Construction of natural biosynthesis pathway of crocins and non-natural biosynthesis pathway of crocetin dialdehyde, in metabolically engineered Escherichia coli
- 주제(키워드) Crocin , Crocetin , novel short chain carotenoid
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 이평천
- 발행년도 2022
- 학위수여년월 2022. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032165
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
목차
Chapter 1. General Introduction 1
1.1 Microbial carotenoid production 2
1.2 MEP pathway 2
1.3 Enzyme engineering 3
1.4 Aims of this study 4
Chapter 2. Microbial synthesis of plant-active pigments crocetin and its glycosylated form, crocin, in metabolic engineered Escherichia coli 5
2.1 Abstract 6
2.2 Introduction 7
2.3 Materials and Methods 10
2.3.1. Strains, mediums, and culture conditions 10
2.3.2. crALDH mining 13
2.3.3. Novel glycosyltransferase (UGT) protein mining 13
2.3.4. Pathway gene cloning and expression plasmid construction for pathway genes 14
2.3.5. Zeaxanthin-biosynthesizing gene integration into E.coli chromosome 23
2.3.6. UGT expression analysis and in vitro activity assay 24
2.3.7. Flask and batch bioreactor fermentations 25
2.3.8. Crocin pathway product extraction and quantification 26
2.3.9. Transcriptional analysis (qRT-PCR) 27
2.4. Results 28
2.4.1. Zeaxanthin-producing E. coli strain construction 28
2.4.2. Crocetin-dialdehyde biosynthetic pathway construction and temperature-dependent crocetin-dialdehyde production 30
2.4.3. Crocetin-dialdehyde dehydrogenase-mediated crocetin biosynthetic pathway construction 34
2.4.4. Crocetin glucosyltransferase-mediated crocin-2 biosynthetic pathway construction 38
2.4.5. Crocin-2 glucosyltransferase-mediated crocin-4 biosynthetic pathway construction 42
2.5 Discussion 47
Chapter 3. Construction of non-natural C20- and C25-carotenoid biosynthetic pathway 51
3.1 Abstract 52
3.2 Introduction 53
3.3 Materials and Methods 56
3.3.1. Strains and media 56
3.3.2. Plasmid construction 56
3.3.3. Site-directed mutagenesis 62
3.3.4. Strain construction 62
3.3.5. Flask cultivation and batch bioreactor fermentations 62
3.3.6. Cultivation and analysis for limonene production 63
3.3.7. Homology modeling and structural analysis of CrtMLIKE 64
3.3.8. Carotenoid isolation 64
3.3.9. Structure analysis of carotenoids 64
3.4. Results 66
3.4.1. Construction of GPP-overproducing platform strain by limonene production 66
3.4.2. CrtMLIKE synthesizes C20- and C25-phytoene in the GPP-overproducing strain 66
3.4.3. Structural analysis of CrtMLIKE 69
3.4.4. Site-directed mutagenesis of CrtMLIKE for non-natural short carotenoid backbone biosynthesis based on protein modeling 71
3.4.5. Desaturation in the non-natural short carotenoid pathway 75
3.4.6. Biosynthesis of crocetin-dialdehyde 77
3.5 Discussion 80
4. Conclusion 83
5. References 85
