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

作为高植物的遗传工具二级代谢工程，由PCR从酵母基因组中分离出了涉及泛氨基酮生物合成的酵母基因COQ2。香烷基转移酶是岩石蛋白生物合成的关键调节酶，在岩石蛋白的生物合成中，采用相同的底物P-羟基苯甲酸（PHB）和Geranyldiphosphate的底物P-Hydroxybenzoic（PHB）。这是我们试图通过表达岩石质细胞中的酵母CoQ2基因来改变湿康蛋白生物合成的。对于植物表达，构建了四个二进制矢量：1）coq2-富含长度（对于线粒体定位），2）ΔCOQ2（胞质），3）COQ2-ERI（ER分类信号），4）COQ2-ER2（用于ER定位，具有ER分类和ER-ER-ER-ER-ER-ER-ER-ER-ER-ER-ER-ER-ER-ER-ER-ERNEINTION）。然后将含有这些经过修改的COQ2基因的二进制载体引入了农杆菌和曲霉杆菌中，分别将烟草（对照）和甲。到目前为止，获得了7至15个inde…获得了显示抗湿霉素耐药性的更多垂直烟草克隆，其中通过基因组PCR证实了引入的COQ2基因的存在，并且还通过北部杂交通过北部通过北部监测了mRNA水平。 PT的酶测定法表明，只有COQ2-ER2转化的线显示出较高的酶活性，然后我们仅将此构造用于岩石质体转化。表达高Pt活性的甲颗粒乳杆菌的毛状根培养物，比对照培养物的直接反应产物和三种相关的苯并呋喃衍生物也积累了更大量的M- geranyl-p-hydroxybenzoic酸，尽管未经对照培养物的直接反应产物和三种相关的苯并育衍生物。这表明在湿康蛋白生物合成中，至少有更多的瓶颈反应。此外，我们尚未在这些培养物中检测到一种新的代谢产物。因为从这些结果中显示出详细函数的重要性和内源性黄烷基转移酶的表达调节，因此我们还进行了该酶的C DNA克隆。较少的