Characterization and properties of G4X mutants of Ralstonia eutropha PHA synthase for poly (3-hydroxybutyrate) biosynthesis in Escherichia coli
Characterization and properties of G4X mutants of Ralstonia eutropha PHA synthase for poly (3-hydroxybutyrate) biosynthesis in Escherichia coli
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DOI：
10.1002/mabi.200400181
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发表时间：
2005-03-15
影响因子：
4.6
通讯作者：
Doi, Y
中科院分区：
文献类型：
--
作者：
Normi, YM;Hiraishi, T;Doi, Y
通过系统的体外进化，对真核Ralstonia eutropha I型聚羟基烷酸合成酶（PhaC(Re)）进行修饰，获得了在重组大肠杆菌中合成聚3-羟基丁酸酯（PHB）活性增强的改良PhaC（Re）。首次发现了一种有益的G4D n端突变，对提高干细胞中PHB含量和体内PhaC（Re）水平都很重要。G4位点定向饱和诱变使我们能够识别其他具有类似增强特性的突变。此外，大多数G4X单突变体合成的PHB均聚物的分子量也高于野生型。纯化的G4D突变体PhaC（Re）的体外酶分析显示，与野生型酶相比，突变体酶的活性和反应效率略低。
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.