Systematic design and evaluation of artificial CO2 assimilation pathways

With the advancement of industry and bio-agriculture, the effective management of CO₂ has emerged as a critical challenge for humanity. This study systematically explores multiple CO₂ assimilation pathways using the comb-FBA algorithm, aiming to identify efficient artificial carbon fixation pathways. By extracting 49 CO₂ and HCO₃⁻ involved reactions and combining them with 6,529 reactions from MetaCyc, we constructed the computational set for analysis. These 16 core reactions give rise to 136 carbon fixation pathways for single C₂ targets (such as acetyl-CoA, glyoxylate, and oxalate) and 576 carbon fixation pathways for single C₃ targets (including glyceraldehyde-3-phosphate or pyruvate). Based on these core reactions, we identified four principal carbon fixation modes. Through systematic assessments, we identified 12 promising CO₂ fixation pathways, each comprising no more than 20 reaction steps and demonstrating thermodynamic feasibility. Through further analysis of enzyme oxygen sensitivity and availability, we identified three novel and promising pathways. By examining metabolite conversion relationships, we also identified alternative carbon fixation reaction modules, offering flexibility for pathway optimization and experimental design. In conclusion, this study provides a diverse library of artificial carbon fixation pathways, demonstrating the power of the comb-FBA algorithm in designing carbon assimilation pathways and laying the foundation for more efficient CO₂ fixation strategies.