Enhancing d-Pantothenic Acid Production in Corynebacterium glutamicum via Integrating Dynamic Regulation With Structure-Guided Protein Engineering of Ketopantoate Reductase.

d-Pantothenic acid (DPA) is an essential vitamin with broad applications. In this study, we engineered Corynebacterium glutamicum for high-titer DPA production by integrating dynamic pathway regulation with structure-guided protein engineering. Functional characterization revealed that endogenous CgPanE and BsPanE2 function as α-hydroxy acid dehydrogenases rather than ketopantoate reductases, whereas heterologous EcPanE and BsPanE, bifunctional activity toward both ketoisovalerate and ketopantoate, possess larger active cavities. A stationary-phase promoter (P4-N14) was employed to delay ketopantoate reductase expression, reducing precursor consumption. Multiple screening strategies were employed to identify candidate residues for alanine scanning and saturation mutagenesis, which yielded two beneficial mutants, T119I and I183S. Notably, I183S exhibited the most prominent improvements, with a 2.25-fold increase in specific activity and a 1.67-fold higher kcat/Km ratio than the wild-type. Molecular dynamics simulations indicated that the mutations enhanced catalytic efficiency by providing a more stable catalytic environment, tighter binding with the catalytic units, expanding the active cavity, and shortening the substrate tunnel length. The final engineered strain achieved a DPA titer of 36.12 g/L in a 5 L bioreactor. This study establishes a robust chassis for sustainable DPA production and provides a generalizable framework for engineering other valuable biochemicals.