Whole‐cell catalysis of N‐ acetylglucosamine into N‐ acetylneuraminic acid using a dual‐pathway conversion in engineered Escherichia coli Nissle 1917

Abstract BACKGROUND N‐ Acetylneuraminic acid (Neu5Ac) is used in food, pharmaceutical, nutraceutical, and cosmetic applications and is a key precursor for the biosynthesis of sialylated human milk oligosaccharides. In the whole‐cell synthesis of Neu5Ac, N‐ acetylmannosamine (ManNAc) is a critical intermediate, and an adequate supply is essential for achieving high‐efficiency production. Given the high cost of cofactors, the establishment of an effective cofactor recycling system is also important for reducing process costs. In this study, the probiotic strain Escherichia coli Nissle 1917 (EcN) Δ pMUT1 Δ pMUT2 (DE3), engineered with a dual metabolic pathway, was used as the chassis cell for Neu5Ac synthesis via a designed whole‐cell catalysis system, with N‐ acetylglucosamine (GlcNAc) and pyruvate as substrates. RESULTS To enhance production of the intermediate ManNAc, two synthetic pathways, with GlcNAc as substrate, were used to improve catalytic efficiency. The inherent capacity of EcN to recycle uridine 5′‐monophosphate (UMP) to uridine 5′‐triphosphate (UTP) met the cofactor requirements for Neu5Ac biosynthesis sufficiently. The synthesis of Neu5Ac by engineered EcN was optimized in several aspects, and whole‐cell catalysis in a 5 L bioreactor achieved a Neu5Ac titer of 71.25 g L −1 . CONCLUSIONS The implementation of a dual pathway improved Neu5Ac production substantially. The inherent cofactor‐recycling capability of the selected chassis strain, EcN, adequately supported the biosynthetic demands of Neu5Ac. These results demonstrate that EcN is a promising engineered platform for efficient Neu5Ac biosynthesis, and the approach developed here provides a cost‐effective strategy by minimizing cofactor‐related expenses. © 2026 Society of Chemical Industry.