High-Efficiency N-acetylneuraminic acid biosynthesis in plasmid-free Escherichia coli MG1655

N-acetylneuraminic acid (Neu5Ac), the predominant sialic acid in human milk oligosaccharides, has garnered substantial interest for infant nutrition and pharmaceutical applications. Microbial production via the N-acetylglucosamine 2-epimerase (AGE) pathway suffers from extensive byproduct accumulation, while plasmid-based systems impose metabolic burden and require costly antibiotics. Here, we engineered a fully chromosomal, plasmid-free Escherichia coli platform for high-efficiency Neu5Ac biosynthesis through the UDP-N-acetylglucosamine 2-epimerase (NeuC) pathway. Chromosomal integration of Neisseria meningitidis neuBC genes, strategic deletion of competing pathways, multi-copy glmM integration, and GlmS deregulation via rapZ deletion established a streamlined metabolic chassis. The optimized strain MGN12 produced 13.15 g/L Neu5Ac in shake-flask cultivation and achieved the highest reported titer of 115.72 g/L in glucose-fed 10-L fed-batch fermentation without antibiotics or inducers. This work establishes a robust chromosomal NeuC-based production strategy and provides a scalable foundation for industrial Neu5Ac biosynthesis.