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Synthetic biology tools for scalable production of medicinal plant terpenes

用于药用植物萜烯大规模生产的合成生物学工具

基本信息

批准号：
10250333
负责人：
JAY D KEASLING
金额：
$ 69.83万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-15 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10250333
关键词：
Address Alleles Anabolism Antineoplastic Agents Artemisinins Candidate Disease Gene Catalysis Cell Culture Techniques Chemicals Chemistry Clinical Complex Coupling Cytochrome P450 Cytochromes Development Digoxin Diterpenes Drug usage Engineering Enzyme Tests Enzymes Equilibrium Etoposide Gene Expression Profiling Genes Genome Health Human Investigational Therapies Lead Malignant Neoplasms Medicinal Plants Medicine Metabolic Pathway Metabolism Mining Natural Products Nature Nicotiana Outcome Oxidation-Reduction Oxidative Stress Oxidoreductase Paclitaxel Pathway interactions Plants Podophyllum Production Protein Engineering Proteins Role Route Saccharomyces cerevisiae Series Sonic Hedgehog Pathway Source Specificity Structure Taxus Terpenes Testing Therapeutic Therapeutic Uses Uncertainty Variant Veratrum Work Yeasts analog biological adaptation to stress chemical synthesis clinical candidate combinatorial cyclopamine design drug candidate electron donor enzyme pathway gene discovery human disease improved inhibitor/antagonist novel novel strategies scaffold scale up sesterterpenes synthetic biology taxadiene therapeutic candidate tool transcriptomics

项目摘要

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.

项目概要/摘要植物萜烯是临床批准的药物和临床候选药物的重要来源，但很少有完整的生物合成途径已得到表征。由于缺乏有效的化学合成路线，许多包括萜烯在内的复杂植物天然产物支架目前仍从生产植物中分离出来或植物细胞培养物，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，植物分子）中生产植物分子的有前途的新方法的使用。制造青蒿素的酵母菌株），以及生物合成工程的有趣可能性更有效地获取类似物和非天然衍生物的途径。鉴于其关键作用药用植物萜烯在人类健康和生物合成基因中的效用，我们在这里提出系统的发现关键的药用植物萜烯生物合成途径、紫杉醇和环杷明，以及用于规模化生产的酵母菌株工程。传统上，植物途径的发现是比细菌途径更慢、更费力；然而，我们的团队展示了两种方法大大加速了完整生物合成路线的鉴定：（1）快速组合测试本塞姆氏烟草异源宿主中的酶，以及（2）转录谱和共表达分析识别途径基因。这种方法发现了六种酶，它们完成了通往在几个月内从未测序的药用植物鬼臼中提取了依托泊苷苷元，并且还导致发现了许多植物萜烯途径，包括一类新型二元萜烯。在这个根据提案，我们优先考虑了宝贵的药用植物萜烯的途径，而众所周知，这些途径很难获取：临床使用的抗癌药紫杉醇和临床候选环巴明。这些化合物是具有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方法，可用于访问许多其他 > 100,000 种不同的植物自然界中的萜烯。除了产生这些高价值植物萜烯的酵母菌株外，一个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，用于高效生产多种系列细胞色素 P450 是植物途径有效转移的主要瓶颈酵母异源宿主。