis, quorum sensing and antibiotics production. In addition, we unveiled that NdgR also controls ABC transport system of glutamate, branched chain amino acid, phosphate and methionine, carbohydrate uptakes, carbohydrate metabolism in PTS, glycerol metabolism, cell stress response, and tolerance to toxic materials. Those roles were confirmed by EMSA, cell viability test, and the observation by confocal microscope and TEM images. From these results, we could accumulate the information about the role of the global regulator, NdgR, to the incomplete transcriptional regulatory network in Streptomyces coelicolor.
In conclusion, we attempted to identify the transcriptional regulatory network and developed a capable method to increase the antibiotics production.
dc.description.tableofcontentsAbstract i

Table of Contents iv

List of Tables ix

List of Figures x

Chapter I. Introduction

1.1 Regulations in Streptomyces 2
1.1.1 Regulatory networks studied in Streptomyces 2
1.1.2 Functional study of transcriptional regulators in Streptomyces 9
1.1.3 Strategies to enhance antibiotic production 10

1.2 Transcriptomics based approach to study TR network 13
1.2.1 DNA microarray 14
1.2.2 Next generation sequencing 16

1.3 The scope of thesis 19

Chapter II. Materials and Methods

2.1 Bacterial strains and plasmids 22

2.2 Growth media and culture conditions 22

2.3 DNA manipulations 26
2.3.1 Plasmids and recombinant preparation 26
2.3.2 Construction of knock-out mutants – PCR targeting 27
2.3.3 Construction of knock-out mutants 30

2.4 Electrophoretic mobility shift assay (EMSA) 30

2.5 Semiquantitative RT-PCR 31

2.6 DNA microarray 31

2.7 DNA array data analysis 38

2.8 Analysis of gene expression data of absB deletion mutants 41

2.9 Analysis of gene expression data of afsS disruption mutants 41

2.10 Antibiotics measurement 43

2.11 In vitro RNA cleavage assay 44
2.11.1 RNA transcripts and proteins preparation 44
2.11.2 RNA cleavage assay and image analysis 45

2.12 Scanning Electron Microscopy (SEM) 45

2.13 Transmission Electron Microscopy (TEM) 46

2.14 Cell viability test 46

2.15 Fluorescence microscopy 47

Chapter III. AbsB-dependent transcriptional regulators involved in antibiotic production

3.1 Identification of novel interaction between known global regulator and new regulator 49
3.1.1 Identification of AbsB-targeted regulators 51
3.1.2 AbsB-targeted regulators control transcription and RNA metabolic processes 52
3.1.3 SCO6808 controls antibiotic production as a direct downstream target of AbsB 65
3.1.4 SCO1839 controls antibiotic production as an indirect downstream target of AbsB 69

3.2 Conclusion 71

Chapter IV. Antibiotic overproduction by the combination of transcriptionally independent regulators

4.1 Effective transcriptional control of antibitotics production 74
4.1.1 Identification of AfsS independent regulators 76
4.1.2 Double mutation of sco4677 and afsS increased antibiotics production 82
4.1.3 Synergy effects of phoU and afsS double mutants in SMM without phosphate 86

4.2 Conclusion 95

Chapter V. Analysis of transcriptional regulatory network of ndgR deletion mutant using time-course microarray

5.1 NdgR, an IclR-like regulator in Streptomyces coelicolor 100

5.2 Time-course microarray experiments of ndgR deletion mutants 103
5.2.1 RNA sample preparation and Array experiments 103
5.2.2 Data analysis 103
5.2.3 Up-regulated genes in BG11 113
5.2.4 Down-regulated genes in BG11 122

5.3 Conclusion 136

Chapter VI. Conclusions and Recommendation 139

References 143

Abstract in Korean 159
dc.format.extent9203537 bytes-
dc.publisher서울대학교 대학원-
dc.subjecttranscriptional regulator-
dc.subjectmass spectrometry-
dc.subjectDNA chip-
dc.subjectregulatory network-
dc.subjectantibiotics overproduction-
dc.titleTranscriptional regulatory network analysis and its application for increasing antibiotics production in Streptomyces coelicolor-
dc.title.alternative방선균에서 전사 조절 네트워크 분석 및 항생제 생산 증가에의 활용-
dc.contributor.AlternativeAuthorBo-Rahm Lee-
dc.citation.pagesx, 161-
dc.contributor.affiliation공과대학 화학생물공학부-
Appears in Collections:
College of Engineering/Engineering Practice School (공과대학/대학원)Dept. of Chemical and Biological Engineering (화학생물공학부)Theses (Ph.D. / Sc.D._화학생물공학부)
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