Artificial cell-free pathway for carbon-efficient glycerol-HCO₃^- conversion to l-aspartate with ATP and cofactor...

Carbon-efficient conversion of waste-derived carbon coupled with HCO₃^- utilization is an attractive goal in sustainable biomanufacturing. Here, a cell-free enzymatic cascade was developed, termed the Artificial Glycerol-HCO₃^- Utilization pathway, for direct conversion of glycerol and HCO₃^- into l-aspartate. The modular pathway integrates glycerol oxidation, pyruvate carboxylation, and reductive amination, enabling carbon-efficient upgrading of a three-carbon substrate into a four-carbon amino acid. Aldehyde dehydrogenase was identified as a major kinetic bottleneck, and structure-guided engineering yielded a variant with 5.4-fold higher activity, substantially enhancing pyruvate formation. In the carbon-fixation module, severe non-productive ATP and NADH hydrolysis by pyruvate carboxylase was uncovered and mitigated through reaction engineering, improving productive cofactor coupling. Under optimized conditions, the integrated system produced 75.4 mM l-Asp from glycerol with a molar conversion yield of 75%. Extension of the pathway toward l-homoserine and R-acetoin further defined the modularity and redox operating boundaries of the platform. However, the high efficiency of the current system comes at the expense of substantial ATP and cofactor consumption, limiting its economic competitiveness under current conditions. These results demonstrate the feasibility of this pathway for efficient conversion of glycerol-HCO₃^- to l-Asp, while highlighting improved ATP and cofactor economy as key challenges for future development.