Metabolic rewiring for enhanced itaconate yield through heterotrophic CO2 fixation in Escherichia coli

It is crucial to obtain high production yields in the microbial production of diverse biochemicals to maximize economic viability. However, microorganisms, particularly heterotrophs, release carbon dioxide (CO2) during the oxidation of organic compounds for energy and building blocks. This metabolism limits the theoretical maximum carbon yield and constrains the development of efficient microbial cell factories. Specifically, metabolite production from tricarboxylic acid (TCA)-cycle intermediates involves major CO2 emission steps, leading to unavoidable carbon loss. Central carbon metabolism in Escherichia coli, a model heterotroph, was synthetically rewired to facilitate CO2 fixation and escalate carbon yields. Specifically, we blocked multiple routes to prevent pyruvate oxidation to acetyl-CoA, which was identified as a major CO2 emission step, and promoted CO2 incorporation into TCA cycle intermediates by reengineering endogenous pathways for co-utilization of glucose and acetate. This strategy was applied to produce itaconate, a promising bioplastic precursor, achieving a yield of 0.81 C mol/C substrate mol, a value near the theoretical maximum yield obtainable from glucose (0.83 C mol/C mol). This heterotrophic CO2 fixation strategy can be used to produce TCA-derived chemicals and enhance the economic viability of microbial production. This study demonstrates that synthetically rewiring central carbon metabolism enables direct incorporation of CO₂ into the product, thereby increasing carbon yields approaching the conventional maximum value. This heterotrophic CO2 fixation strategy not only provides a breakthrough for efficient itaconate production but also serves as a versatile platform for engineering microbial cell factories to produce various TCA-derived chemicals with amplified carbon yield.