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Title Page 1

Abstract 4

Contents 5

Technical Terms and Abbreviation 14

Chapter 1. Current Advances ion carbon dioxide utilization - bio catalytic milestone for carbon capture utilization 15

1.1. Introduction 15

1.1.1. Carbon dioxide conversion 15

1.1.2. Formate as a valuable chemical 15

1.2. Biological conversion of carbon dioxide 18

1.2.1. Role of Formate Dehydrogenase 18

1.2.2. Metal-dependent FDHs 18

1.2.3. Unique characteristics of W-FDHs 18

1.3. Challenges in production of W-FDHs 22

1.3.1. Heterologous expression 22

1.3.2. Characterization of W-FDHs 22

1.4. Significance of developing W-FDHs 23

1.5. Current limitation in application of W-FDHs 23

1.6. Objectives and scope of this study 23

1.7. References 25

Chapter 2. Development of Cupriavidus necator H16 as a Host for Heterologous Production of Formate Dehydrogenase 7 (MeFDHI) of Methylorubrum extorquens: Possibilities and Limitations 27

2.1. Introduction 27

2.2. Materials and methods 0

2.2.1. Materials 28

2.2.2. Strains, plasmids and primers 29

2.2.3. Media composition 29

2.2.4. Culture experiments 31

2.2.5. Enzyme production and purification 31

2.2.6. Enzyme activity assay and protein quantification by SDS-PAGE 31

2.2.7. Analytical methods 32

2.3. Results and Discussion 32

2.3.1. Genome analysis of C. necator H16 32

2.3.2. Expression and purification of MeFDH1 33

2.3.3. Comparison between C. necator H16 and M. extorquens as recombinant hosts for MeFDH1 40

2.3.4. Effect of metal ions availability on enzyme activity and production 48

2.3.5. Bioreactor cultivation of C. necator H16 for large-scale enzyme production 54

2.4. Conclusion 56

2.5. References 57

Chapter 3. Production and characterization of CO₂ reducing and tungsten-containing formate dehydrogenases (CnFDW) of Cupriavidus necator H16 60

3.1. Introduction 60

3.2. Materials and Methods 61

3.2.1. Materials 61

3.2.2. Plasmids and strains construction 61

3.2.3. Culture of microorganisms and production of recombinant FDHs 63

3.2.4. SDS-PAGE and Western blot 63

3.2.5. Enzyme purification 63

3.2.6. Determination of enzyme activity 64

3.2.7. Analysis of metal content 64

3.2.8. Molecular docking in silico analysis 64

3.3. Results and Discussion 65

3.3.1. CwFDW expression in host strains 65

3.3.2. Effect of W and Mo in culture medium on the production of CnFDW and its activity 67

3.3.3. Determination of W and Mo content 72

3.3.4. Enzyme kinetics 74

3.3.5. Oxygen tolerance 78

3.3.6. In silico comparison between MeFDH1 and CnFDW 80

3.4. Conclusion 91

3.5. References 92

Chapter 4. Engineering of Tungsten-Dependent Formate Dehydrogenase of Cupriavidus necator H16 (CnFDW) for Enhanced Activity and Oxygen Stability 95

4.1. Introduction 95

4.2. Materials and Methods 96

4.2.1. Materials 96

4.2.2. Plasmid and strain construction 96

4.2.3. Media composition and enzyme preparation 98

4.2.4. Analytical method 98

4.2.5. In silico structural analysis 98

4.3. Results and Discussion 99

4.3.1. Swapping of alpha and beta subunits 99

4.3.2. In silico modeling for key residues identification in cofactor binding pocket 102

4.3.3. Determination of amino acids for protecting B1-SF4 from oxygen damage 106

4.4. Conclusion 111

4.5. References 112

Chapter 5. Conclusion and Future Perspectives 113

List of Tables 13

Table 1.1. Study on characterization of formate dehydrogenases in prokaryotes 21

Table 2.1. Strains, plasmids and primers used in this study 30

Table 2.2. Homology in amino acid sequences of synthesis enzymes for tungsto-bis-(metalopterin… 37

Table 2.3. Comparison of cell growth between C. necator H16 and M. extorquens PA1a 43

Table 2.4. Determination of tungsten (W) in purified MeFDH1 a subunit 53

Table 3.1. Strains, plasmids and primers used in this study 62

Table 3.2. Metal contents in purified CnFDW a subunit 73

Table 3.3. Kinetic parameters of CwFDW and MeFDHl expressed in M. extorquens PA1 76

Table 3.4. Homology of amino acid sequences in tungsten-containing formate dehydrogenase between M. extorquens PA1 and C. necator H16 81

Table 3.5. Comparison of W-bis-MGD docking in the corresponding binding pockets of MeFDH1 and... 90

Table 4.1. Strains, plasmids and primers used in this study 97

Table 4.2. Docking simulation of CnFDW mutant enzymes for 3 different amino acids appearing in W-bis-MGD pocket surface 105

Table 4.3. Amino acid residues participating hydrogen-bonding between α-subunit and β-subunit 108

List of Figures 8

Figure 1.1. Formate global market and industrial application. Three general strategies for formic acid production using methanol or carbon... 17

Figure 1.2. Classification of formate dehydrogenases and their physiological role in cellular metabolism 20

Figure 2.1. Comparative gene arrangement of formate dehydrogenase and tungsto-bis-... 34

Figure 2.2. Amino acid sequence comparison of formate dehydrogenase 1 orthologs with M 35

Figure 2.3. Metabolic pathway of tungsto-bis-(metalopterin guanine dinucleotide) (W-bis-MGD)... 36

Figure 2.4. SDS-PAGE analysis and enzyme activity of MeFDH1 produced by recombinant C... 39

Figure 2.5. Growth comparison between wild type and mutant strain of M. extorquens PA1 41

Figure 2.6. Growth comparison between C necator H16 and M extorquens PA1 42

Figure 2.7. Comparison of MeFDH1 activity produced by recombinant C necator H16 (Cn2) and M. extorquens PA1 (Me3) 46

Figure 2.8. Comparison of MeFDH1 α and β subunits produced by recombinant C. necator H16 (Cn2) and M. extorquens PA1 (Me3) 47

Figure 2.9. Production and activity of MeFDH1 by recombinant C. necator H16 (Cn2) and M. extorquens PA1 (Me3) grown in the tungsten and... 49

Figure 2.10. Production and activity of MeFDH1 by recombinant C. necator H16 (Cn2) and M.... 50

Figure 2.11. Schematic representations of MeFDH1 possibly present during overexpression inside... 52

Figure 2.12. Bioreactor experiment for the production of MeFDH1 by recombinant C. necator H16... 55

Figure 3.1. Enzyme expression study of CnFDW produced by recombinant M. extorquens PA1... 66

Figure 3.2. The effect of cofactor metals on CnFDW expression 69

Figure 3.3. Enzymatic activities of purified CnFDW produced by M extorquens PA1 (Me8)... 70

Figure 3.4. In vivo enzyme functionality test of M. extorquens PA1 on fomate under different... 71

Figure 3.5. The effect of temperature and pH on formate oxidation and formate formation... 77

Figure 3.6. Effect of formate on oxygen tolerance of CnFDW and MeFDH1 79

Figure 3.7. Sequence alignment of MeFDH1 and CnFDW alpha subunit 82

Figure 3.8. Sequence alignment of MeFDH1 and CnFDW beta subunit 83

Figure 3.9. Structure analysis between MeFDH1 and CnFDW 84

Figure 3.10. W-bis-MGD docking mode analysis in MeFDH1 85

Figure 3.11. Binding pocket (gray colored) comparison of pterin 2 adjacent to the SF4 cluster (A4) 86

Figure 3.12. W-bis-MGD docking mode analysis in CnFDW 88

Figure 3.13. Comparison of W-bis-MGD docking in the corresponding binding pockets of... 89

Figure 4.1. Expression confirmation of chimeric FDHs in M. extorquens PA1 101

Figure 4.2. Location of three amino acids different between CnFDW and MeFDHl: M636E... 103

Figure 4.3. Loop and its amino acid residues affecting the tt-tt interaction with pterin 2 and... 104

Figure 4.4. Region within 15Å distance from B1 SF4 and their amino acid residues present in the protein surface 107

Figure 4.5. Prediction of O2 Docking Region 109

Figure 4.6. Expected O2 tunnels near a-Arg63 and b-Arg356 110

Figure 5.1. Overall challenges on future perspectives to achieve large scale productin of W-FDHs 117

초록보기

 The identification of the formate dehydrogenase MeFDH1 from Methylorubrum extorquens represents a significant advance in sustainable CO₂ fixation and utilization. However, large-scale production of MeFDH1 is complicated by its dependence on the unique tungsto-bis-metalopterin guanine dinucleotide(W-bis-MGD) cofactor, which constrains its practical application. This study investigates Cupriavidus necator H16 as a viable host for the large-scale production of MeFDH1, leveraging fructose as a carbon source and the host's intrinsic cofactor synthesis capabilities. In minimal salt medium, C. necator H16 successfully produced active MeFDH1, achieving a specific activity of 80 to 100 U/㎎ for converting CO₂ to formate. Fed-batch bioreactor experiments yielded approximately 50g/L of cell dry weight(CDW) and 10,000U/L of MeFDH1 within 50 hours. These results establish C. necator H16 as a promising recombinant host for MeFDH1 production, offering a pathway to efficient and scalable methods for generating this vital enzyme.

Along with the host investigation, CnFDW from C. necator H16, similar to MeFDH1, was expressed in M. extorquens and characterized. The heterodimeric CnFDW exhibits high formate-forming activity(kcat＝120 s⁻¹) when using reduced ethyl viologen. The presence of tungstate during cell growth was essential for activity, although inactive apo-CnFDW was completely synthesized in its absence. High-resolution inductively coupled plasma mass spectrometry(HR-ICP-MS) was used to determine the maximal tungsten content at 0.56 ㏖/㏖ enzyme, implying that about 44% of the produced recombinant enzymes lack the cofactor. The optimal activity was observed at 30℃ and pH 6.0 for formate production, and pH 8.0 for oxidation. It remained stable in air without formate, yet displayed oxygen sensitivity, with a half-life of 3.0 h in the presence of both oxygen and formate. Docking studies, informed by the MeFDH1 structure, suggest that the reduced activity and W-content in CnFDW compared to MeFDH1 are likely due to steric hindrance in cofactor binding. This study presents CnFDW as a promising enzyme candidate for formate formation from CO₂ and provides valuable insights into enhancing the performance of this important enzyme.

In silico structural analysis of CnFDW was also conducted to get insight on how to improve its activity and stability. Using the known x-ray structure of MeFDH1 and the Alphafold program, the structure of CnFDW was predicted, and docking modes for cofactors and oxygen were determined using SITEID and SYBYL molecular modeling software. Potential amino acid residues for improving enzyme activity and stability were predicted based on simulation results, and initial wet experiments were conducted to validate in silico findings. Ongoing research will enhance the understanding of W-dependent FDH enzymes withexcellent performance in formate formation.

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