Thermostable Whole-Cell Biocatalysts Enable Sustainable and Practical Synthesis of N-Acetylneuraminic Acid

N-Acetylneuraminic acid (Neu5Ac), a naturally occurring monosaccharide, has diverse applications in food, cosmetics, and pharmaceuticals. A two-enzyme cascade involving N-acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic acid aldolase (NanA) offers a viable strategy for the synthesis of Neu5Ac from N-acetylglucosamine (GlcNAc) and pyruvate, although improving yield and substrate-to-product conversion remains a challenge. Here, thermostable AGE and NanA with high specific activity and a high soluble expression level were identified via sequence similarity networks. Compared with free enzymes, thermostable whole-cell biocatalysts enabled efficient Neu5Ac production without exogenous ATP supplementation. By optimizing the pyruvate feeding, reaction temperature, and cell ratios, whole-cell catalysts produced 768.3 mM (237.6 g/L) Neu5Ac in 36 h, corresponding to a 76.8% GlcNAc-to-Neu5Ac conversion. Immobilized whole-cells in calcium–diatomite–alginate beads retained 46.9% of the initial conversion rate after nine cycles. Collectively, the whole-cell biosystem demonstrates the feasibility of sustainable Neu5Ac synthesis and underscores its potential for industrial-scale production.