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

本项目的目标是在一个具有良好特性的模式植物组织培养系统中提高吲哚生物碱的生产率。总体目标是确定吲哚和非甲氧戊酸萜类途径中一些关键酶的遗传操作对吲哚生物碱合成途径通量的代谢影响，特别强调初级和次级代谢对最终生物碱生产率的相互作用。其具体目标是：1)在最近鉴定的可诱导启动子系统的控制下，构建和鉴定高表达关键酶如ORCA3、G10H、DXS、DXR和AS-β的转基因毛状根系；2)开发基于核磁共振的代谢流图方法；3)对这些第一代转基因株系进行代谢鉴定；4)开发允许多基因导入的共转化技术；5)构建和鉴定第二代转基因株系，其表达根据上述第一代转基因株系的结果而确定的策略基因组合。

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.