Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst.
An 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 alpha2,3-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 alpha2,3-sialyltransferase.
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000138:chemical synthesis / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D005240:Feasibility Studies; D015964:Gene Expression Regulation, Bacterial; D009097:Multienzyme Complexes / Q000378:metabolism*; D000216:N-Acylneuraminate Cytidylyltransferase / Q000378:metabolism; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D012799:Sialyltransferases / Q000378:metabolism*; D013045:Species Specificity
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D012799:Sialyltransferases; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D000216:N-Acylneuraminate Cytidylyltransferase; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase