Eukaryotic Expression of MeFDH1 Enables Formate Production from CO2 in Engineered Pichia pastoris
- 발행기관 서강대학교 일반대학원
- 지도교수 이진원
- 발행년도 2025
- 학위수여년월 2025. 8
- 학위명 석사
- 학과 및 전공 일반대학원 화공생명공학과
- 실제 URI http://www.dcollection.net/handler/sogang/000000082049
- UCI I804:11029-000000082049
- 본문언어 영어
- 저작권 서강대학교 논문은 저작권 보호를 받습니다.
초록 (요약문)
The formate dehydrogenase MeFDH1, derived from Methylobacterium extorquens, catalyzes the reduction of CO₂ to formate and represents a promising biocatalyst for carbon capture and utilization. However, its heterologous expression has been limited due to its structural complexity, including a heterodimeric α/β-subunit architecture and a requirement for the tungsten-containing cofactor W-bis-MGD. In this study, we established a eukaryotic expression system for functional MeFDH1 production using Pichia pastoris as the host. To address the challenges of subunit co-expression, we compared three strategies: dual vector, dual promoter, and self-cleaving systems. Among them, the self-cleaving strategy not only enabled the most balanced expression of α- and β-subunit, but also resulted in the highest level of soluble expression of MeFDH1. Furthermore, we reconstituted the tungsten cofactor biosynthesis pathway by integrating TupABC and MobA into the P. pastoris genome. Enzyme activity was detectable only when both genes were present and tungsten was supplemented, resulting in a total activity of 4.85 U per gram of wet cell weight. The engineered strain exhibited stable growth (OD₆₀₀ = 36.45) and sustained MeFDH1 expression over 120 h of methanol induction. Furthermore, CO2 conversion using the recombinant lysate as a biocatalyst resulted in the production of 703 μM formate within 3 hours. These results demonstrate the feasibility of functionally expressing MeFDH1 in a eukaryotic host and provide a platform for future development of biocatalysts for CO₂ valorization.
more목차
1. Introduction 12
2. Material and Methods 16
2.1. Strains and Plasmids 16
2.2. Gene Cloning and Strain Construction 16
2.3. Culture Conditions and Induction 18
2.4. RNA Isolation and RT-PCR 19
2.5. Cell Lysis and Protein Extraction 22
2.6. SDS-PAGE and Western Blot 23
2.7. Tungsten Concentration Analysis 23
2.8. Enzyme Activity Assay 24
2.9. CO2 Reduction to Formate and Quantification 24
3. Results and Discussion 26
3.1. Expression of MeFDH1 α and β Subunits in P. pastoris 26
3.2. Design and Evaluation of Co-Expression Strategies 28
3.2.1. Dual Vector System 28
3.2.2. Dual Promoter System 31
3.2.3. Self-Cleaving System 34
3.3. Integration of Cofactor Genes and Tungsten Supplementation 36
3.4. Functional Validation of MeFDH1 Activity in Engineered P. pastoris 40
3.5. Cultivation and Enzyme Production 42
3.6. CO₂-to-Formate Conversion by Engineered P. pastoris 44
4. Conclusion 46
5. Acknowledgement 47
6. References 48
