高等植物叶绿体异源合成抗疟药物青蒿素的研究

The unique anti-malarial and other new pharmaceutical effects bring artemisinin a huge market and high price. To solve this problem, application of artemisinin metabolic gene engineering is an effective route, but needs to be reinforced to a large degree especially in the aspect using different plant host, as implied by its current studies. After a comprehensive analysis, we propose that the chloroplasts of higher plants might be an ideal place for heterologous biosynthesis of artemisinin. In this program, two effectual strategies for plant multigene expression (i.e., the techniques of chloroplast “polycistron” and 2A peptide-mediated nuclear “fusion gene”) are utilized parallelly to introduce the entire pathway of artemisinin biosynthesis into tobacco chloroplasts. This effort aims to achieve a great amount of artemisinin synthesized directly in the chloroplasts of transgenic tobacco plants, mainly regarding a conceivable advantage of adequate supply in chloroplasts of isopentenyl pyrophosphate and singlet oxygen that are two key components involved in artemisinin biosynthesis. Ultimately, we hope to establish a new heterologous system for potent production of artemisinin, and clarify whether or not a scenario of efficient artemisinin biosynthesis can take place in heterogeneous plant species. Meanwhile, the technical scheme of this program can serve as a useful reference for high in planta production of other important secondary metabolites using synthetic biology approaches. Furthermore, from the appraisal of the influence of artemisinin biosynthesis in chloroplasts on plant stress resistance, a new strategy for the associated trait improvements can be envisioned. Overall, this program is of notable novelty and significance in both practical and fundamental studies.

青蒿素的抗疟奇效及诸多新功能使得它的市场需求巨大和价格高企。青蒿素代谢基因工程是这一问题的有效解决之道，但目前的研究现状表明其在异种植物平台上深入开展仍有大的必要和突破空间。经综合分析，本课题认为高等植物叶绿体是青蒿素异源合成的理想场所，拟同时采用两种有效的植物多基因表达策略（叶绿体“多顺反子”和2A寡肽介导的细胞核“融合基因”技术）将青蒿素生物合成途径整体引入到烟草叶绿体中，期望主要利用青蒿素合成的两关键要素（异戊烯基焦磷酸和单线态氧）在叶绿体的供应优势，直接在转基因烟草叶绿体内大量合成青蒿素并建立一个新的青蒿素异源高效生产体系，同时也从根本上阐明异种植物能否高效合成青蒿素。另外，本课题在技术方案上也为其它重要次生代谢物在植物中的合成生物学研究提供有益借鉴。而且，通过分析叶绿体合成青蒿素对植物抗逆性的影响，为植物抗逆性改良研究提供新的思路。总之，本课题具有显著的创新性和应用基础研究意义。

青蒿素因其抗疟特效及诸多新功能而需求巨大，异种植物平台上的代谢基因工程可为其规模化生产提供潜在有效解决途径。本项目研究内容主要是通过多基因表达策略向烟草叶绿体引入黄花青蒿特有的青蒿素生物合成途径，建立基于叶绿体底盘异源生产青蒿素的技术体系。取得的结果主要包括：获得整合青蒿素合成途径的细胞核转基因烟草植株；构建了基于底物通道策略可形成青蒿素合成酶复合体的新型植物叶绿体表达载体并推进了烟草叶绿体转化；验证了叶绿体载体中各个融合蛋白模块（含青蒿素合成相关的所有重要酶/蛋白组分，包括被视为关键瓶颈的细胞色素P450单氧酶CYP71AV1）在大肠杆菌中的成功表达，为大肠杆菌底盘异源从头合成青蒿素的原创性探索行进了一大步，而且可以成为本项目叶绿体合成青蒿素的极大弥补和印证（基于细菌与叶绿体的高度相似性）。同时，项目实施中出现的主要问题和原因以及下一步的工作设想也给予了说明。另外，在项目期间基于多方面考虑还开展了其他一些与本项目总体相关的工作并取得一定进展，包括：国内外首次成功在大肠杆菌中异源从头合成降胆固醇药物洛伐他汀；建立了基于亚磷酸盐的植物遗传转化/磷利用/杂草控制系统；利用超酸化蛋白融合策略改善功能蛋白的分子特性和生物活性。这些额外工作及其包含的研究思路和方法应该会有助于本项目研究的完善和拓展。