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N-acetyl-D-neuraminic acid (Neu5Ac) is a costly precursor for many drugs such as anti-influenza antivirals. In a previous study, a whole-cell process for Neu5Ac production was developed using a combination of two Escherichia coli cells expressing Anabaena sp. CH1 N-acetyl-D-glucosamine-2-epimerase (bage) and E. coli N-acetyl-D-neuraminic acid aldolase (nanA), respectively. In this study, we constructed a bAGE and NanA co-expression system to improve Neu5Ac production. Two recombinant E. coli strains, E. coli BL21 (DE3) pET-bage-nanA (HA) and E. coli BL21 (DE3) pET-bage-2nanA (HAA), synchronously expressing bAGE and NanA were used as biocatalysts to generate Neu5Ac from N-acetyl-D-glucosamine (GlcNAc) and pyruvate. The HA biocatalysts produced 187.5 mM Neu5Ac within 8 h. The yield of GlcNAc was 15.6%, and the Neu5Ac production rate was 7.25 g/L/h. The most active HAA biocatalysts generated 412.6 mM Neu5Ac and a GlcNAc yield of 34.4%. HAA achieved a Neu5Ac production rate of 15.9 g/L/h, which surpassed those for all reported Neu5Ac production processes so far. The present study demonstrates that using recombinant E. coli cells synchronously expressing bAGE and NanA as biocatalysts could potentially be used in the industrial mass production of Neu5Ac.