Monoterpene indole alkaloid gene discovery and evolution in the Apocynaceae

夹竹桃科单萜吲哚生物碱基因的发现和进化

• 批准号: RGPIN-2015-05151
• 负责人: Stout, Jake
• 金额: $ 2.11万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=683176
• 关键词: Monoterpene; indole; alkaloid; gene; discovery

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.**

总体而言，植物生产用于开发和防御的40万种天然产品。这些化合物中有许多与人类生物学功能相互作用，因此可以用作药物剂。鉴于植物衍生的天然产物数量大量，尚未将大量植物天然产品作为潜在药物进行测试。****用作药物的天然产品在某些植物科中特别丰富。例如，Dogbane（apocynaceae）和相关植物家族产生超过3000多个单烷吲哚生物碱（MIAS）。 Catharanthus Roseus积累了葡萄碱和长春新碱，它们被用作化学治疗药物来对抗许多癌症。最近的基因组学研究确定了许多产生这些药物的基因和酶。在这项研究中，我将利用这些资源来开发一个基因捕获阵列，该阵列将用于分离编码MIA生物合成基因的DNA片段。然后，该阵列将用于捕获来自其他产生医学MIA的其他物种的新基因，我将使用下一代测序技术对这些基因进行测序。这些生物酮和ibogaine中的两个具有治疗优化添加的潜力。我的实验室将进行实验，以鉴定，克隆和表征MIA生物合成基因和产生这些化合物的编码酶。我们将同时开发IBOGA“毛茸茸的根”细胞培养系统。使用该系统，我们可以引入希望转移并纳入IBOGA基因组的DNA片段。我们将添加DNA序列，以减少产生Iboga生物碱的基因的表达。一个长期计划是引入新的MIA生物合成基因，这些基因从基因捕获实验中鉴定出来，以筛选新的生物合成能力。****这些实验将扩大我们对MIA生物合成基因和酶多样性的了解。工程化的iboga毛茸茸的根培养物的发展有可能生产出无数的新型MIA，并将为制药学家提供活动筛查。在毛根培养系统中优化MIA生物合成可能允许扩大这些化合物的生产。未来的目标是在该系统中生产葡萄碱和长春新碱，这将使这些药物的可持续供应与从玫瑰念珠菌植物中提取相比，其成本可能更低。**