【學術亮點】Carbon fluxes rewiring in engineered E. coli via reverse tricarboxylic acid cycle pathway under chemolithotrophic condition
Recycling Agriculture: Agricultural Waste Recycling: Research and Development of Low-Carbon Agricultural Technologies【Department of Food Science Biotechnology, NCHU / Chiang, En-Pei Isabel / Professor】
循環農業：農業廢棄物再資源化—研發低碳農業技術【食品暨應用生物科技學系/蔣恩沛教授】
| 論文篇名 | 英文：Carbon fluxes rewiring in engineered E. coli via reverse tricarboxylic acid cycle pathway under chemolithotrophic condition 中文：化能無機自營條件下以逆向三羧酸循環路徑建構之轉殖 E. coli 的碳流導向 |
| 期刊名稱 | Journal of Biological Engineering |
| 發表年份,卷數,起迄頁數 | 2025, 19, no. 20 |
| 作者 | Peng, Jian-Hau; Lo, Shou-Chen; Yu, Yu-Ning; Yang, Ya-Tang; Chen, Yu-Chieh; Tsai, An-, I; Wu, Dong-Yan; Huang, Chu-Han; Su, Tien-Tsai; Huang, Chieh-Chen(黃介辰)*; Chiang, En-Pei Isabel(蔣恩沛)* |
| DOI | 10.1186/s13036-025-00489-w |
| 中文摘要 | 本研究成功建立一株可於化能無機自營 (chemolithotrophic) 條件下，利用氫氣驅動厭氧呼吸直接將氣態 CO2 固碳為生物質量的轉殖 Escherichia coli。此轉殖菌株透過表現來自 Chlorobium tepidum 之逆向三羧酸循環 (reverse tricarboxylic acid cycle, rTCA) 中關鍵酵素，包括 α-酮戊二酸：鐵氧還蛋白氧化還原酶 (α-ketoglutarate: ferredoxin oxidoreductase, KOR) 與 ATP 依賴型檸檬酸裂解酶 (ATP-dependent citrate lyase, ACL)，以實現 CO2 固定與進入中心碳代謝路徑。研究結果顯示，於化能無機自營條件下，單獨表現 KOR 即足以維持細胞基本代謝與能量平衡；當進一步共同表現 ACL 時，CO2 同化效率顯著提升。利用 13CO2 穩定同位素標記實驗證實，KOR 單獨表現即可促進 CO2 進入三羧酸循環中間代謝產物；然而，KOR 與 ACL 的共同表現則使碳流由三羧酸循環重新導向至多種生合成路徑，特別是與蛋白質與核苷酸合成相關之代謝路徑。與僅表現 KOR 相比，共同表現 ACL 顯著提升多種胺基酸 (如甲硫胺酸、蘇胺酸、甘胺酸) 與核苷酸 (如去氧胸苷、去氧胞苷) 中碳骨架的同位素富集 (enrichment) 程度。此結果顯示，在無機氮供應充足的情況下，ACL 有助於含氮代謝物之合成，而 KOR 則能在化能無機自營條件下支撐核心代謝運作。本研究提出一項僅需導入一至兩種異源酵素即可促進 E. coli 於化能無機自營條件下進行 CO2 固定的新策略，揭示了 CO2 固定的最低基因與營養需求，並釐清轉殖菌株中碳流分配的代謝特徵。此成果為永續固碳技術及生物技術應用提供了新的設計概念與未來潛在方向性。 |
| 英文摘要 | A transgenic strain of Escherichia coli has been engineered to directly assimilate gaseous CO2 into its biomass through hydrogen-powered anaerobic respiration. This was achieved by expressing key components of the reverse tricarboxylic acid (rTCA) cycle, including genes encoding α-ketoglutarate: ferredoxin oxidoreductase (KOR) and ATP-dependent citrate lyase (ACL) from Chlorobium tepidum. These enzymes were selected for their essential roles in enabling CO2 fixation and integration into central metabolism. This study found that KOR alone can support cellular maintenance under chemolithotrophic conditions, while additional expression of ACL enhances CO2 assimilation. Using isotopic 13CO2 tracing, it was demonstrated that KOR alone facilitates CO2 assimilation into TCA metabolites. However, co-expression of ACL with KOR redirected carbon fluxes from TCA cycle toward essential metabolic pathways, particularly those involved in protein and nucleotide biosynthesis. Compared to KOR alone, ACL co-expression significantly increased isotopic enrichments in amino acids (e.g., methionine, threonine, glycine) and nucleotides (e.g., deoxythymidine, deoxycytidine). These results suggest that ACL supports the synthesis of nitrogen-containing metabolites when inorganic nitrogen is sufficient, while KOR alone sustains core metabolic functions under chemolithotrophic conditions. This study demonstrates a novel strategy to engineer E. coli for CO2 fixation using only one or two heterologous enzymes under chemolithotrophic conditions. These findings reveal the minimal genetic and nutritional requirements for CO2 assimilation and provide insights into metabolic flux partitioning in engineered strains. This research paves the way for sustainable applications in carbon fixation and biotechnological innovation. |
| 發表成果與本中心研究主題相關性 | 本研究成功提建立一株可固定二氧化碳的轉殖大腸桿菌，同時藉由同位素標定實驗分析得知，可藉由不同代謝工程策略調整二氧化碳的碳流流向，導流至重要代謝產物的生成，包含蛋白質、核苷酸，達到環境保護、增加碳匯及廢棄物回收轉換具有經濟價值副產品之多重功效。具永續循環、環境友善之研究成果。本成果同時成功申請專利獲證 (證號：I857496) 與多項國際研討會/競賽獲獎之肯定，包含：2021 國際生物技術與生化工程學會研討會海報優等獎、 2022 臺灣表基因體學研討會及生物科技暨健康產業國際學術研討會海報第一名、2023 第十七屆生物催化與農業生物科技國際研討會英文口語競賽博士組優勝、 2024 第六屆綠點子國際發明暨設計競賽鈦金獎 (百組隊伍中總分前 10% 獲獎)、2025 台灣創新技術博覽會發明競賽銀獎。顯示本研究之重要性。 |
