Recombinant Escheichia coli-Catalyzed Production of Cytidine 5′-Triphosphate from Cytidine 5′-Monophosphate

Sun-Gu Lee; Byung-Gee Kim

Journal of Industrial and Engineering Chemistry, Vol.12, No.5, 757-761, September, 2006

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Recombinant Escheichia coli-Catalyzed Production of Cytidine 5′-Triphosphate from Cytidine 5′-Monophosphate

Sun-Gu Lee^†, and Byung-Gee Kim¹

Department of Chemical and Biochemical Engineering, Pusan National University, Busan 609-735, Korea
¹School of Chemical Engineering, and Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea

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A recombinant Escherichia coli overexpressing CMP-kinase was constructed and employed as a whole cell biocatalyst for the conversion of cytidine 5′-monophosphate to cytidine 5′-triphosphate. In the whole cell biocatalysis, recombinant CMP kinase catalyzed the conversion of CMP to CDP, and endogenous acetate kinase of the E. coli was utilized for the ATP regeneration as well as for the conversion of CDP to CTP. A conversion yield of ca. 88 % CTP was obtained when starting with 20 mM CMP, 1 mM ATP, and 80 mM acetyl phosphate based on the initial CMP concentration. Endogenous pyruvate kinase and polyphosphate kinase were inefficient in the process. The CTP production system was applied to the production of CMP-NeuAc by additionally introducing the CMP-NeuAc synthetase gene into the recombinant E. coli.

Keywords:CMP-kinase;whole cell biocatalysis;ATP regeneration;cytidine 5′-monophosphate

[References]

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[Related Articles]

[1] Bulter T, Elling L, Glycoconjugate J., 16, 147 (1999)

[2] Simon ES, Bednarski MD, Whitesides GM, J. Am. Chem. Soc., 110, 7159 (1988)

[3] Fujio T, Maruyama A, Biosci. Biotechnol. Biochem., 61, 956 (1997)

[4] Simon ES, Grabowski S, Whitesides GM, J. Org. Chem., 55, 1834 (1990)

[5] Hashimoto S, Ozaki A, Curr. Opin. Biotechnol., 10, 604 (1999)

[6] Hirschbein BL, Mazenod FP, Whitesides GM, J. Org. Chem., 47, 3765 (1982)

[7] Ryabova LA, Vinokurov LM, Shekhovtsova EA, Alakhov YB, Spirin AS, Anal. Biochem., 226, 184 (1995)

[8] Kim DM, Swartz JR, Biotechnol. Bioeng., 66, 180 (1999)

[9] Sambrook J, Fritsch EF, Maniatis T, Molecular Cloning, 2nd Edn. Cold Spring Harbor Laboratory Press (1989)

[10] Bucurenci N, Sakamoto HH, Briozzo P, Palibroa N, Serina L, Sarfati RS, Labesse G, Briand G, Danchin A, Barzu O, Gilles AM, J. Biol. Chem., 271, 2856 (1996)

[11] Gilbert M, Watson DC, Wakarchuk WW, Biotechnol. Lett., 19(5), 417 (1997)

[12] Schupp JM, Travis SE, Price LB, Shand RF, Keim P, Biotechniques, 19, 18 (1995)

[13] Skarstedt MT, Silverstein E, J. Biol. Chem., 251, 6775 (1976)

[14] Ryll T, Wagner R, J. Chromatogr., 570, 77 (1991)

[15] Hoffman RC, Wyman PL, Moe OA, Biotechnol. Appl. Biochem., 10, 107 (1988)

[16] Ahn K, Kornberg A, J. Biol. Chem., 265, 11734 (1990)