Reprogramme the E. coli metabolism by engineering a functional carbon-fixation pathway

Background Rising atmospheric CO₂ levels and their impact on climate change have intensified the need for innovative carbon capture and fixation strategies. The Calvin-Benson-Bassham (CBB) cycle, a central metabolic pathway in all photoautotrophic organisms and many autotrophic bacteria, plays a pivotal role in global carbon assimilation but is limited by the low catalytic efficiency of Rubisco. Results Here, we engineered a complete, functional CBB cycle in Escherichia coli , by heterologously expressing up to 13 genes encoding phosphoribulokinase, α-carboxysomes, and inorganic carbon pumps. This bioengineering approach allowed E. coli to utilize atmospheric CO ₂ and led to upregulation of sugars such as ribose (4.94-fold) and xylitol (8.94-fold). Detailed metabolomic profiling of central carbon metabolism using gas chromatography-mass spectrometry (GC-MS) demonstrated that installation of the CBB cycle has a notable impact on the metabolic landscape of E. coli , resulting in substantial alterations in central carbon and amino acid metabolism. These findings deepen our understanding of the natural biological carbon-fixation pathway and its engineering in heterotrophic hosts. Furthermore, this work provides a versatile platform for evaluating and selecting efficient carbon-fixation modules, as well as assessing metabolic bottlenecks in engineered systems. Conclusion These advances offer practical guidance for rational metabolic engineering in diverse organisms for biotechnological applications, including carbon sequestration, sustainable bioproduction, and crop improvement.