Metabolic engineering toward production of novel acive secondary metabolites.

作为高等植物次生代谢工程的遗传工具，利用PCR技术从酵母基因组中克隆了一个编码泛醌生物合成相关的异戊二烯基转移酶（PT）的酵母基因coq 2。香叶基转移酶是紫草中紫草素生物合成的关键调控酶，其底物与酵母PT相同，都是对羟基苯甲酸和香叶基二磷酸。因此，我们试图通过在紫草细胞中表达酵母coq 2基因来改变紫草素的生物合成。对于植物表达，构建了四个二元载体：1）coq 2-全长（用于线粒体定位），2）Δ coq 2（胞质），3）coq 2-ERI（具有ER分选信号），4）coq 2-ER 2（用于ER定位，具有ER分选和ER保留信号）。然后将含有这些修饰的coq 2基因的双元载体导入根癌农杆菌和A. rhizogenes，烟草（对照）和L.刺桐属植物进行转化。到目前为止，7至15个印度 ...更多信息 获得了表现出潮霉素抗性的悬垂烟草克隆，其中通过基因组PCR证实了引入的coq 2基因的存在，并且通过北方杂交通过北方监测mRNA水平。PT的酶活性测定表明，只有coq 2-ER 2转化的株系表现出较高的酶活性，因此我们仅将该构建体用于紫草的转化。采用毛状根培养技术，对L.表达高PT活性的刺桐积累了大量的间香叶基对羟基苯甲酸，PT的直接反应产物，以及三个相关的苯并呋喃衍生物，虽然没有观察到显着增加的紫草素。这表明紫草素生物合成中异戊二烯化后至少还有一个瓶颈反应。此外，我们在这些培养物中未检测到新的代谢物。由于内源性香叶基转移酶的详细功能和表达调控在L.结果表明，该酶为赤藓糖醇，并进行了cDNA克隆。少

As the genetic tool secondary metabolic engineering of higher plant, a yeast gene coq2 which encoded prenyltransferase (PT) involved in ubiquinone biosynthesis was isolated from yeast genome by PCR. Geranyltransferase, a critical regulatory enzyme of shikonin biosynthesis in Lithospermum erythrorhizon, takes the same substrates P-hydroxybenzoic acid (PHB) and geranyldiphosphate as the yeast PT. Thus, we made an attempt to alter the shikonin biosynthesis by expressing the yeast coq2 gene in Lithospermum cells. For plant expression, four binary vectors were constructed : 1) coq2-full-length (for mitochondrial localization), 2) Δcoq2 (cytosolic), 3) coq2-ERI (having a sorting signal for ER), 4) coq2-ER2 (for ER localization, with ER-sorting and ER-retention signals). Binary vectors containing these modified coq2 genes were then introduced into Agrobacterium tumefaciens and A. rhizogenes, with which tobacco (control) and L. erythrorhizon were transformed, respectively. So far, 7 to 15 inde … More pendent tobacco clones showing hygromycin resistant were obtained, in which the existence of introduced coq2 genes were confirmed by genomic PCR, and the mRNA levels were also monitored by Northern by Northern hybridization. The enzyme assay of PT revealed that only coq2-ER2-transformed lines showed high enzyme activity, then we used only this construct for Lithospermum transformation. The hairy root cultures of L. erythrorhizon expressing high PT activity accumulated much larger amount of m-geranyl-p-hydroxybenzoic acid, the direct reaction product of PT, than the control cultures, and three related benzofuran derivatives as well, although no significant increase of shikonin was observed. This suggests that there is at least on more bottle-neck reaction after prenylation step in shikonin biosynthesis. In addition, we have not detected a new metabolite in those cultures. Because the importance of the detailed function and the expressional regulation of endogenous geranyltransferase in L. erythrorhizon was shown from these results, we also carried out the c DNA cloning of this enzyme. Less