Centre for Biospectroscopy, School of Chemistry, Monash University, Clayton Campus, 3800, Victoria, Australia.
Analyst. 2019 Jan 28;144(3):901-912. doi: 10.1039/c8an01477a.
Cyclopropane fatty acids (CFAs) are a group of lipids with unique physical and chemical properties between those of saturated and monounsaturated fatty acids. The distinctive physicochemical characteristics of CFAs (e.g. oxidative stability, self-polymerization at high temperatures, etc.) results from the presence of a cyclopropane ring within their structure making them highly useful in industrial applications. CFAs are present in several species of plants and bacteria and are typically detected with standard lipid profiling techniques, such as gas or liquid chromatography. In this work we investigated several strains of S. cerevisiae, genetically modified to introduce the production of CFAs, in comparison to control strain using confocal Raman spectroscopy (CRS). The aim of our work was to demonstrate the potential of CRS not only to detect changes introduced due to the CFAs presence, but also to track CFAs within the cells. We present for the first time Raman and IR spectra of CFA standard (cis-9,10-methyleneoctadecanoic acid), completed with quantum chemical calculations and band assignment. We identified marker bands of CFA (e.g. 2992, 1222, 942 cm-1) attributed to the vibrations of the cyclopropyl ring. Furthermore, we analysed lipid bodies (LBs) from modified and control yeast using CRS imaging and identified multiple changes in size, number and composition of LBs from engineered strains. We observed a significant reduction in the degree of unsaturation of LBs using the ratio of bands located at 1660 cm-1 (ν(C[double bond, length as m-dash]C)) and 1448 cm-1 (δ(CH2)) in the modified cell lines. In addition, we were able to detect the presence of CFAs in LBs, using the established marker bands. CRS shows tremendous potential as technique to identify CFAs in lipid bodies providing a new way to track lipid production in genetically modified single yeast cells.
环丙烷脂肪酸（CFAs）是一组具有独特物理化学性质的脂质，其性质介于饱和脂肪酸和单不饱和脂肪酸之间。CFAs 的独特物理化学特性（例如氧化稳定性、高温下的自聚合等）源于其结构中存在环丙烷环，这使得它们在工业应用中非常有用。CFAs 存在于几种植物和细菌中，通常使用标准脂质分析技术（如气相或液相色谱法）进行检测。在这项工作中，我们使用共焦拉曼光谱（CRS）研究了几种经过基因改造以引入 CFAs 生产的酿酒酵母菌株，与对照菌株进行了比较。我们的工作目的是不仅证明 CRS 不仅可以检测由于 CFAs 存在而引入的变化，而且可以跟踪细胞内的 CFAs。我们首次展示了 CFA 标准品（顺式-9,10-亚甲基十八烷酸）的拉曼和红外光谱，同时进行了量子化学计算和带分配。我们确定了 CFA 的标记带（例如 2992、1222、942 cm-1），归因于环丙基环的振动。此外，我们使用 CRS 成像分析了经过修饰和对照酵母的脂滴（LBs），并确定了工程菌株的 LB 大小、数量和组成的多个变化。我们观察到，在用位于 1660 cm-1（ν（C[双键，长度为 m-dash]C））和 1448 cm-1（δ（CH2））处的带的比率表示的经修饰细胞系中，LB 的不饱和度显着降低。此外，我们能够使用已建立的标记带检测 LB 中 CFAs 的存在。CRS 作为在脂滴中识别 CFAs 的技术具有巨大的潜力，为跟踪遗传修饰的单个酵母细胞中的脂质生产提供了一种新方法。
