Institute of Molecular Biosciences, Massey University, Private Bag 11222, Palmerston North, New Zealand.
J Biotechnol. 2010 May 3;147(1):31-6. doi: 10.1016/j.jbiotec.2010.02.021. Epub 2010 Mar 7.
Polyhydroxyalkanoates are biodegradable biocompatible polymers naturally produced by various bacteria and archaea. Biotechnological production in transgenic plants has already been demonstrated with efficient polyhydroxybutyrate production requiring targeting of the enzymes to the chloroplasts. Three enzymes are required to establish the polyhydroxybutyrate biosynthesis pathway in non-naturally producing microorganisms or plants. To facilitate production of biopolyesters in plants, a gene encoding a translational fusion of the polyhydroxybutyrate biosynthesis enzymes PhaA (beta-ketothiolase), PhaB (acetoacetyl-CoA reductase) and PhaC (PHA synthase) was constructed. Escherichia coli harboring a plasmid encoding this fusion protein (PhaA-PhaB-PhaC) under control of the lac promoter accumulated polyhydroxybutyrate contributing to 0.4% (w/w) of cellular dry weight. Insertion of an extended linker between PhaA and PhaB increased polyhydroxybutyrate accumulation to 3.9% (w/w) of cellular dry weight. Introduction of a second plasmid encoding PhaA and PhaB restored polyhydroxybutyrate accumulation to wildtype levels of about 35% (w/w) of cellular dry weight suggesting that the functions of PhaA and/or PhaB were limiting factors. Deletion of PhaA in trans led to significantly reduced polyhydroxybutyrate production suggesting that the PhaA activity in the fusion protein is reduced. This study showed that a single-chain translational fusion protein comprising the three enzymes essential for polyhydroxybutyrate synthesis can be engineered which will strongly facilitate the establishment of recombinant polyhydroxybutyrate production organisms particularly requiring targeting to sub-cellular compartments such as the chloroplasts in plants.
聚羟基烷酸酯是可生物降解的生物相容性聚合物，天然由各种细菌和古菌产生。在转基因植物中的生物技术生产已经通过高效的聚羟基丁酸酯生产得到证明，需要将酶靶向叶绿体。在非天然产生微生物或植物中建立聚羟基丁酸酯生物合成途径需要三种酶。为了促进植物中生物聚酯的生产，构建了编码聚羟基丁酸酯生物合成酶 PhaA（β-酮硫解酶）、PhaB（乙酰乙酰辅酶 A 还原酶）和 PhaC（PHA 合酶）翻译融合物的基因。在乳糖启动子控制下，携带编码该融合蛋白（PhaA-PhaB-PhaC）的质粒的大肠杆菌积累了聚羟基丁酸酯，占细胞干重的 0.4%（w/w）。在 PhaA 和 PhaB 之间插入扩展接头可将聚羟基丁酸酯积累增加到细胞干重的 3.9%（w/w）。引入编码 PhaA 和 PhaB 的第二个质粒可将聚羟基丁酸酯积累恢复到野生型水平，约为细胞干重的 35%（w/w），表明 PhaA 和/或 PhaB 的功能是限制因素。反式缺失 PhaA 导致聚羟基丁酸酯产量显著降低，表明融合蛋白中 PhaA 的活性降低。这项研究表明，可工程设计包含聚羟基丁酸酯合成所需的三种酶的单链翻译融合蛋白，这将极大地促进重组聚羟基丁酸酯生产生物体的建立，特别是需要靶向亚细胞区室如植物中的叶绿体。
