Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: Enhancement of PHA production and alteration of polymer molecular weight
Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: Enhancement of PHA production and alteration of polymer molecular weight
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DOI：
10.1021/bm049650b
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发表时间：
2005-01-01
影响因子：
6.2
通讯作者：
Taguchi, S
中科院分区：
文献类型：
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作者：
Matsumoto, K;Takase, K;Taguchi, S
在体外进化的聚羟基链烷酸酯（PHA）合酶基因从假单胞菌61-3（phaC 1 p，）已进行产生高活性的酶。本研究对PHA合成酶突变体Glu 130 Asp（E130 D）进行了体内外研究。与重组大肠杆菌相比，携带E130 D突变基因的重组大肠杆菌菌株JM 109从葡萄糖中积累高10倍（1.0wt%）的聚（3-羟基丁酸酯）[P（3 HB）]。大肠杆菌（0.1重量%）。重组大肠在十二烷酸酯上生长的具有E130 D PHA合酶基因的大肠杆菌菌株LS 5218比具有野生型PHA合酶基因的LS 5218菌株（13重量%）产生更多的聚（3 HB-共-3-羟基链烷酸酯）[P（3 HB-co-3 HA）]共聚物（20重量%）。E130 D突变也导致共聚物的产生，与野生型PHA合酶产生的共聚物相比，3 HB组成略有增加。E130 D PHA合成酶对各种3-羟酰基-CoA（长度为4-10个碳）的体外酶活性均高于野生型酶。E130 D突变与其他有益突变如Ser 325 Thr和Gln 481 Lys的组合对体内PHA产生和体外酶活性表现出协同效应。有趣的是，凝胶渗透色谱分析表明，E130 D突变也有协同作用的分子量的聚合物在体内产生。
In vitro evolution of the polyhydroxyalkanoate (PHA) synthase gene from Pseudomonas sp. 61-3 (phaC1p,) has been performed to generate highly active enzymes. In this study, a positive mutant of PHA synthase, Glu130Asp (E130D), was characterized in detail in vivo and in vitro. Recombinant Escherichia coli strain JM109 harboring the E130D mutant gene accumulated 10-fold higher (1.0 wt %) poly(3-hydroxybutyrate) [P(3HB)] from glucose, compared to recombinant E. coli harboring the wild-type PHA synthase gene (0.1 wt %). Recombinant E. coli strain LS5218 harboring the E130D PHA synthase gene grown on dodecanoate produced more poly(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] (20 wt %) copolymer than an LS5218 strain harboring the wild-type PHA synthase gene (13 wt %). The E130D mutation also resulted in the production of copolymer with a slight increase in 3HB composition, compared to copolymer produced by the wild-type PHA synthase. In vitro enzyme activities of the E130D PHA synthase toward various 3-hydroxyacyl-CoAs (4-10 carbons in length) were all higher than those of the wild-type enzyme. The combination of the E130D mutation with other beneficial mutations, such as Ser325Thr and Gln481Lys, exhibited a synergistic effect on in vivo PHA production and in vitro enzyme activity. Interestingly, gel-permeation chromatography analysis revealed that the E130D mutation also had a synergistic effect on the molecular weight of polymers produced in vivo.