| 摘要 | 第10-12页 |
| ABSTRACT | 第12-13页 |
| Chapter 1 INTRODUCTION | 第20-37页 |
| 1.1 Synthetic Biology and Metabolic Engineering | 第20-21页 |
| 1.2 Preface of Promoters | 第21-23页 |
| 1.2.1 Function and Structure of Promoter | 第21-22页 |
| 1.2.2 Application of Promoters in Metabolic Engineering | 第22-23页 |
| 1.3 Strategies Applied for Promoter Engineering | 第23-25页 |
| 1.3.1 Engineering Yeast Promoters via Tuning Nucleosome Architecture | 第23-24页 |
| 1.3.2 Promoter Engineering through Directed Evolution | 第24-25页 |
| 1.3.3 Engineering Hybrid Promoters via UAS | 第25页 |
| 1.4 Isoprenoids and Isoprenoid Biosynthesis Pathway | 第25-33页 |
| 1.4.1 Structure and Classification of Terpenes | 第25-26页 |
| 1.4.2 Terpenoid Backbone Biosynthesis Pathway | 第26页 |
| 1.4.3 Mevalonate Pathway | 第26-30页 |
| 1.4.4 Enzyme of Non-mevalonate Pathway | 第30-33页 |
| 1.5 Squalene and Squalene Production | 第33-35页 |
| 1.5.1 Natural Sources, Applications and Biosynthesis of Squalene | 第33-34页 |
| 1.5.2 Squalene Production via Metabolic Engineering | 第34-35页 |
| 1.6 Motivation and Research Significance | 第35-37页 |
| Chapter 2 Cloning and Characterization of Promoters of Saccharomyces cerevisiae | 第37-62页 |
| Background | 第37-38页 |
| 2.1 Materials and Methods | 第38-45页 |
| 2.1.1 Strains, Vectors and Reagents | 第38-39页 |
| 2.1.2 Chemical and Biochemical Reagents | 第39-40页 |
| 2.1.3 Bio-scientific Instruments | 第40页 |
| 2.1.4 Recipe of Growth Media | 第40-41页 |
| 2.1.5 Yeast Genomic Extraction Protocol | 第41页 |
| 2.1.6 Plasmid Digestion with Restriction Enzyme | 第41-42页 |
| 2.1.7 Preparation of Saccharomyces cerevisiae for Transformation | 第42页 |
| 2.1.8 Yeast DNA Transformation with Electroporator | 第42页 |
| 2.1.9 Extraction of Total RNA from Yeast | 第42-43页 |
| 2.1.10 Fluorescence Microscopy | 第43页 |
| 2.1.11 Flow cytometry | 第43-44页 |
| 2.1.12 β-galactosidase Assay | 第44-45页 |
| 2.2 Results and Discussion | 第45-61页 |
| 2.2.1 Genomic Data Mining to Retrieve Promoters Sequence | 第45-46页 |
| 2.2.2 Construction of DNA Assembler of e GFP and Promoters | 第46-51页 |
| 2.2.3 Microscopic Analysis of eGFP Transformants | 第51页 |
| 2.2.4 Measurement of Strength of Constitutive Promoters | 第51-57页 |
| 2.2.5 Characterization of Strength of Repressible Promoters | 第57-59页 |
| 2.2.6 Enhance Strength of Promoters with Loan TFBS | 第59-61页 |
| 2.3 Summary | 第61-62页 |
| Chapter 3 Engineering of Terpenoid Backbone Biosynthesis Pathway in S. cerevisiae for SqualeneOverproduction | 第62-91页 |
| Background | 第62-63页 |
| 3.1 Materials and Methods | 第63-70页 |
| 3.1.1 Strains, Vectors and Reagents | 第63页 |
| 3.1.2 Preparation and Construction of Expression Cassettes | 第63-66页 |
| 3.1.3 Preparation of Saccharomyces cerevisiae for Transformation | 第66-67页 |
| 3.1.4 Extraction of Total RNA from Yeast | 第67-68页 |
| 3.1.5 Reverse Transcription of Total RNA | 第68页 |
| 3.1.6 Inhibition of Squalene Monooxygenase with Terbinafine | 第68-69页 |
| 3.1.7 Extraction, Identification, and Quantification of Metabolites | 第69-70页 |
| 3.1.8 In-vitro Production of Longifolene | 第70页 |
| 3.2 Results and Discussion | 第70-88页 |
| 3.2.1 Integration of Terpenoid Backbone Biosynthesis Pathway Genes | 第70-73页 |
| 3.2.2 Effect of Terpenoid Backbone Biosynthesis Pathway (TBBP) on Cell Growth and MetabolitesProduction | 第73-79页 |
| 3.2.3 Effect of Ergosterol Overproduction on Oleic Acid Biosynthesis | 第79-81页 |
| 3.2.4 Conversion of Farnesol into Longifolene in Acidic Conditions | 第81-82页 |
| 3.2.5 Conversion of Squalene into Fatty acids in Acidic Conditions | 第82-84页 |
| 3.2.6 Effect of Overexpression of TBBP on Ethanol Production | 第84-85页 |
| 3.2.7 Accumulation of Squalene Monooxygenase by Inhibition with Terbinafine | 第85-87页 |
| 3.2.8 Inhibition of 14-alpha-demethylase with Ketoconazole | 第87-88页 |
| 3.3 Summary | 第88-91页 |
| Chapter 4 CONCLUSIONS | 第91-94页 |
| REFERENCES | 第94-107页 |
| Acknowledgements | 第107页 |
