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.