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Title Page

국문 초록

ABSTRACT

Contents

Abbreviations 14

CHAPTER Ⅰ. Introduction 15

1. Introduction 16

1.1. Motivation of this study 16

1.2. Overview of photosynthesis 18

1.3. Previous studies for carbon utilization 26

1.4. Objectives of this study 30

CHAPTER Ⅱ. Application of Dark Reaction: CO₂ Assimilation Element of C4 to C5 in rTCA Cycle 32

2.1. Introduction 33

2.2. Materials and methods 35

2.2.1. Strains and plasmid 35

2.2.2. Construction of plasmid 36

2.2.3. Alignment of amino acid sequence 39

2.2.4. Screening condition of CtOGOR mutants 39

2.2.5. Protein expression and purification 39

2.2.6. Structure analysis 40

2.2.7. Decarboxylation and carboxylation activity assay for CtOGOR 41

2.3. Results and discussion 44

2.3.1. Amino acid sequence comparison with OFORs 44

2.3.2. Verification of purified CtOGOR 46

2.3.3. Structural analysis of CtOGOR 48

2.3.4. Validation of icd-base edited E. coli BL21 (DE3) 50

2.3.5. Expression of CtOGOR in icd-BE E. coli BL21 (DE3) with minimal condition 53

2.3.6. Isolation of CtOGOR mutants 55

2.3.7. Determination of decarboxylation activity of CtOGORs 56

2.3.8. CO₂ reduction ability of CtOGORs 59

CHAPTER Ⅲ. Application of Dark Reaction: C1 conversion Element in CO₂ to Methanol Pathway 65

3.1. Introduction 66

3.2 Materials and methods 67

3.2.1. Structure analysis 67

3.2.2. Construction of mutants screening strain 67

3.2.3. Construction of formaldehyde sensor 71

3.2.4. Construction of BmfaldDH mutant library and mutant plasmids 73

3.2.5. Fluorescence assay for GFP and mCherry 73

3.2.6. Isolation of BmfaldDH mutants and sample preparation for FACS 73

3.2.7. Oxidation and Reduction activity assay 74

3.2.8. Determination of formaldehyde 77

3.3. Results and discussion 77

3.3.1. Structure analysis by cryo-TEM 77

3.3.2. Validation of formaldehyde sensor in one vector system 79

3.3.3. Validation of mutant screening strain 82

3.3.4. Final 10 mutants were selected in BmFaldDH mutants 84

3.3.5 Analysis of activity in BmFaldDH varients 88

3.3.6. Production of formaldehyde by formaldehyde dehydrogenase 91

3.3.7. Identification of amino acid variation critical for activity at mutation 65 93

CHAPTER Ⅳ. Application of Light Reaction: Enhancing Protoporphyrin IX Production through Intracellular ATP-Increasing Element 97

4.1. Introduction 98

4.1.1. Proteorhodopsin 98

4.1.2. Applications and biosynthesis of protoporphyrin IX 101

4.2. Materials and methods 104

4.2.1. Genome-scale metabolic model (GEM) analysis 104

4.2.2. Construction of plasmid 104

4.2.3. Strains, culture condition 106

4.2.4. Protein expression and purification 106

4.2.5. Enzymatic reaction of ALAS and 5-aminolevulinic acid (ALA) assay 107

4.2.6. ATP and PPIX analysis 108

4.3. Results and discussion 109

4.3.1. GEM analysis for heme synthesis by using E. coli through expression of PR and ALAS 109

4.3.2. Succinyl-CoA-dependent production on intermediate metabolites of heme biosynthesis 114

4.3.3. Expression of heterologous proteins in E. coli 116

4.3.4. Influence of PR on the intracellular ATP changes with light irradiation 118

4.3.5. Evaluation of light enhanced production of PPIX 120

CONCLUDING REMARKS 123

REFERENCES 124

List of Tables

Table 1. Primers used in CtOGOR study 38

Table 2. Kinetic properties of 2-oxoglutarate oxidation in OGORs 58

Table 3. List of primers used for construdction of BmFaldDH screening strain 70

Table 4. Primers used in this study to construct one vector system 72

Table 5. List of final ten mutants of BmFaldDH 86

Table 6. The normalized activity of various BmFaldDHs 90

Table 7. The normalized activity of sub-mutants of BmFaldDH 65 96

Table 8. List of primers used for constructing expression vector of PR and ALAS 105

List of Figures

Figure 1. Mechanism of photosynthesis in light reaction 20

Figure 2. An overview dark reaction of photosynthesis illustration. 25

Figure 3. Schematic diagrams of CtOGOR decarboxylation and carboxylation activity assay 43

Figure 4. Alignment of amino acid sequences of OFORs 45

Figure 5. Preparation of CtOGOR for cryo-EM structural analysis 47

Figure 6. 3-Dimensional structure of CtOGOR analyzed by cryo-EM 49

Figure 7. Sequence analysis of base-editing induced in icd region 51

Figure 8. Validation of CtOGOR screening strain 52

Figure 9. Expression of CtOGOR in icd-deficient strain 54

Figure 10. 2-Oxoglutarate decarboxylation activity in dependence of pH 57

Figure 11. CO₂ reduction ability of CtOGORs 61

Figure 12. Analysis of distance between TPP and α subunit 342 62

Figure 13. Rotation of CtOGORs about 90 the vertical axis relative 63

Figure 14. Accessibility test of carbon substrate in carboxylation reaction 64

Figure 15. Scheme of BmFaldDH activity assay 76

Figure 16. 3-D Structure of BmFaldDH by cryo-TEM 78

Figure 17. One vector system plasmid for protein expression and formaldehyde sensing. 80

Figure 18. Validation of GFP and mCherry fluorescence in one vector system plasmid 81

Figure 19. Formaldehyde sensitivity of formaldehyde screening candidate strains. 83

Figure 20. GFP Analysis for validation of obatained 473 BmFaldDH variants. 85

Figure 21. FACS analysis of ten BmFaldDH mutants 87

Figure 22. Activity of formaldehyde oxidation and formate reduction in various BmFaldDHs 89

Figure 23. Determination of formaldehyde by formate reduction reaction in BmFaldDH wild type and mutant 65 92

Figure 24. Activity of formaldehyde oxidation and formate reduction in sub-mutants of BmFaldDH 65 95

Figure 25. Structure of proteorhodopsin (PR) with retinal choromophore (PDB entry 2L6X). 100

Figure 26. Heme biosynthetic pathways with C4 and C5 103

Figure 27A. Overall scheme of PR introduced PPIX production 112

Figure 27B-C. Flux balance analysis of heme biosynthesis with PR expression 113

Figure 28. Correlation of succinyl-CoA and 5-aminolevulinic acid (ALA) production 115

Figure 29. Expression of heterologous proteins in E. coli C41 (DE3). 117

Figure 30. Comparison of ATP level in non-C4 heme biosynthetic pathway and C4 heme biosynthetic pathway introduced group. 119

Figure 31. Comparison of PPIX production in non-C4 heme biosynthetic pathway and C4 heme biosynthetic pathway introduced group with light 122

초록보기

 지구온난화로 인해 주목받고 있는 기후 위기 문제가 전 세계적 관심으로 대두됨에 따라 이를 해결하기 위한 연구가 절실히 필요한 실정이다. 본 연구에서는 지구 온난화 문제 해결에 일조하기 위해 합성생물학적 기법을 이용하여 자연계에 존재하는 광합성 요소를 모사, 기능을 개발하였다. 광합성 요소의 개발은 복잡한 자연계의 시스템을 집약하여 보다 효율적으로 광합성을 실현할 수 있는 연구이다.

1장에서는 광합성에 대한 전반적인 이해 및 생물학적 광합성 요소의 선행 연구, 그리고 본 논문에 대한 목적에 대해 정리하였다. 2, 3장에서는 광합성 암반응 요소의 기능 개량 연구, 4장에서는 광합성 명반응을 모사한 연구에 대해 기술하였다. 광합성 암반응 요소로써 탄소 환원에 관여하는 2-oxoglutarate oxidoreductase와 formaldehyde dehydrogenase 의 돌연변이 연구를 통해 야생형 효소보다 각각 최대 2.8배, 5.5배 증가한 돌연변이를 개발하였다. 광합성 명반응 요소 연구에서는 빛 에너지를 이용할 수 있는 프로테오로돕신 광기구 모듈과 프로토포르피린 IX 생합성 경로를 도입하여 세포 내 ATP 레벨을 90 ~ 120 %, 프로토포르피린 IX 생성량을 45 % 증가시킨 연구를 기술하였다.

본 연구는 무한한 에너지원인 빛과 이산화탄소의 이용을 활성화하기 위해 광합성 요소를 적용, 및 활성 개량을 한 연구이다. 이와 같은 광합성 요소 기술의 연구는 생물학적 인공 광합성의 실현 가능성을 제시하므로 탄소 이용 연구 및 산업계에도 기여할 수 있을 것으로 기대한다.

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