Model-Driven Cell-Free Synthesis of the C4 Amino Acid Aspartate Directly from CO2

The synthesis of amino acids from CO2 as the sole carbon source is a critical goal for sustainable biomanufacturing, offering a green alternative to conventional production methods plagued by greenhouse gas emissions and resource competition. Here, we construct a highly efficient cell-free enzymatic platform for the direct biosynthesis of aspartate from CO2. Our integrated platform couples a carbon-fixing module using the modified reductive glycine pathway (rGlyP) with a custom-designed synthesis cascade that condenses two glycine molecules into aspartate, driven by glycine oxidase and a redesigned b-hydroxyaspartate cycle (BHAC). A key challenge in this multienzyme system is achieving pathway synergy. To address this, we employed an enzyme-constrained static model to rationally design the optimal stoichiometry of the five-enzyme cascade. This computational approach not only boosted the final aspartate yield by over 25% but was also essential in identifying glycine oxidase (Bs-ThiO) as the primary rate-limiting bottleneck. Through systematic optimization of enzymes, reaction conditions, and feeding strategies, the system finally achieved accumulation of 17.3 mM aspartate (2.3 g/L) within 2 h, reaching an exceptional 92% of the model-predicted theoretical yield. This study establishes a powerful and sustainable platform for green amino acid production and showcases the significant potential of combining computational design with cell-free engineering to advance the frontier of CO2-based biomanufacturing.