Synergistic Use of Multiple Regulatory Strategies for Efficient N-Acetylneuraminic Acid Biosynthesis in <i>Escherichia coli</i>

N-Acetylneuraminic acid (NeuAc), a sialic acid derivative with critical roles in antiviral drug synthesis and infant nutrition, faces biosynthesis bottlenecks due to insufficient phosphoenolpyruvate (PEP) precursor flux and byproduct accumulation in the microbial chassis. Here, we present a multitiered engineering strategy to enhance the NeuAc production in <i>Escherichia coli</i>: dynamic control of PEP metabolism-related genes, global transcription machinery engineering, and biosensor-guided screening of the carbon starvation regulator CsiR mutant. The engineered strain DN20 achieved 28.17 g/L NeuAc in shake-flask fermentation using glucose─a 1.6-fold increase over the parent strain DN11 and the highest shake-flask titer for glucose-based <i>E. coli</i> systems. Transcriptomic analysis was performed to investigate the potential impact of CsiR overexpression on NeuAc biosynthesis. This work establishes a multisynthetic biology strategy fusion for metabolic reprogramming and provides a reference for optimizing high-value biochemical production.