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 NeuAc production with a molar conversion rate of 26.55% from N-acetylglucosamine (GlcNAc). Next， NeuAc... More
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 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 ， 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 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 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.
