title: Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase.
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.
authors.affiliation: Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, 351-0198, Wako-shi, Saitama, 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: 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 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods; D020118:Cupriavidus necator / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic; D012542:Scattering, Radiation; D000094122:Polyhydroxybutyrates
chemical_list: D000217:Acyltransferases; D006885:Hydroxybutyrates; D010649:Phenylalanine; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C077225:poly(3-hydroxyalkanoic acid) synthase