Combinatorial Biosynthesis of New Alkaloids in Periwinkle

智力价值：本项目的总体目标是启动Catharanthus roseus（马达加斯加长春花）的代谢工程工作，以生产非天然生物碱。组合生物合成需要将野生型酶与改变底物特异性的酶交换，已经成功地应用于各种原核生物合成途径，以产生天然产物的非天然版本。本研究描述了如何组合生物合成可以完成在玫瑰玫瑰，以产生复杂的萜烯吲哚生物碱类似物。红玫瑰中萜烯吲哚生物碱生物合成机制的底物灵活性已被证明对非天然底物具有耐受性。此外，该途径的第一个承诺酶（strictosidine合成酶）已被重新设计以显示改变的底物特异性。这些重组酶将被转化到玫瑰玫瑰细胞培养中，并在转基因玫瑰玫瑰菌株中监测萜烯吲哚生物碱类似物的生产水平。更广泛的影响：研究生，本科生和博士后研究人员接触化学合成，蛋白质表达和诱变以及分子生物学协议。由于本研究采用多学科方法来解决天然产物生物合成领域的问题，学生有很多机会与其他具有互补专业领域的实验室互动。此外，自然产物生物合成的研究生水平课程已经开发并正在完善。本课程材料可通过麻省理工学院的公开课程网站URL向公众开放。本文还介绍了加强化学导论教学的策略。

Intellectual Merit:The overall goal of this project is to initiate metabolic engineering efforts in Catharanthus roseus madagascar periwinkle) to produce non-natural alkaloids. Combinatorial biosynthesis, which entails the swapping of wild type enzymes with enzymes having altered substrate specificity, has been successfully applied to a variety of prokaryotic biosynthetic pathways to yield nonnatural versions of natural products. This research describes how combinatorial biosynthesis can be accomplished in C. roseus to yield complex terpene indole alkaloid analogues. The substrate flexibility of the terpene indole alkaloid biosynthetic machinery in C. roseus has been shown to be tolerant to non-natural substrates. Additionally, the first committed enzyme of the pathway (strictosidine synthase) has been reengineered to display altered substrate specificity. These reengineered enzymes will be transformed into C. roseus cell culture and the production levels of terpene indole alkaloid analogues will be monitored in the transgenic C. roseus strains.Broader Impact:Graduate, undergraduate and post-doctoral researchers are exposed to chemical synthesis, protein expression and mutagenesis and molecular biology protocols. Since this research uses a multidisciplinary approach to solve problems in the area of natural product biosynthesis, students have numerous opportunities to interact with other labs with complementary areas of expertise. Furthermore, a graduate level course in natural product biosynthesis has been developed and is being refined. This course material is made available to the general public through MIT's open courseware site URL. Strategies to enhance the teaching of an introductory chemistry course are also described.