ME: Collaborative Research: Metabolic Engineering of Hairy Roots for Alkaloid Production

本项目的目的是提高吲哚类生物碱在一个具有良好特征的模式植物组织培养系统中的生产效率。总体目标是确定吲哚和非甲羟戊酸萜类途径中一些关键酶的遗传操作对通过吲哚生物碱合成途径的通量的代谢影响，特别强调初级和次级代谢对最终生物碱产量的相互作用。具体目标是：1)在新发现的诱导启动子系统控制下，构建并表征过表达ORCA3、G10H、DXS、DXR和as - β等关键酶的转基因毛状根系；2)开发基于核磁共振的代谢通量图方法；3)对这些第一代转基因品系进行代谢鉴定；4)开发允许引入多个基因的共转化技术；5)构建和表征第二代转基因系，表达从上述第一代转基因系的结果确定的基因的战略组合。

The objective of this project is to improve the productivity of indole alkaloids in a well-characterized model plant tissue culture system, C. roseus. The overall goals are to determine the metabolic effects of genetic manipulations of some key enzymes in the indole and non-mevalonate terpenoid pathways on flux through the indole alkaloid synthesis pathways, with special emphasis on the interplay between primary and secondary metabolism on the final alkaloid productivity. The specific objectives are to: 1) construct and characterize transgenic hairy root lines overexpressing key enzymes, such as ORCA3, G10H, DXS, DXR and AS-beta under the control of a recently characterized inducible promoter system; 2) develop methods for NMR- based metabolic flux maps; 3) perform metabolic characterization of these first generationtransgenic lines; 4) develop techniques for co-transformation that allow the introduction ofmultiple genes; 5) construct and characterize a second generation of transgenic lines expressing strategic combinations of genes as determined from the results of the first generation transgenic lines above.