Metabolomics Australia, Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia.
Novo Nordisk Foundation Centre for Biosustainability, Technical University of Denmark, Lyngby, Denmark.
Methods Enzymol. 2022;670:235-284. doi: 10.1016/bs.mie.2022.03.018. Epub 2022 May 14.
Isoprenoids, also known as terpenes or terpenoids, are a very large and diverse group of natural compounds. These compounds fulfil a myriad of critical roles in biology as well as having a wide range of industrial uses. Isoprenoids are produced via two chemically distinct metabolic pathways, the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Downstream of these two pathways is the shared prenyl phosphate pathway. Because of their importance in both basic physiology and industrial biotechnology, extraction, identification, and quantification of isoprenoid pathway intermediates is an important protocol. Here we describe methods for extraction and analysis of intracellular metabolites from the MVA, MEP, and prenyl phosphate pathways for five key model microbes: the yeast Saccharomyces cerevisiae, the bacterium Escherichia coli, the diatom Phaeodactylum tricornutum, the green algae Chlamydomonas reinhardtii, and the cyanobacterium Synechocystis sp. PCC 6803. These methods also detect several central carbon intermediates. These protocols will likely work effectively, or be readily adaptable, to a variety of related microorganisms and metabolic pathways.
异戊二烯类化合物，也称为萜类或萜烯类化合物，是一大类非常多样化的天然化合物。这些化合物在生物学中具有多种关键作用，同时在工业上也有广泛的用途。异戊二烯类化合物通过两条化学上截然不同的代谢途径产生，即甲羟戊酸（MVA）途径和甲基赤藓醇磷酸（MEP）途径。这两条途径的下游是共同的 prenyl phosphate 途径。由于它们在基础生理学和工业生物技术中的重要性，提取、鉴定和定量异戊二烯途径中间产物是一个重要的方案。在这里，我们描述了从 MVA、MEP 和 prenyl phosphate 途径中提取和分析五种关键模型微生物（酵母酿酒酵母、细菌大肠杆菌、硅藻三角褐指藻、绿藻莱茵衣藻和蓝藻集胞藻 sp. PCC 6803）细胞内代谢物的方法。这些方法还可以检测几种中心碳中间产物。这些方案可能会有效地适用于多种相关微生物和代谢途径，或者可以很容易地进行适应性调整。
