Murphy Katherine M, Chung Siwon, Fogla Shruti, Minsky Hana B, Zhu Karen Yong, Zerbe Philipp
Department of Plant Biology, University of California Davis.
Department of Plant Biology, University of California Davis;
J Vis Exp. 2019 Oct 4(152). doi: 10.3791/59992.
Diterpenoids form a diverse class of small molecule natural products that are widely distributed across the kingdoms of life and have critical biological functions in developmental processes, interorganismal interactions, and environmental adaptation. Due to these various bioactivities, many diterpenoids are also of economic importance as pharmaceuticals, food additives, biofuels, and other bioproducts. Advanced genomics and biochemical approaches have enabled a rapid increase in the knowledge of diterpenoid-metabolic genes, enzymes, and pathways. However, the structural complexity of diterpenoids and the narrow taxonomic distribution of individual compounds in often only a single species remain constraining factors for their efficient production. Availability of a broader range of metabolic enzymes now provide resources for producing diterpenoids in sufficient titers and purity to facilitate a deeper investigation of this important metabolite group. Drawing on established tools for microbial and plant-based enzyme co-expression, we present an easily operated and customizable protocol for the enzymatic production of diterpenoids in either Escherichia coli or Nicotiana benthamiana, and the purification of desired products via silica chromatography and semi-preparative HPLC. Using the group of maize (Zea mays) dolabralexin diterpenoids as an example, we highlight how modular combinations of diterpene synthase (diTPS) and cytochrome P450 monooxygenase (P450) enzymes can be used to generate different diterpenoid scaffolds. Purified compounds can be used in various downstream applications, such as metabolite structural analyses, enzyme structure-function studies, and in vitro and in planta bioactivity experiments.
二萜类化合物构成了一类多样的小分子天然产物，广泛分布于生物界，在发育过程、生物体间相互作用和环境适应中具有关键的生物学功能。由于这些多样的生物活性，许多二萜类化合物作为药物、食品添加剂、生物燃料和其他生物制品也具有重要的经济价值。先进的基因组学和生化方法使得人们对二萜类代谢基因、酶和途径的认识迅速增加。然而，二萜类化合物的结构复杂性以及单个化合物往往仅在单一物种中的狭窄分类分布，仍然是其高效生产的制约因素。现在，更广泛的代谢酶的可获得性为以足够的产量和纯度生产二萜类化合物提供了资源，以便更深入地研究这一重要的代谢物组。利用已建立的基于微生物和植物的酶共表达工具，我们提出了一种易于操作且可定制的方案，用于在大肠杆菌或本氏烟草中酶促生产二萜类化合物，并通过硅胶柱色谱和半制备高效液相色谱法纯化所需产物。以玉米（Zea mays）多勒巴林二萜类化合物组为例，我们强调了二萜合酶（diTPS）和细胞色素P450单加氧酶（P450）酶的模块化组合如何用于生成不同的二萜类骨架。纯化的化合物可用于各种下游应用，如代谢物结构分析、酶结构功能研究以及体外和植物体内生物活性实验。
