목차

LIST OF TABLES i
LIST OF FIGURES ii
ABSTRACT iv

I. Introduction 1
II. Materials and Methods 6
1. Cloning (genes and plasmids) 6
2. Directed evolution 7
3. Purification of wild-type and mutant CrtN clones 8
4. Western blot analysis 8
5. In vitro assay 9
6. Culture Growth and Isolation of Carotenoids 9
7. Analysis of Carotenoids 10
III. Results and Discussion 13
1. Reconstruction of C40 carotenoid biosynthesis pathway in E. coli 13
2. Reconstruction of C30 carotenoid biosynthesis pathway in E. coli 16
3. Directed evolution of CrtN 19
3.1 Directed evolution 19
3.2 site-directed mutagenesis 22
3.3 in vitro assay 24
4. Extension of carotenoid biosynthesis pathway 27
4.1 Extended C40 carotenogenic pathway 27
4.1.1 Extension of didehydrolycopene pathway with a mutant CrtY2 in
E. coli 27
4.1.2 Extension of didehydrolycopene pathway with a spheroidene monooxygenase CrtA 27
4.1.3 Directed extension of B. linens β-carotene synthetic module in E. coli 28
4.1.4 Assembly of B. linens aromatic isorenieratene synthetic module in E. coli 29
4.2 Extended C30 carotenogenic pathway 34
4.2.1 Rational assembly of diapotorulene pathway and directed extension 34
4.2.2 Assembly of diapo-β-carotene pathway with in vitro evolved carotenogenic enzyme 35
VI. Conclusion 40
V. References 43
ABSTRACT IN KOREAN 47
ACKNOWLEDGEMENTS 49