AbstractAn Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5′‐monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5′‐monophosphate N‐acetylneuraminic acid (CMP‐NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP‐NeuAc. Both recombinant E. coli extract and whole cells catalyzed the production of CMP‐NeuAc from CMP (20 mM), N‐acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used. It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP. On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently. The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP‐NeuAc by about 1.5‐fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction. This E. coli strain could also be employed as an enzyme source for in situ regeneration of CMP‐NeuAc in a sialyltransferase catalyzed reaction. About 90% conversion yield of α2,3‐sialyl‐N‐acetyllactosamine was obtained from N‐acetyllactosamine (20 mM), CMP (2 mM), N‐acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E. coli extract and α2,3‐sialyltransferase. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 516–524, 2002.