Synthetic biology tools for scalable production of medicinal plant terpenes

项目概要/摘要 植物萜烯是临床批准的药物和临床候选物的重要来源，但很少有完整的植物萜烯。 生物合成途径已被表征。由于缺乏有效的化学合成途径，许多 包括萜烯的复杂的植物天然产物支架目前仍从生产植物中分离 或植物细胞培养，然后通过半合成途径转化为临床使用的药物（例如地高辛和紫杉醇 2015年世界卫生组织基本药物清单）。缺乏有关萜烯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。考虑到 药用植物萜烯在人类健康和生物合成基因的效用，我们在这里提出系统 发现了关键的药用植物萜类生物合成途径，紫杉醇和环巴胺， 工程化酵母菌株以用于可规模化生产。传统上，植物途径的发现 比细菌途径更慢，更费力;然而，我们的团队已经证明了两种方法， 这大大加速了完整生物合成途径的鉴定：（1）快速组合测试 酶在N.本塞姆氏异源宿主，和（2）转录谱分析和共表达分析， 识别通路基因。这种方法使六种酶的发现成为可能，这些酶完成了通往蛋白质的途径。 在几个月的时间里，从未测序的药用植物鬼臼属中提取出依托泊苷元， 发现了许多植物萜途径，包括一类新的二倍半萜。在这 我们已经优先考虑了有价值的药用植物萜类化合物的途径，这些萜类化合物是出了名的难以分离的。 获取：临床使用的抗癌药紫杉醇和临床候选药物环巴胺。这些化合物 是代表性的药用植物萜烯，将用于证明我们的发现的广泛用途 和酵母工程的方法，可用于访问许多其他> 100，000不同的植物 自然界中的萜烯。除了生产这些高价值植物萜烯的酵母菌株外， 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系列，这代表了植物途径有效转移到 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.