N-Acetylneuraminic acid (NeuAc) is the predominant sialic acid found in human cells and a human-identical milk monosaccharide. Due to its numerous health benefits, it has great commercial potential in the pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering strategies is an important approach to its large-scale production. In this study, a NeuAc synthetic pathway was constructed in Escherichia coli BL21(DE3) by deleting the competitive pathway genes and introducing two genes encoding UDP-N-acetylglucosamine (GlcNAc) 2-epimerase (NeuC) and NeuAc synthase (NeuB). UDP-GlcNAc pathway genes, glmS, glmM, and glmU, were overexpressed to strengthen precursor supply for enhancement of NeuAc synthesis. The microbial source of neuC and neuB was optimized, and their expression was fine-tuned. In addition, glycerol as the carbon source showed a much better effect on NeuAc synthesis than glucose. The final engineered strain produced 7.02 g/L NeuAc by shake-flask cultivation. The titer was enhanced to 46.92 g/L by fed-batch cultivation, with the productivity of 0.82 g/L/h and 1.05 g/g DCW.
Keywords: N-acetylneuraminic acid; fed-batch cultivation; metabolic engineering; microbial synthesis; sialic acid.