Improvement of polyhydroxyalkanoate synthase from Wautersia eutropha by in vitro evolution

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Date
2006
Authors
Mohd Yahaya, Normi
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Abstract
The key enzyme in PHB biosynthesis of the bacterium Wautersia eutropha is PHA synthase. In this study, in vitro evolution of PHA synthase of W. eutropha (PhaCWe) was performed to obtain “evolved” PhaCWe exhibiting improved characteristics. Suppression-type mutagenesis was performed via error-prone PCR to obtain beneficial mutations. The PHA synthase gene (phaCWe) of the E11 mutant harboring an S80P mutation conferring low PHB accumulation was used as a template. As a result, a G4D mutation conferring high PHB accumulation and molecular weight and in vivo level of PhaCWe enzyme was obtained. Most of other amino acid substitutions at the G4 position conferred similar phenotypes. Site-directed mutagenesis was also performed at position F420 to investigate the effect of amino acid substitutions on PHB content. This position was chosen as previous F420S substitution resulted in a PhaCWe enzyme with enhanced activity (Taguchi et al., 2002a). Among all the substitutions, the F420S substitution conferred the highest PHB content. Interestingly, the lag phase of PhaCWe enzyme was significantly reduced by this single F420S substitution. This finding strengthened previous postulation reporting the possibility of the F420 substitution in the dimerization of PhaCWe enzyme (Taguchi et al., 2002a). The G4D and F420S mutations were then combined. The G4D/F420S mutant had high PHB accumulation and in vivo concentration of PhaCWe enzyme, similar to the G4D mutant. However, the molecular weight of the polymer and the kinetic properties of PhaCWe of G4D/F420S mutant were similar to that of the F420S mutant. These findings suggested the possible dominance of the G4D mutation in determining the PHB content and in vivo level of PhaCWe enzyme and the F420S mutation in determining the molecular weight of the polymer and the kinetic properties of PhaCWe enzyme of the G4D/F420S mutant. Since all the above studies were done using Escherichia coli JM109 as the host, the effect of the mutations using native W. eutropha PHB-4 as the host strain was also investigated. All the recombinants gave similar PHB content when grown on fructose. However, the F420S recombinant gave a slightly higher PHA accumulation than the wild-type when grown on soybean oil. All recombinants had comparable 3HHx mole fractions. Interestingly, the in vivo levels of PhaCWe enzyme were elevated in most G4 and G4D/F420S recombinants, indicating that the effect of G4 substitution on in vivo levels of PhaCWe enzyme is not specific to E. coli strains only.
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PhD
Keywords
Chemical science , Polyhydroxyalkanoate synthase , In vitro evolution
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