Biotechnology Letters

, Volume 27, Issue 10, pp 705–712

Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase

  • Yahaya M. Normi
  • Tomohiro Hiraishi
  • Seiichi Taguchi
  • Kumar Sudesh
  • Nazalan Najimudin
  • Yoshiharu Doi
Article

Abstract

The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaCRe). We have now carried out site-directed saturation mutagenesis of F420 of PhaCRe and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaCRe enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaCRe enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant.

Keywords

combined mutations in vitro evolution PHA synthase poly(3-hydroxybutyrate) saturation mutagenesis 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Gerngross, TU, Snell, KD, Peoples, OP, Sinskey, AJ, Csuhai, E, Masamune, S, Stubbe, J 1994Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activityBiochemistry3393119320CrossRefPubMedGoogle Scholar
  2. Junker B, York G, Park C, Jia Y, Rha C, Stubbe J, Sinskey AJ (2000) Genetic manipulation of polyhydroxybutyrate synthase activity in Ralstonia eutropha. In: The 8th International Symposium of Biological Polyester, September 11–15, Cambridge, MassachusettesGoogle Scholar
  3. Karr, DB, Waters, JK, Emerich, DW 1983Analysis of poly-β-hydroxybutyrate in Rhizobium japonicum bacteroids by ion-exclusion high-pressure liquid chromatography and UV detectionAppl. Environ. Microbiol4613391344Google Scholar
  4. Kawaguchi, Y, Doi, Y 1992Kinetics and mechanism of synthesis and degradation of poly(3-hydroxybutyrate) in Alcaligenes eutrophusMacromolecules2523242329CrossRefGoogle Scholar
  5. Kichise, T, Taguchi, S, Doi, Y 2002Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthaseAppl. Environ. Microbiol6824112419CrossRefPubMedGoogle Scholar
  6. Kusaka, S, Iwata, T, Doi, Y 1998Microbial synthesis and physical properties of ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate]J. Macromol. Sci., Pure Appl. ChemA35319335Google Scholar
  7. Madison, L, Huisman, GW 1999Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plasticMicrobiol. Mol. Biol. Rev632153PubMedGoogle Scholar
  8. Normi, YM, Hiraishi, T, Taguchi, S, Abe, H, Sudesh, K, Najimudin, N, Doi, Y 2005Characterization and properties of G4X mutants of Ralstonia eutropha PHA synthase for poly(3-hydroxybutyrate) biosynthesis in Escherichia coliMacromol. Biosci5197206CrossRefPubMedGoogle Scholar
  9. Sudesh, K, Abe, H, Doi, Y 2000Synthesis, structure and properties of polyhydroxyalkanoates: biological polyestersProg. Polym. Sci2515031555CrossRefGoogle Scholar
  10. Taguchi, S, Doi, Y 2004Evolution of polyhydroxyalkanoate (PHA) production system by ‘enzyme evolution’: successful case studies of directed evolutionMacromol. Biosci4145156CrossRefGoogle Scholar
  11. Taguchi, S, Maehara, A, Takase, K, Nakahara, M, Nakamura, H, Doi, Y 2001Analysis of mutational effects of polyhydroxybutyrate (PHB) polymerase on bacterial PHB accumulation using an in vivo assay systemFEMS Microbiol. Lett1986571CrossRefPubMedGoogle Scholar
  12. Taguchi, S, Nakamura, H, Hiraishi, T, Yamato, I, Doi, Y 2002In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesisJ. Biochem131801806PubMedGoogle Scholar
  13. Takase, K, Matsumoto, K, Taguchi, S, Doi, Y 2004Alteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by invitro evolution: in vivo and in vitro enzyme assaysBiomacromolecules5480485CrossRefPubMedGoogle Scholar
  14. Takase, K, Taguchi, S, Doi, Y 2003Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis, and in vitro recombination of the type II polyhydroxyalkanoate synthase geneJ. Biochem133139 145CrossRefPubMedGoogle Scholar
  15. Tsuge, T, Saito, Y, Kikkawa, Y, Hiraishi, T, Doi, Y 2004Biosynthesis and compositional regulation of poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] in recombinant Ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genesMacromol. Biosci4238242CrossRefPubMedGoogle Scholar
  16. Zhang, S, Yasuo, T, Lenz, RW, Goodwin, S 2000Kinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutrophaBiomacromolecules1244251CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Yahaya M. Normi
    • 1
    • 2
  • Tomohiro Hiraishi
    • 1
  • Seiichi Taguchi
    • 3
  • Kumar Sudesh
    • 2
  • Nazalan Najimudin
    • 2
  • Yoshiharu Doi
    • 1
  1. 1.Polymer Chemistry LaboratoryRIKEN Institute Wako-shiJapan
  2. 2.School of Biological SciencesUniversiti Sains MalaysiaMindenMalaysia
  3. 3.Division of Molecular Chemistry, Graduate School of EngineeringHokkaido UniversitySapporoJapan

Personalised recommendations