Nawrath C, Poirier Y, Somerville C
Department of Plant Biology, Carnegie Institution of Washington, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12760-4. doi: 10.1073/pnas.91.26.12760.
In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plants accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-micron granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB that accumulated. We conclude that there does not appear to be any biological barrier to high-level production of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand.
在真养产碱杆菌中，有三个基因编码从乙酰辅酶A催化合成聚(R)-(-)-3-羟基丁酸酯所需的酶。为了将这些酶靶向高等植物的质体，通过添加编码豌豆叶绿体转运肽、组成型植物启动子和聚(A)添加序列的DNA片段对这些基因进行了修饰。通过农杆菌介导的转化将每个修饰的细菌基因导入拟南芥，并通过有性杂交获得含有所有三个基因的植株。这些植株在质体内积累了高达干重14%的PHB，以0.2至0.7微米的颗粒形式存在。与早期在细胞质中表达PHB生物合成途径对生长产生有害影响的实验不同，在质体中表达PHB生物合成途径对转基因植物的生长或育性没有明显影响，并且导致积累的PHB量增加了100倍。我们得出结论，高等植物中高水平生产PHB似乎不存在任何生物学障碍。高水平的PHB积累还表明，质体乙酰辅酶A的合成受一种响应代谢需求的机制调控。
