Discovery and Engineering of Plant Natural Product Pathways

项目总结/摘要 植物天然产物（NPs）是临床批准的药物和膳食营养素的重要来源，但很少 完整的生物合成途径已被表征。因此，许多复杂的植物自然 产物支架目前仍从生产植物或植物细胞培养物中分离， 通过半合成途径临床使用的药物（例如依托泊苷、地高辛、吗啡、长春碱和紫杉醇， 2015年世卫组织基本药物清单）。缺乏关于其生物合成途径的信息 严重限制了在异源宿主（例如， 制造青蒿素的酵母菌株），以及设计生物合成青蒿素的有趣可能性。 获得类似物和非天然衍生物的途径具有更大的功效。更少有人知道 这些途径可能是可食用植物工程或育种工作的目标，以提高营养含量。 鉴于植物天然产物在人类健康中的关键作用和生物合成基因的实用性，我们建议 在这里，开发和应用一个广泛的通用平台，以加速发现 和关键植物天然产物途径的工程。传统上，植物途径的发现 比细菌途径更慢，更费力;然而，我们最近表明， 三种技术大大加快了快速植物途径的发现，我们将进一步扩大作为这一计划的一部分， 努力：（1）烟草异源宿主中酶的快速组合测试，（2）转录谱分析 和共表达分析，以确定途径基因，和（3）非靶向代谢组学作为在线分析 工具.这种方法使得发现了六种酶，完成了依托泊苷的十步途径 在几个月的时间里，从未测序的药用植物鬼臼中提取出糖苷配基（以前的工作是阐明 前四个步骤报道了十几年）。在本提案中，我们对以下途径进行了优先排序： 临床上使用的NP（依托泊苷）、在可食用植物中丰富的分子（芸苔属吲哚植物抗毒素和 来自胡萝卜和番茄的falcarindiol），或临床候选人，其评估将通过以下能力来实现： 以产生天然化合物或类似物（柠檬苦素）。这些化合物代表了一组不同的NP 类，并将用于展示我们的发现方法的广泛实用性。在每种情况下，我们也将 重点发展烟草作为新型生产平台。这项工作的一个主要成果将是 生物合成基因，其可用于工程化异源宿主以制备植物NP和类似物， 有效的生物活性。

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. Classically, the discovery of plant pathways has been slower and more painstaking than bacterial pathways; however, we have recently shown that combining three technologies greatly accelerates rapid plant pathway discovery that we will further expand as part of this effort: (1) rapid combinatorial testing of enzymes in a tobacco heterologous host, (2) transcriptional profiling and co-expression analysis to identify pathway genes, and (3) untargeted metabolomics as an in-line analytical tool. This approach enabled the discovery of six enzymes that complete the ten step pathway to the etoposide aglycone from the unsequenced medicinal plant Podophyllum in a matter of months (previous work to elucidate the first four steps was reported over ~ a dozen years). In this proposal we have prioritized pathways for clinically used NPs (etoposide), molecules abundant in edible plants (Brassica indolic phytoalexins and falcarindiol from carrot and tomato), or clinical candidates whose assessment would be enabled by the ability to generate the native compound or analogs (limonoids). These compounds represent a diverse set of NP classes and will be used to demonstrate the broad utility of our discovery approach. In each case we will also focus on development of tobacco as a novel production platform. 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.