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.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
TU Gerngross KD Snell OP Peoples AJ Sinskey E Csuhai S Masamune J Stubbe (1994) ArticleTitleOverexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: evidence for a required posttranslational modification for catalytic activity Biochemistry 33 9311–9320 Occurrence Handle10.1021/bi00197a035 Occurrence Handle8049232
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, Massachusettes
DB Karr JK Waters DW Emerich (1983) ArticleTitleAnalysis of poly-β-hydroxybutyrate in Rhizobium japonicum bacteroids by ion-exclusion high-pressure liquid chromatography and UV detection Appl. Environ. Microbiol 46 1339–1344
Y Kawaguchi Y Doi (1992) ArticleTitleKinetics and mechanism of synthesis and degradation of poly(3-hydroxybutyrate) in Alcaligenes eutrophus Macromolecules 25 2324–2329 Occurrence Handle10.1021/ma00035a007
T Kichise S Taguchi Y Doi (2002) ArticleTitleEnhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase Appl. Environ. Microbiol 68 2411–2419 Occurrence Handle10.1128/AEM.68.5.2411-2419.2002 Occurrence Handle11976116
S Kusaka T Iwata Y Doi (1998) ArticleTitleMicrobial synthesis and physical properties of ultra-high-molecular-weight poly[(R)-3-hydroxybutyrate] J. Macromol. Sci., Pure Appl. Chem A35 319–335
L Madison GW Huisman (1999) ArticleTitleMetabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic Microbiol. Mol. Biol. Rev 63 21–53 Occurrence Handle10066830
YM Normi T Hiraishi S Taguchi H Abe K Sudesh N Najimudin Y Doi (2005) ArticleTitleCharacterization and properties of G4X mutants of Ralstonia eutropha PHA synthase for poly(3-hydroxybutyrate) biosynthesis in Escherichia coli Macromol. Biosci 5 197–206 Occurrence Handle10.1002/mabi.200400181 Occurrence Handle15768438
K Sudesh H Abe Y Doi (2000) ArticleTitleSynthesis, structure and properties of polyhydroxyalkanoates: biological polyesters Prog. Polym. Sci 25 1503–1555 Occurrence Handle10.1016/S0079-6700(00)00035-6
S Taguchi Y Doi (2004) ArticleTitleEvolution of polyhydroxyalkanoate (PHA) production system by ‘enzyme evolution’: successful case studies of directed evolution Macromol. Biosci 4 145–156 Occurrence Handle10.1002/mabi.200300111
S Taguchi A Maehara K Takase M Nakahara H Nakamura Y Doi (2001) ArticleTitleAnalysis of mutational effects of polyhydroxybutyrate (PHB) polymerase on bacterial PHB accumulation using an in vivo assay system FEMS Microbiol. Lett 198 65–71 Occurrence Handle10.1016/S0378-1097(01)00125-2 Occurrence Handle11325555
S Taguchi H Nakamura T Hiraishi I Yamato Y Doi (2002) ArticleTitleIn vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis J. Biochem 131 801–806 Occurrence Handle12038975
K Takase K Matsumoto S Taguchi Y Doi (2004) ArticleTitleAlteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by in vitro evolution: in vivo and in vitro enzyme assays Biomacromolecules 5 480–485 Occurrence Handle10.1021/bm034323+ Occurrence Handle15003009
K Takase S Taguchi Y Doi (2003) ArticleTitleEnhanced 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 gene J. Biochem 133 139– 145 Occurrence Handle10.1093/jb/mvg015 Occurrence Handle12761209
T Tsuge Y Saito Y Kikkawa T Hiraishi Y Doi (2004) ArticleTitleBiosynthesis and compositional regulation of poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] in recombinant Ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes Macromol. Biosci 4 238–242 Occurrence Handle10.1002/mabi.200300077 Occurrence Handle15468213
S Zhang T Yasuo RW Lenz S Goodwin (2000) ArticleTitleKinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha Biomacromolecules 1 244–251 Occurrence Handle10.1021/bm005513c Occurrence Handle11710107
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Normi, Y.M., Hiraishi, T., Taguchi, S. et al. Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase. Biotechnol Lett 27, 705–712 (2005). https://doi.org/10.1007/s10529-005-5186-z
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s10529-005-5186-z