Pathway Engineering of Bacillus subtilis for Enhanced N-Acetylneuraminic Acid Production via Whole-Cell Biocatalysis.

N-acetylneuraminic acid (NeuAc) is a common sialic acid that has a wide range of applications in nutraceuticals and pharmaceuticals. However, low production efficiency and high environmental pollution associated with traditional extraction and chemical synthesis methods constrain the supply of NeuAc. Here, a biological approach is developed for food-grade NeuAc production via whole-cell biocatalysis by the generally regarded as safe (GRAS) bacterium Bacillus subtilis (B. subtilis). Promoters for controlling N-acetylglucosamine 2-epimerase (AGE) and NeuAc adolase (NanA) are optimized, yielding 32.84 g L-1 NeuAc production with a molar conversion rate of 26.55% from N-acetylglucosamine (GlcNAc). Next, NeuAc production is further enhanced to 46.04 g L-1 , which is 40.2% higher than that of the strain with promoter optimization, by expressing NanA from Staphylococcus hominis instead of NanA from Escherichia coli. To enhance the expression level of ShNanA, the N-terminal coding sequences of genes with high expression levels are fused to the 5'-end of the ShNanA gene, resulting in 56.82 g L-1 NeuAc production. Finally, formation of the by-product acetoin from pyruvate is blocked by deleting the alsS and alsD genes, resulting in 68.75 g L-1 NeuAc production with a molar conversion rate of 55.57% from GlcNAc. Overall, a GRAS B. subtilis strain is demonstrated as a whole-cell biocatalyst for efficient NeuAc production.