Metabolically Modifying the Central and Competitive Metabolic Pathways for Enhanced D-Pantoic Acid Synthesis

D-Pantoic acid is an essential precursor for the synthesis of vitamin B₅. However, the microbial synthesis of D-pantoic acid suffers from a low yield. Herein, to improve D-pantoic acid biosynthesis in Escherichia coli, the central metabolic and byproduct-forming pathways were first engineered, increasing the D-pantoic acid titer to 1.55 g/L from 0.75 g/L. Subsequently, the modification was focused on preventing the accumulation of α-ketoglutarate (α-KG). Six genes (ppc, mdh, icd, sucA, kgtP, and dcuA) related to α-KG metabolism and transport were screened by the CRISPRi system and further genetically manipulated. Ultimately, significantly improved D-pantoic acid biosynthesis (2.03 g/L in a shake flask and 14.78 g/L in a 5-L bioreactor) with dramatically reduced formation of byproducts was achieved. To our best knowledge, this is the first attempt to modify the key metabolic targets related to α-KG accumulation for enhanced D-pantoic acid biosynthesis. These findings would also offer valuable insights into the metabolic regulation of other related metabolites.