Synthetic biology tools for scalable production of medicinal plant terpenes

项目摘要/摘要 植物萜类是临床批准的药物和临床候选药物的重要来源，但很少是完全的 生物合成途径已经被描述。由于缺乏有效的化学合成路线，许多 目前，包括萜类化合物在内的复杂植物天然产物支架仍与生产工厂分离。 或植物细胞培养，然后通过半合成路线(如地高辛和紫杉醇)转化为临床使用的药物 在2015年世卫组织基本药物清单上)。缺乏关于萜类生物合成途径的信息 严重限制了在异源宿主中生产植物分子的前景看好的新方法的使用(例如 产生青蒿素的酵母菌株)，以及设计生物合成的耐人寻味的可能性 获得更有效的类似物和非天然衍生物的途径。鉴于网络的关键作用 药用植物萜类化合物在人类健康和生物合成基因方面的应用，我们在这里提出了系统的 药用植物萜类生物合成关键途径紫杉醇和环多巴胺的发现 可规模化生产的酵母菌工程。传统上，植物途径的发现一直是 比细菌途径更慢、更费力；然而，我们的团队已经演示了两种方法 这大大加快了对完整生物合成路线的识别：(1)快速组合测试 异源寄主中的酶，以及(2)转录图谱和共表达分析 确定途径基因。这种方法使六种酶的发现成为可能，它们完成了通向 在几个月的时间里从未测序的药用植物鬼臼中提取了依托泊苷苷元，而且还导致了 发现了许多植物萜类化合物的途径，包括一类新的七萜类化合物。在这 提案中，我们优先考虑了有价值的药用植物萜类化合物的路径，这些路径是出了名的难以 访问：临床使用的抗癌药物紫杉醇和临床候选环丙胺。这些化合物 具有代表性的药用植物萜类化合物将被用来证明我们的发现的广泛用途 和酵母工程方法，可用于访问其他100,000种不同的植物 自然界中的萜类化合物。除了产生这些极有价值的植物萜类的酵母菌株外，一种主要的 这项工作的成果将是广泛适用的酵母合成生物学工具，用于高效生产多种 细胞色素P450的串联，这是有效转移植物途径到 酵母异源宿主。

PROJECT SUMMARY/ABSTRACT Plant terpenes are a critical source of clinically approved drugs and clinical candidates, yet very few complete biosynthetic pathways have been characterized. Due to the lack of efficient chemical synthesis routes, many complex plant natural product scaffolds including terpenes 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. digoxin and taxol on the 2015 WHO list of essential medicines). Lack of information regarding terpene 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. Given the critical role of medicinal plant terpenes in human health and utility of biosynthetic genes, we propose here systematic discovery of key medicinal plant terpene biosynthesis pathways, taxol and cyclopamine, and the engineering of yeast strains for scalable production. Classically, the discovery of plant pathways has been slower and more painstaking than bacterial pathways; however, our team has demonstrated two approaches that greatly accelerates identification of complete biosynthetic routes: (1) rapid combinatorial testing of enzymes in a N. benthamiana heterologous host, and (2) transcriptional profiling and co-expression analysis to identify pathway genes. This approach enabled the discovery of six enzymes that complete the pathway to the etoposide aglycone from the unsequenced medicinal plant Podophyllum in a matter of months and has also led to the discovery of numerous plant terpene pathways including a novel class of sesterterpenes. In this proposal, we have prioritized pathways for valuable medicinal plant terpenes that are notoriously difficult to access: the clinically used anticancer agent taxol and the clinical candidate cyclopamine. These compounds are representative medicinal plant terpenes that will be used to demonstrate the broad utility of our discovery and yeast engineering approach that can be applied for accessing many of the other >100,000 different plant terpenes in nature. In addition to yeast strains that produce these highly valuable plant terpenes, a major outcome of this work will be broadly applicable yeast synthetic biology tools for efficient production of multiple cytochromes P450s in series which represents a major bottleneck for efficient transfer of plant pathways to yeast heterologous hosts.