A Synthetic Acetyl-CoA Bi-cycle Synergizes the Wood-Ljungdahl Pathway for Efficient Carbon Conversion in Syngas Fermentation

The Wood-Ljungdahl pathway (WLP) is a natural carbon fixation pathway capable of converting one-carbon (C1) compounds (CO2, CO, formate) to two-carbon (C2) metabolite acetyl-CoA or coordinating with canonical glycolysis to convert sugar feedstocks to acetyl-CoA with high carbon yield. The catalytic inefficiency and engineering difficulty in key enzymes, however, limit the biosynthetic potential of this pathway. Here we design a synthetic acetyl-CoA bi-cycle to synergize the WLP for efficient C2 metabolite synthesis. This pathway produces an acetyl-CoA by fixation of two CO2 equivalents via three functional modules acting in series: carbon fixation, gluconeogenesis, and non-oxidative glycolysis. We examine the pathway through comprehensive in silico thermodynamic and kinetic analyses. The prototypic pathway is implemented in a syngas-fermenting Clostridium ljungdahlii DSM 13528 by expressing a heterologous phosphoketolase and coordinating with native enzymes in the host acetogen. We demonstrated the effectiveness of this synthetic pathway in carbon conversion under various growth conditions, which complements the WLP for valorization of syngas as well as sugar feedstocks with high catalytic efficiency. This study underscores the reductive acetyl-CoA bi-cycle as a practical strategy to improve carbon conversion and redox homeostasis in the acetogenic host.