Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae.
Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae.
复制标题
DOI：
10.1021/bm050113g
复制
发表时间：
2005-05
影响因子：
6.2
通讯作者：
Ken’ichiro Matsumoto;Rina Nagao;T. Murata;Y. Arai;T. Kichise;H. Nakashita;S. Taguchi;H. Shimada;Y. Doi
中科院分区：
文献类型：
--
作者：
Ken’ichiro Matsumoto;Rina Nagao;T. Murata;Y. Arai;T. Kichise;H. Nakashita;S. Taguchi;H. Shimada;Y. Doi
在这项研究中，提高光合PHA的生产实现了高活性突变体的PHA合成酶的体外进化技术创建。将野生型和突变型的来自豚鼠气单胞菌的PHA合酶基因与来自真养罗尔斯通氏菌的NADPH依赖性乙酰乙酰辅酶A还原酶基因一起引入拟南芥。与携带野生型PHA合酶基因的植物相比，高活性突变PHA合酶基因N149 S和D171 G的表达导致T1转基因拟南芥中PHA含量增加8-10倍。在纯合T2后代中，PHA含量进一步增加至6.1 mg/g细胞干重。经GC/MS分析表明，这些PHA是由0.2- 0.8mol%3HV组成的聚（3-羟基丁酸酯-co-3-羟基戊酸酯）[P（3 HB-co-3 HV）]共聚物。由野生型和突变的PHA酶合成的P（3 HB-共-3HV）共聚物的单体组成反映了在大肠杆菌中观察到的底物特异性。这些结果表明，在体外进化的PHA酶可以提高PHA的生产力和调节单体组成的转基因植物。
In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.