体外无细胞紫杉醇及其前驱体生物合成系统的构建

With the development of synthetic biotechnology on genome and protein enzyme engineering, re-designed metabolism reaction networks showed great potential for mass manufacturing chemical with complicated structures, which is very difficult to be synthesized commercially using traditional chemical method. Facing the more and more serous environmental problem, reengineering bio-metabolism brougt us an alternative way for synthesis of material of high value. Although, many progressed has been made in engineering cell, such as E.coli and yeast, for biosynthesis of chemcial compound in the past few years. However, it is still very challenging to tame the cell for artifical purposes, because compolicated endogenic metabolism network for maintenaning cell. Here, we propose a new strategy in which a complete new cell free metabolism network will be constructed from purified metabolism enzymes for chemical synthesis in test tube. Furthermore, DNA origami will be used to regulate spacial position of the metabolims enzymes in vitro. Generally, one metabolism network was comprised from many enzymes and chemical was synthesized through multiple steps in a series circuits. For mimicking and regulating the reaction flow in the in vitro system, enzymes will be imobilized on the designed DNA scaffold in the order, by which they get involed in the metabolims flow. It will be a completed new artifical complicate biomolecular machine, which we called as metabolism robotic. In this proposal, we will focus on P450 enzyme system related part in Taxol metablosim, which is the bottleneck restricting mass biosynthesis of taxol in engineered cell. There is lots of advantage in this new technology in comparison with in vivo metabolism engineering. Because only components that are necessary for the target chemical synthesis are included, there should no interference with other bioreaction that happened in the cell in vivo. Similarly, it should be easy to connect metabolism reaction module from different cell sources. Therefore, the in vitro system will be a flexiable platform for various bioengineering purposes.

针对复杂代谢产物如紫杉醇生物合成困难问题,申请者提出了开发介于"死"的化学合成与"活"的生物合成之间的体外代谢合成系统, 结合先进的人工DNA折叠纳米结构技术，开发具有精密空间可控性的体外串联代谢分子机器。主要研究内容为：1）在体外构建紫杉醇合成中包括基于P450酶的，IPP聚合、母核成环以及下游的羟化、酰基转移反应的生物合成系统，2）通过设计DNA折叠纳米结构体，精密地控制代谢酶的空间位置，实现串联代谢分子机器。该研究思路和研究方案具有明显创新性，本研究可望成为体外合成生物系统的一个经典案例，为系统生物学的研究提供全新的技术，为实现产业化提供基础。还可以加深对紫杉醇的生物合成途径反应机理理解，成为减少生物工程中对细胞过渡依赖的崭新技术，可预计此技术可转化应用到更多的生物工程研究中。

本项目以开发基于DNA纳米结构的生化分子机器为核心研究目标。基于DNA本身的生化特性，DNA折纸技术为纳米级别的复杂三维结构体的构建提供了最佳技术平台，各种不同的纳米颗粒或者生物元件可以精准的被固化到特定的空间位置，形成功能可设计的人工分子机器。这种分子机器不论在合成生物学、分子检测等领域都具有极大的应用潜力。由于现有的DNA折纸技术具有依赖现有的细菌噬菌体基因组的单链DNA进行构建，极大地限制了其应用，因此在本项目的执行中，我们首先对DNA折纸技术的基本长单链DNA模板制备技术进行了探索。我们工程改造了革兰氏阳性菌中常见的一种特定的DNA滚环扩增反应，利用此反应的环状单链DNA的中间产物在常用的革兰氏阴性工程菌株中进行长单链DNA的生成，通过融合特定革兰氏阳性菌和革兰氏阴性菌质粒DNA的复制子机制，成功地实现了细胞内可控的单链DNA的生成，此外我们也开发了记忆无细胞的体外酶法单链DNA的生成方法，这里结合单点内切酶和外切酶III的特定活性在试管内，通过双链环状DNA的特定降解得到了环状单链DNA。在此基础上我们以RNA转录反应为模型在100nm尺寸的DNA纳米母板上固化了RNA转录酶和荧光报告基因，并通过精准控制其间的相对空间位置，成功地展示了具有人工调控活性的DNA分子机器。至此，虽然我们在制备植物源紫杉醇代谢酶方面遇到了很大的困难，但是本项目的研究成果成功地展示了大片段单链DNA分子的制备新方法的可能性，以及基于DNA纳米结构的生化分子机器。