Efficient Production of N-Acetylneuraminic Acid in Escherichia coli Based on the UDP-N-Acetylglucosamine Biosynthetic Pathway

Abstract

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.

Sialic acids in infection and their potential use in detection and protection against pathogens

“…Translational impact for sialic acids faces a number of challenges going forwards. While the scalable enzymatic synthesis of sialic acid [155][156][157][158] and sialylated glycans [159][160][161] has been achieved, the correct presentation of this key sugar recognition element is critical to achieving physiologically or therapeutically relevant biological recognition. For instance, the valency of NeuAc presentation 97 as well as secondary interactions from the glycan chain to which it is attached 140 can have a profound impact on target engagement.…”

Construction of an Escherichia coli chassis for efficient biosynthesis of human-like N-linked glycoproteins

“…To alleviate this metabolic burden, we propose to further optimize gene expression levels through engineering promoters and ribosome-binding sites to better balance the protein expression, LLO production, and glycosylation processes. As improving the terminal sialylation efficiency remains a challenge and other studies have suggested that overexpression of neuA , to supplement CMP-Neu5Ac, and alkaline phosphatase can decrease the efficiency of the sialidase enzyme, thereby increasing its sialyltransferase efficiency ( Kang et al, 2015 ; Zhang et al, 2022 ; Zhao et al, 2023 ), we will focus on incorporating a stronger promoter into the neuBCA gene cluster, as well as screening other α-2,3-sialyltransferases to optimize sialylation.…”