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, Chiang En-Pei Isabel
Doctoral Program in Microbial Genomics, National Chung Hsing University and Academia Sinica, Taichung City and Taipei City, 402 and 115, Taiwan.
Advanced Plant and Food Crop Biotechnology Center, National Chung Hsing University, Taichung City, 402, Taiwan.
J Biol Eng. 2025 Feb 26;19(1):20. doi: 10.1186/s13036-025-00489-w.
A transgenic strain of Escherichia coli has been engineered to directly assimilate gaseous CO 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 CO 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 CO assimilation. Using isotopic CO tracing, it was demonstrated that KOR alone facilitates CO 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 CO fixation using only one or two heterologous enzymes under chemolithotrophic conditions. These findings reveal the minimal genetic and nutritional requirements for CO 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.
已构建出一种转基因大肠杆菌菌株，通过氢驱动的厌氧呼吸作用将气态一氧化碳直接同化为其生物质。这是通过表达反向三羧酸（rTCA）循环的关键组分实现的，包括编码来自嗜热绿菌的α-酮戊二酸：铁氧化还原蛋白氧化还原酶（KOR）和ATP依赖性柠檬酸裂解酶（ACL）的基因。选择这些酶是因为它们在使一氧化碳固定并整合到中心代谢中发挥着重要作用。
本研究发现，仅KOR就能在化能无机营养条件下维持细胞存活，而额外表达ACL可增强一氧化碳同化作用。使用同位素一氧化碳示踪法表明，仅KOR就能促进一氧化碳同化为三羧酸代谢物。然而，ACL与KOR共表达会使碳通量从三羧酸循环转向基本代谢途径，特别是那些参与蛋白质和核苷酸生物合成的途径。与仅表达KOR相比，ACL共表达显著增加了氨基酸（如蛋氨酸、苏氨酸、甘氨酸）和核苷酸（如脱氧胸苷、脱氧胞苷）中的同位素富集。这些结果表明，当无机氮充足时，ACL支持含氮代谢物的合成，而仅KOR能在化能无机营养条件下维持核心代谢功能。
本研究展示了一种新策略，即在化能无机营养条件下仅使用一两种异源酶对大肠杆菌进行工程改造以实现一氧化碳固定。这些发现揭示了一氧化碳同化所需的最低遗传和营养要求，并为工程菌株中的代谢通量分配提供了见解。这项研究为碳固定的可持续应用和生物技术创新铺平了道路。
