Metabolic Engineering of Escherichia coli for Increased Bioproduction of N -Acetylneuraminic Acid.

N -Acetylneuraminic acid (NeuAc) is widely used in the food and pharmaceutical industries. Therefore, it is important to develop an efficient and eco-friendly method for NeuAc production. Here, we achieved de novo biosynthesis of NeuAc in an engineered plasmid-free Escherichia coli strain, which efficiently synthesizes NeuAc using glycerol as the sole carbon source, via clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9-based genome editing. NeuAc key precursor, N -acetylmannosamine (ManNAc; 0.40 g/L), was produced by expressing UDP- N -acetylglucosamine-2-epimerase and glucosamine-6-phosphate synthase (GlmS) mutants and blocking the NeuAc catabolic pathway in E. coli BL21 (DE3). The expression levels of GlmM and GlmU-GlmS A metabolic modules were optimized, significantly increasing the ManNAc titer to 8.95 g/L. Next, the expression levels of NeuAc synthase from different microorganisms were optimized, leading to the production of 6.27 g/L of NeuAc. Blocking the competing pathway of NeuAc biosynthesis increased the NeuAc titer to 9.65 g/L. In fed-batch culture in a 3 L fermenter, NeuAc titer reached 23.46 g/L with productivity of 0.69 g/L/h, which is the highest level achieved by microbial synthesis using glycerol as the sole carbon source in E. coli . The strategies used in our study can aid in the efficient bioproduction of NeuAc and its derivatives.