Enhanced d-Pantothenic Acid Biosynthesis in Corynebacterium glutamicum by the CPSPC System and Spatial Organization Strategy.

d-Pantothenic acid (D-PA) is an essential precursor of coenzyme A and the acyl carrier protein. Its biosynthesis requires abundant precursors and precise regulation of the multifunctional enzyme, ketopantoate reductase. Here, we engineered Corynebacterium glutamicum for enhanced D-PA production through metabolic engineering, spatial enzyme organization, and a dynamically controlled CPSPC system (Coenzyme and Product Synthesis Pathway Coupling System). Competing pathways were suppressed, while target synthesis routes and 5,10-methylenetetrahydrofolate supply were strengthened. A RIAD-RIDD-mediated substrate channel between dihydroxy acid dehydratase and ketopantoate hydroxymethyltransferase was used to optimize enzymatic stoichiometry and localization. After functionalizing a glycine riboswitch in C. glutamicum, we developed the CPSPC system that dynamically couples one-carbon metabolism with D-PA synthesis. Fluorescence tracking confirmed its glycine-responsive regulation. The engineered strain DPAj-2-tuf achieved 23.06 g/L in a 5 L bioreactor over 63 h, productivity of 0.37 g/L/h, and glucose yield of 0.17 g/g. These outcomes offer an efficient and scalable strategy for industrial D-PA production using C. glutamicum.