Discovery and Engineering of Plant Natural Product Pathways

项目摘要/摘要 植物天然产物(Np)是临床批准的药物和饮食营养的重要来源，但很少。 完整的生物合成途径已经被表征。因此，许多复杂的植物天然 产品支架目前仍从生产植物或植物细胞培养物中分离出来，然后转化为 临床使用的半合成药物(如依托泊苷、地高辛、吗啡、长春花碱和紫杉醇-ALL) 在2015年世卫组织基本药物清单上)。严重缺乏有关其生物合成途径的信息 限制在异源宿主(例如酵母菌株)中生产植物分子的有前景的新方法的使用 这使得青蒿素)，以及耐人寻味的工程生物合成途径的可能性 类似物和非天然衍生品具有更好的疗效。更不知道可能是 在可食用植物中进行工程或育种努力以提高营养含量的目标。鉴于网络的关键作用 植物天然产物对人类健康和生物合成基因的利用，我们在此提出发展建议 以及可广泛推广的平台的应用，以加速关键字的发现和工程 植物天然产物途径。 到目前为止，限制发现植物途径的最具挑战性的步骤之一是确定候选 生物合成基因。在这里，我们提出了三种互补的途径来阐明我们 预期将使人们能够获得与人类健康相关的具有不同生物活性的小分子：(1) 植物天然产物分支家族的比较转录组学，(2)基因与代谢物的相关性 发现生物合成需要全植物协调的途径，以及(3)以基因为中心的发现 以特权途径酶为目标。这些方法最近使8基因的发现成为可能 获得秋水仙碱生物碱的途径，以及将该途径工程到异源生产宿主中。在这 建议我们为临床使用的NPs(高三尖杉酯碱[Synribo]和加兰他明)确定优先途径 [Razadyne])，可食用植物中具有免疫调节活性的分子(番茄糖苷生物碱)，以及临床 其评估将通过接触天然化合物或类似物(柠檬苦素， 石杉碱类和吲哚里西定生物碱)。这些复合词代表了一组不同的NP类别，并将被使用 以展示我们发现方法的广泛用途。这项工作的一个主要成果将是 生物合成基因，可用于设计异源宿主，使植物NPs及其类似物具有强大的 与人类健康相关的生物活动。

PROJECT SUMMARY/ABSTRACT Plant natural products (NPs) are a critical source of clinically approved drugs and dietary nutrients, yet very few complete biosynthetic pathways have been characterized. As a consequence, many complex plant natural product scaffolds are currently still isolated from the producing plant or plant cell culture and then converted to a clinically-used drug by semisynthetic routes (e.g. etoposide, digoxin, morphine, vinblastine, and paclitaxel – all on the 2015 WHO list of essential medicines). Lack of information regarding their biosynthetic pathways severely limits the use of promising new approaches to produce plant molecules in heterologous hosts (e.g. yeast strains that make artemisinin), as well as the intriguing possibility of engineering the biosynthetic pathways to access analogs and non-natural derivatives with greater efficacy. Even less is known about pathways that could be the target of engineering or breeding efforts in edible plants to improve nutrient content. Given the critical role of plant natural products in human health and utility of biosynthetic genes, we propose here the development and application of a broadly generalizable platform to accelerate the discovery and engineering of key plant natural product pathways. One of the most challenging steps limiting the discovery of plant pathways to date is the identification of candidate biosynthetic genes. Here we propose three complementary approaches for pathway elucidation that we anticipate will enable access to small molecules with diverse biological activities relevant to human health: (1) comparative transcriptomics for branching families of plant natural products, (2) gene-to-metabolite correlation to uncover pathways that require whole-plant coordination for biosynthesis, and (3) gene-centric discovery targeting privileged pathway enzymes. These approaches have recently enabled the discovery of an 8-gene pathway to colchicine alkaloids, and engineering of this pathway into a heterologous production host. In this proposal we have prioritized pathway for clinically used NPs (homoharringtonine [Synribo] and galantamine [Razadyne]), molecules with immune modulatory activity in edible plants (tomato glycoalkaloids), and clinical candidates whose assessment would be enabled by access to the native compound or analogs (limonoids, huperzines, and indolizidine alkaloids). These compounds represent a diverse set of NP classes and will be used to demonstrate the broad utility of our discovery approach. A major outcome of this work will be sets of biosynthetic genes that can be used to engineer heterologous hosts to make plant NPs and analogs with potent biological activity of relevance to human health.