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In Vitro Evolution of a Polyhydroxybutyrate Synthase by Intragenic Suppression-Type Mutagenesis

2002; Oxford University Press; Volume: 131; Issue: 6 Linguagem: Inglês

10.1093/oxfordjournals.jbchem.a003168

1756-2651

Seiichi Taguchi, Hiro Nakamura, Tetsuya Hiraishi, Ichiro Yamato, Y. Doi,

Biofuel production and bioconversion

In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbCRe), which is a key enzyme catalyzing the formation of polyhy-droxybutyrate (PHB) from (f?)-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-medi-ated 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 a⊘p 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.