title: In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis.
abstract: In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbC(Re)), which is a key enzyme catalyzing the formation of polyhydroxybutyrate (PHB) from (R)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-mediated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27% of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved alpha/beta hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.
authors.affiliation: Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa, Wako, Saitama 351-0198, Japan. staguchi@isc.meiji.ac.jp
authors.forename: Seiichi
authors.initials: S
authors.lastname: Taguchi
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nakamura
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hiraishi
authors.forename: Ichiro
authors.initials: I
authors.lastname: Yamato
authors.forename: Yoshiharu
authors.initials: Y
authors.lastname: Doi
mesh_terms: D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D019943:Amino Acid Substitution; D020118:Cupriavidus necator / Q000201:enzymology* / Q000235:genetics; D019143:Evolution, Molecular; D016296:Mutagenesis; D017422:Sequence Analysis, DNA
chemical_list: D000217:Acyltransferases; C060970:poly-beta-hydroxybutyrate polymerase