Enhanced Activity of Cytidine Deaminase by Gene Family Shuffling.

Gene Family Shuffling을 이용한 Cytidine Deaminase 활성 증가

  • Hong, Sik (Department of Microbiology, Kyungpook National University) ;
  • Kim, Kyung-Dong (Department of Microbiology, Kyungpook National University) ;
  • Song, Bang-Ho (Department of Biology Education, Kyungpook National University) ;
  • Jung, Kyung-Hwa (Genomics Laboratory, Amicogen Inc.) ;
  • Kim, Sa-Yeol (Department of Microbiology, Kyungpook National University)
  • 홍식 (경북대학교 미생물학과) ;
  • 김경동 (경북대학교 미생물학과) ;
  • 송방호 (경북대학교 생물교육과) ;
  • 정경화 (아미코젠(주) 제노믹스연구실) ;
  • 김사열 (경북대학교 미생물학과)
  • Published : 2002.12.01

Abstract

A family shuffling associating PCR-based and in vitro recombination and expression in Escherichia coli cdd mutant was carried out. Two cdd genes encoding cytidine deaminases (CDase) from thermophilic Bacillus caldolyticus and B. stearothermophilus were shuffled. Around 150 viable mutant colonies screened on AB minimal medium without uracil by E. coli cdd complementation were selected for cytidine deaminase assay and 4 candidates (SH1067, SH1077, SH1086, and SH1118) were chosen for the detailed study. The nucleotide sequence analyses of 4 selected mutants revealed that they have several point mutations and recombinations. Surprisingly, the SH 1067 showed 770 fold more specific CDase activity at $80^{\circ}C$ than that of T101 from parental B. stearothermophilus.

Keywords

Bacillus caldolyticus;B. stearothermophilus;cytidine deaminase;family shuffling.

References

  1. Eur. J. Biochem. v.19 Induction of enzymes involved in the catabolism of deoxyribonucleosides in Escherichia coli K12 Hammer Jespersen,K.;A.Munch Petersen;P.Nygaard;M.Schwarz https://doi.org/10.1111/j.1432-1033.1971.tb01345.x
  2. Trends Biotechnol. v.16 Artificial evolution by DNA shuffling Harayama,S. https://doi.org/10.1016/S0167-7799(97)01158-X
  3. Enzyme Microb. Technol. v.30 Purification and characterization of thermostable cytidine deaminase encoded by the Bacillus caldolyticus cdd gene Woo,J.H.;N.J.Heo;S.Y.Ghim;J.G.Kim;B.H.Song https://doi.org/10.1016/S0141-0229(01)00480-X
  4. Biochemistry v.31 Cloning and nucleotide sequence of the Escherichia coli cytidine deaminase (cdd) gene Yang,C.;D.Carlow;R.Wolfenden;S.A.Short https://doi.org/10.1021/bi00132a003
  5. Mol. Gen. Genet. v.216 Chromosomal location cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase Song,B.H.;J.Neuhard https://doi.org/10.1007/BF00334391
  6. Biochemistry v.38 Cytidine deaminase from Bacillus subtilis and Escherichia coli compensating effects of changing zinc coordination and quatermary structure Carlow,D.C.;C.W.Carter;N.Jr.Mejlhed;J.Neuhard;R.Wolfenden https://doi.org/10.1021/bi990819t
  7. Nature v.391 DNA shuffling of a family of gene from diverse species accelerates directed evolution Crameri,A.;S.A.Raillard;E.Bermudez;W.P.Stemmer https://doi.org/10.1038/34663
  8. J. Bacteriol. v.179 Functional analysis of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally Kimura,N.;A.Nishi;M.Goto;K.Furukawa https://doi.org/10.1128/jb.179.12.3936-3943.1997
  9. J. Mol. Biol. v.23 DNA replication and the division cycle in Escherichia coli Lark,D.J.;O.Maaloe https://doi.org/10.1016/S0022-2836(67)80070-6
  10. Biochemistry v.14 Crystal structure of the tetrameric cytidine deaminase from Bacillus subtilis at 2.0 Å resolution Eva,J.;M.Nina;N.Jan;L.Sine
  11. Nucleic Acids Res. v.23 Random recombination of antibody single chain Fv sequences after fragmentation with DNaseI in the presence of $Mn^{2+}$ Lorimer,I.A.J.;I.Pastan https://doi.org/10.1093/nar/23.15.3067
  12. Nature v.370 Rapid evolution of a protein in vitro by DNA shuffling Stemmer,W.P. https://doi.org/10.1038/370389a0
  13. Biotechnol. Lett. v.23 Lamivudine production via enantioselective deamination by thermostable Bacillus caldolyticus cytidine deaminase Woo,J.h.;H.J.Shin;T.H.Kim;S.Y.Ghim;L.S.Jeong;J.G.Kim;H.J.Shin;B.H.Song https://doi.org/10.1023/A:1010353502346
  14. J. Mol. Biol. v.235 Cytidine deaminase, the 2.3 Å crystal structure of an enzyme transition state analog complex Betts,L.;S.Xiang;S.A.Short;R.Wolfenden;C.W.Carter https://doi.org/10.1006/jmbi.1994.1018
  15. Nat. Biotechnol. v.17 DNA shuffling of subgenomic sequences of subtilisin Ness,J.E.;M.Welch;L.Giver;M.Bueno;J.R.Cherry;T.V.Borchert;W.P.Stemmer;J.Minshull https://doi.org/10.1038/12884
  16. Proc. Natl. Acad. Sci. USA v.93 Identifying functional domains within terpene cyclases using a domain-swapping strategy Back,K.;J.Chappell https://doi.org/10.1073/pnas.93.13.6841
  17. Nat. Biotechnol. v.15 Chimeric proteins can exceed the sum of their parts: Implications for evolution and protein design Campbell,R.K.;E.R.Bergert;Y.Wang;J.C.Morris;W.R.Moyle https://doi.org/10.1038/nbt0597-439
  18. Molecular cloning and nucleotide spquence of the Bacillus stearothermophilus cdd gene encoding thermostable cytidine/deoxycytidine deaminase (personal communication) Chang,J.S.;H.K.Bae;S.O.Kim;S.M.Lee;B.H.Song
  19. Nat. Biotechnol. v.17 Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling Christians,F.C.;L.Scapozza;A.Crameri;G.Folkers;W.P.Stemmer https://doi.org/10.1038/7003
  20. J. Bacteriol. v.62 Studies of lysogenesis.Ⅰ. The mode of phage liberation by lysogenic Escherichia coli Bertani,G.
  21. Nat. Biotechnol. v.14 Directed evolution of a para-nitrobenzyl esterase for aqueous organic solvents Moore,J.C.;F.H.Arnold