Synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetylglucosamine (GlcNAc) and pyruvate was carried out by constructing and expressing a polycistronic plasmid encoding an N-acetylglucosamine 2-epimerase (AGE) gene and an N-acetylneuraminic acid lyase (Nal) gene simultaneously. Nal from Escherichia coli K12 and AGEs from Synechocystis sp. PCC 6803 (snAGE) and Anabaena sp. CH1 (anAGE) were used. And four polycistronic plasmids were constructed in which the positions of AGE gene differed with respect to Nal gene. Among these plasmids, pET-28a-Nal-anAGE with anAGE gene located next to Nal gene caused the production of the highest amount of Neu5Ac, generating 61.3 g/L in 60 h by whole-cell catalysis without the addition of ATP as AGE activator. And pET-28a-Nal-anAGE lowered anAGE's expression level, allowing it to fold properly. Thus, an inclusion-body-free E. coli strain capable of producing Neu5Ac by whole-cell catalysis with high yield and low cost was constructed in the present study. (C) 2012 Elsevier Ltd. All rights reserved.