To develop a strategy for producing N-acetyl-d-neuraminic acid (Neu5Ac), which is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and pyruvate, but without using pyruvate. An efficient three-module whole-cell biocatalyst strategy for Neu5Ac production by utilizing intracellular phosphoenolpyruvate was established. In module I, the synthetic pathway was constructed by coexpressing GlcNAc 2-epimerase from Anabaena sp. CH1 and Neu5Ac synthase from Campylobacter jejuni in Escherichia coli. In module II, the Neu5Ac degradation pathway of E. coli was knocked out, resulting in 2.6 +/- 0.06 g Neu5Ac l(-1) after 72 h in Erlenmeyer flasks. In module III, the transmembrane mode of GlcNAc was modified by disruption of GlcNAc-specific phosphotransferase system and Neu5Ac now reached 3.7 +/- 0.04 g l(-1). In scale-up catalysis with a 1 l fermenter, the final Neu5Ac yield was 7.2 +/- 0.08 g l(-1). A three-module whole-cell biocatalyst strategy by manipulating synthetic, degradation and transmembrane pathways in E. coli was an economical method for Neu5Ac production.