Development and Application of Synthetic Regulatory Apparatus for Metabolite Production in Saccharomyces cerevisiae
- 주제(키워드) Synthetic biology , Metabolic engineering , Saccharomyces cerevisiae
- 발행기관 고려대학교 대학원
- 지도교수 오민규
- 발행년도 2016
- 학위수여년월 2016. 8
- 학위구분 박사
- 학과 대학원 화공생명공학과
- 원문페이지 156 p
- 실제URI http://www.dcollection.net/handler/korea/000000068363
- 본문언어 영어
- 제출원본 000045881685
초록/요약
Since the beginning of the genetic engineering, systematic modification of microbial metabolism became possible. Metabolic engineering, emerged in 1990s, tried to optimize cell for engineering purpose, and their methodologies are focused on overproduction of value-added metabolite and improved resistance against various chemicals. Production of value-added metabolite, such as biofuels and pharmaceuticals in microbial hosts required efficient methodologies for optimizing microbial metabolism. Recent advance in biotechnology enabled novel artificial modifications on cell regulation system by introducing exogenous regulatory apparatus to microbial host. Synthetic regulatory apparatus, therefore, can expand the principles and methodologies of metabolic engineering. In this doctoral thesis, I proved that advanced metabolic engineering is possible by using synthetic regulatory apparatus. First, synthetic transcription regulator library was examined and its characteristic for isolating mutant with various transcription profile including multiple gene repression, gene modulation, and activation was proven, which are advantageous over conventional methods. Also, I found obstacles in isolating mutant with inconspicuous metabolite production through evolutionary engineering methods. Therefore, I tried to overcome those limits by designing synthetic suicide riboswitch which can recue growth defect in response to intracellular metabolite, so that growth selection can be used for isolating mutants with inconspicuous metabolite production. Lastly, advanced engineering of cell metabolism based on knowledge from those studies was conducted and metabolite production was further improved by reducing glycolytic flux, proving that development and application of synthetic regulatory apparatus is a competitive approach for metabolic production in eukaryotic host system.
more목차
ABSTRACT iv
LIST OF FIGURES ix
LIST OF TABLES xii
CHAPTER 1. INTRODUCTION 1
1.1 Golden era of genetic engineering 2
1.2 Metabolic engineering aided by synthetic biology 3
1.3 Aim of this thesis 5
CHAPTER 2. APPLICATION OF SYNTHETIC APPARATUS 9
2.1 Introduction 12
2.2 Material and methods 23
2.3 Results and discussion 28
2.3.1 Introduction of ZFP-TF and screening of the sec14ts strain as a model system 28
2.3.2 DNA microarray and quantitative PCR analysis of SG1 and SG9 31
2.3.3 Validation of the growth defect suppression in the strains SG1 and SG9 35
2.3.4. qPCR analysis of all isolated strains 38
2.3.5 Prediction of the Binding sequences for synthetic ZFP-TF 40
2.3.6 The NTE1 gene in the SG23 strain is not activated by ZFP-TF directly 45
2.4 Conclusion 48
CHAPTER 3. DEVELOPMENT OF SYNTHETIC APPARATUS 50
3.1 Introduction 53
3.2 Material and methods 62
3.3 Results 67
3.3.1 Design and examination of synthetic suicide riboswitch 67
3.3.2 Screening of S. cerevisiae GFA1 mutant library 73
3.3.3 Screening of heterologous HAD phosphatases originated from E. coli 77
3.3.4 Desgining and examination of suicide riboswitch for flavin mononucleotide 80
3.4 Discussion 82
3.5 Conclusions 89
CHAPTER 4. ADVANCED ENGINEERING OF METABOLISM 91
4.1 Introduction 94
4.2 Materials and Methods 98
4.3 Results and discussion 102
4.3.1 Strong overexpression of GFA1 mutant and YqaB for GlcNAc production 102
4.3.2. Disruption of PFK-2 to reduce glycolytic flux 105
4.3.3 Effect of carbon sources and GNA1 overexpression 109
4.4 Conclusion 112
OVERALL CONCLUSION 115
LIST OF PUBLICATIONS 117
REFERENCES 118
