Deciphering the Molecular Mechanism of Formate Dehydrogenase in the CO2-reducing Acetogen, Eubacterium callanderi KIST612 = CO2 환원 아세트산균 Eubacterium callanderi의 포름 산 탈수소효소 분자 메커니즘 규명 KIST612

다국어 초록 (Multilingual Abstract)

Metal-dependent formate dehydrogenase （FDH） is a promising biocatalyst for bioelectrocatalytic CO2 reduction. Owing to its metabolic diversity, FDH exhibits various structural and functional types across various microorganisms. However, studying this enzyme remains challenging due to factors such as oxygen sensitivity and the complexity of its structure, which hinder detailed functional characterization and make it difficult to identify the specific molecules involved in its mechanism. In this study, we aimed to characterize FDH from Eubacterium callanderi KIST612 and to identify the key molecules and cofactors involved in its mechanism. As a result, we specifically identified two fdh genes （ELI_0994 and ELI_3306） that encode the characteristic sequence from the putative gene cluster and categorized them into different types based on three points of consideration. i.e., subunit composition, presence of chaperon in the operon, and cofactor. Evaluations classified ELI_0994 as type 1, while ELI_3306 falls under type 6. Gene-cluster analysis suggests that these two FDH systems may operate through distinct mechanisms. The major difference between them appears to lie in the location of the electron-bifurcation center: a hydrogenase in the case of ELI_0994 （supported by similarity to A.woodii）, and FMN for ELI_3306 （tentatively proposed）. Across both systems, the key molecules implicated in their function include NADH, ferredoxin, FMN, the molybdenum cofactor, and hydrogenase. These predicted mechanisms may aid in selecting suitable FDH candidates for future applications. Keywords: Formate dehydrogenase; CO2 reduction; Molecular mechanism; Eubacterium callanderi KIST612

목차 (Table of Contents)

• Abstract i
• Contents ii
• List of abbreviations iv
• List of tables v
• List of figures vi
• I. INTRODUCTION 1
• 1.1. Motivation and significance of this study 1
• 1.2. Acetogen 2
• 1.2.1 Electron Bifurcation 3
• 1.3. The Wood-Ljungdahl pathway（WLP） 3
• 1.4. Formate dehydrogenase （CO2 reductase） 6
• 1.4.1. Metal-independent formate dehydrogenase 6
• 1.4.2 Metal-dependent formate dehydrogenase 7
• 1.4.3 Classification of metal-dependent formate dehydrogenase 8
• 1.5. Formate dehydrogenase in acetogen 10
• 1.6. Model Strain Used: Eubacterium callanderi KIST612 11
• 1.7. Research objective 12
• II. MATERIALS AND METHODS 13
• 2.1. Reagents and chemical 13
• 2.2. Bacterial strains and cultivation 13
• 2.3. Bioinformatics 13
• 2.4. Preparation of cell-free from E. callanderi KIST612 14
• 2.5. Protein expression of FDH 14
• 2.5.1. Plasmid construction 14
• 2.5.2. Transformation into E. coli BL21（DE3） 14
• 2.5.3. Protein expression and purifications 15
• 2.5.4. SDS-PAGE and Western blot 15
• 2.5.5. Enzyme assay 16
• III. RESULTS AND DISCUSSION 19
• 3.1. Bioinformatics identification of two fdh genes in E. callanderi KIST612 19
• 3.2. Classification of two fdh genes in E. callanderi KIST612 24
• 3.3. Protein expression and purification of two fdh genes 26
• 3.4. Enzyme activity 30
• 3.5. Predicted mechanism of fdh genes in E. callanderi KIST612 32
• IV. CONCLUSION 35
• SUMMARY 36
• Acknowledgement 37
• References 38