Thymidine Production by Corynebacterium ammoniagenes Mutants

  • Song, Kyung-Hwa (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Kwon, Do-Young (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies) ;
  • Kim, Sang-Yong (BioNgene Co., Ltd.) ;
  • Lee, Jung-Kul (BioNgene Co., Ltd.) ;
  • Hyun, Hyung-Hwan (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
  • 발행 : 2005.06.01

초록

Corynebacterium ammoniagenes ATCC 6872, which does not accumulate pyrimidine nucleoside or nucleotide, was metabolically engineered to secrete a large amount of thymidine. Characteristics of 5-fluorouracil resistance ($FU^r$), hydroxyurea resistance ($HU^r$), trimethoprim resistance ($TM^r$), thymidylate phosphorylase deficiency ($deoA^-$), inosine auxotrophy ($ino^-$), 5-fluorocytosine resistance ($FC^r$), thymidine kinase deficiency, and thymidine resistance ($thym^r$) were successively introduced into mutant strains KR3 and DY5T9-5, and shake-flask cultures were able to accumulate 408.1 mg/l and 428.2 mg/l of thymidine, respectively, as a major product. The mutant strains did not accumulate thymine at all and accumulated less than 10 mg/l of other pyrimidine nucleosides, such as cytosine, cytidine, and deoxycytidine, as byproducts.

키워드

참고문헌

  1. Asahi, S. and Y. Tsueni. 1989. Method for production of cytidine and/or deoxycytidine. US patent 4,839,285
  2. Freisheim J. H., C. C. Smith, and P. M. Guzy. 1972. Dihydrofolate reductase and thymidylate synthetase in strains of Streptococcus faecium resistant to pyrimethamine, chlorguanide triazine, trimethoprim, and amethopterin. Arch. Biochem. Biophys. 148: 1-9 https://doi.org/10.1016/0003-9861(72)90108-7
  3. Hammer, J. K. 1983. Nucleotide catabolism, pp. 203-258 In Munch-Petersen, A. (ed.), Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganism. Academic Press, London
  4. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. BioI. Chem. 193: 265-275
  5. Neuhard, J. 1983. Utilization of preformed pyrimidine bases and nucleoside, pp. 95-148. In Munch-Petersen, A. (ed.) Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganism. Academic Press, London
  6. Roland, K. L., F. E. Powell, and C. L. Jr. Turnbough. 1985. Role of translation and attenuation in the control of pyrBl operon expression in Escherichia coli K-12. J. Bacteriol. 163: 991-999
  7. Potvin, B. W., R. J. Jr. Kelleher, and H. Gooder. 1975. Pyrimidine biosynthetic pathway of Bacillus subtilis. J. Bacteriol. 123: 604-615
  8. Santi, D. V. and C. S. McHenry. 1972. 5-Fluoro-2'deoxyuridylate: Covalent complex with thymidylate synthetase. Proc. Natl. Acad. Sci. USA 69: 1855-1857
  9. Saunders P. P., B. A. Wilson, and G. F. Saunders. 1969. Purification and comparative properties of a pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus. J. BioI. Chem. 244: 3691-3697
  10. Schwartz, M. 1976. Thymidine phosphorylase from Escherichia coli, pp. 442-443. In A. H. Patricia and E. J. Mary (eds.). Methods in Enzymology, vol. 51, Academic Press, Avenue, New York, U.S.A
  11. Scocca, J. J. 1971. Purification and substrate specificity of pyrimidine nucleoside phosphorylase from Haemophilus irifluenzae. J. BioI. Chem. 246: 6606-6610
  12. Timson J. 1975. Hydroxyurea. Mutat. Res. 32: 115-132 https://doi.org/10.1016/0165-1110(75)90002-0
  13. Tsen, S. D. 1994. Chemostat selection of Escherichia coli mutants secreting thymidine, cytosine, uracil, guanine, and thymine. Appl. Microbiol. Biotechnol. 41: 232-238