Metabolic engineering toward production of novel acive secondary metabolites.

用于生产新型活性次生代谢物的代谢工程。

基本信息

批准号：
10680565
负责人：
YAZAKI Kazufumi
金额：
$ 1.66万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

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

作为高等植物二次代谢工程的遗传工具，通过PCR从酵母基因组中分离到编码参与泛醌生物合成的异戊烯基转移酶(PT)的酵母基因coq2，该基因是紫草紫草素生物合成的关键调控酶。对羟基苯甲酸（PHB）和香叶基二磷酸作为酵母PT。尝试通过在紫草细胞中表达酵母 coq2 基因来改变紫草素生物合成对于植物表达，构建了四个二元载体：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 分选和然后将含有这些修饰的 coq2 基因的二元载体引入根癌农杆菌和发根农杆菌中，并分别转化烟草（对照）和红根草，到目前为止，已转化了 7 至 15 个独立的烟草。获得显示潮霉素抗性的克隆，其中通过基因组PCR证实导入的coq2基因的存在，并且还通过Northern by监测mRNA水平Northern杂交表明，只有coq2-ER2转化株系表现出高酶活性，因此我们仅使用该构建体进行紫草转化，表达高PT活性的红草毛状根培养物积累了更多的m。 -牻牛儿基-对羟基苯甲酸（PT 的直接反应产物）比对照培养物以及三种相关的苯并呋喃衍生物有所增加，但紫草素没有显着增加。观察到，紫草素生物合成中至少存在更多的瓶颈反应，因为内源香叶基转移酶的详细功能和表达调节的重要性。从这些结果表明，我们还进行了该酶的c DNA克隆。