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分离出一个编码泛素酮合成过程中戊烯基转移酶（PT）的酵母菌基因coq2。Geranyltransferase是紫草草中紫草素合成的关键调控酶，其底物对羟基苯甲酸（PHB）和香叶苷二磷酸与酵母PT相同，因此，我们尝试通过在紫草草细胞中表达酵母coq2基因来改变紫草素的生物合成。对于植物表达，构建了4个二元载体：1)coq2-全长（用于线粒体定位），2)Δcoq2（细胞质），3)coq2-ERI（具有ER分选信号），4)coq2-ER2（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coq2基因的二元载体导入农杆菌和根状芽孢杆菌，分别转化对照烟草和红僵菌。目前已获得7 ~ 15个耐潮霉素的烟草悬垂无性系，通过基因组PCR证实了引入coq2基因的存在，并通过Northern by Northern杂交技术对其mRNA水平进行了监测。PT酶分析结果显示，只有coq2- er2转化系具有较高的酶活性，因此我们只使用这个构建体对紫草进行转化。表达高PT活性的赤藓菌毛状根培养物中PT直接反应产物间香叶酰-对羟基苯甲酸的积累量远高于对照培养物和3种相关苯并呋喃衍生物，但紫草素的含量没有显著增加。说明在紫草素生物合成过程中，戊酰化后至少存在一个瓶颈反应。此外，我们没有在这些培养物中检测到新的代谢物。由于这些结果显示了内源性香叶基转移酶在赤藓菌中的详细功能和表达调控的重要性，我们也对该酶进行了c DNA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