title: Characterization and properties of G4X mutants of Ralstonia eutropha PHA synthase for poly(3-hydroxybutyrate) biosynthesis in Escherichia coli.
abstract: Modification of the type I polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) was performed through systematic in vitro evolution in order to obtain improved PhaC(Re) having an enhanced activity of poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli. For the first time, a beneficial G4D N-terminal mutation important for the enhancement of both PHB content in dry cells and PhaC(Re) level in vivo was identified. Site-directed saturation mutagenesis at the G4 position enabled us to identify other mutations conferring similar enhanced characteristics. In addition, the PHB homopolymer synthesized by most G4X single mutants also had higher molecular weights than that of the wild-type. In vitro enzymatic assays of purified G4D mutant PhaC(Re) revealed that the mutant enzyme exhibited slightly lower activity and reaction efficiency compared to the wild-type enzyme. [diagram in text].
authors.affiliation: Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Yahaya M
authors.initials: YM
authors.lastname: Normi
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hiraishi
authors.forename: Seiichi
authors.initials: S
authors.lastname: Taguchi
authors.forename: Hideki
authors.initials: H
authors.lastname: Abe
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
authors.forename: Nazalan
authors.initials: N
authors.lastname: Najimudin
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Doi
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D020118:Cupriavidus necator / Q000201:enzymology*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006885:Hydroxybutyrates / Q000378:metabolism*; D016296:Mutagenesis; D009154:Mutation
chemical_list: D006885:Hydroxybutyrates; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase