Monoterpene indole alkaloid gene discovery and evolution in the Apocynaceae

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Collectively, plants produce over 400k natural products that they use for development and defence. Many of these compounds interact with human biological functions, and thus may serve as pharmaceutical agents. Given the high number of plant-derived natural products, a large number of plant natural products have not been tested for efficacy as potential drugs.***     *The natural products used as drugs are particularly abundant in certain plant families. For example, the Dogbane (Apocynaceae) and related plant families produce over 3000 monoterpene indole alkaloids (MIAs). Catharanthus roseus accumulates vinblastine and vincristine, which are used as chemotherapeutic drugs to combat a number of cancers. Recent genomics studies identified many of the genes and enzymes that produce these drugs. In this study, I will utilize these resources to develop a gene capture array that will be used to isolate DNA fragments that encode MIA biosynthetic genes. This array will then be used to capture new genes from other species that produce medicinal MIAs, and I will sequence these genes using next-generation sequencing techniques.***     I will focus on the plant Tabernanthe iboga (iboga), which accumulates MIAs in its roots in quantities up to 6% by weight. Two of these alkaloids, coronaridine and ibogaine, have potential to treat opiate addictions. My laboratory will perform experiments to identify, clone and characterize the MIA biosynthetic genes and encoded enzymes that produce these compounds. We will concurrently develop an iboga `hairy root' cell culture system. Using this system, we can introduce DNA fragments that we wish to be transferred and incorporated into the iboga genome. We will add DNA sequences to reduce the expression of the genes that produce the iboga alkaloids. A long term plan is to introduce the new MIA-biosynthetic genes that were identified from the gene capture experiments to screen for new biosynthetic capabilities.***     *These experiments will expand our knowledge of the diversity in MIA biosynthetic genes and enzymes. The development of the engineered iboga hairy root cultures has the potential to produce a myriad of novel MIAs which will be made available to pharmacologists for activity screening. Optimization of MIA biosynthesis in the hairy root culture system may allow for scaled-up production of these compounds. A future goal is to produce vinblastine and vincristine in this system, which will result in a sustainable supply of these drugs at a potentially lower cost compared to their extraction from C. roseus plants.**