Hybrid Embden–Meyerhof–Parnas Pathway for Reducing CO₂ Loss and Increasing the Acetyl-CoA Levels during Microbial Fermentation

The fundamental Embden–Meyerhoff–Paranas (EMP) pathway for sugar catabolism, anabolism, and energy metabolism has been reconstituted with nonoxidative glycolysis (NOG) for sustainable biosystems. Although carbon conservation was achieved via NOG, the energy metabolism was significantly limited. Herein, we showed the construction of a hybrid EMP that replaced the first phase of the EMP in an industrial bacterium, Corynebacterium glutamicum, with NOG in the absence of an evolutionary process and observed a metabolic link of carbon and phosphorus metabolism. In accordance with synthetic glucose kinase activity and phosphoketolase on the hybrid EMP, cell growth was completely recovered in the C. glutamicum pfkA mutant strain, where the first phase of EMP was eliminated. By analyzing the carbon metabolism of the various mutants, we have revealed a carbon flux of acetyl phosphate to acetyl-CoA and a phosphate-replenishing pathway that involved trehalose biosynthesis for the generation of inorganic phosphate (P_i) sources in the hybrid EMP when the external P_i supply was limited. As a result, an engineered strain with the hybrid EMP pathway reduced CO₂ emission by 10% and increased the acetyl-CoA levels by 19% in comparison to the wild type. Thus, the redesigned hybrid EMP pathway with balanced carbon and phosphorus states provides an efficient microbial platform for sustainable biochemical production.