Triacylglycerol (TAG) is a storage lipid with high energy density that is widely present in animals, plants, and algae. The fact that microalgae are able to accumulate TAGs has attracted scientific and public interest due to the potential to provide biofuel feed stocks. In addition, algal TAG is able to serve as a source of polyunsaturated fatty acids that show benefits for human health. Moreover, experimental manipulation of algal TAG metabolism is relatively easy, especially in the single-cellular alga Chlamydomonas reinhardtii, making this organism a good model for studying TAG related events in multi-cellular organisms.Chlamydomonas reinhardtii is the most investigated green alga, with a full set of molecular tools available. Another microalga, Nannochloropsis oceanica, has received research interests as it has been specifically considered for biofuel production because of its naturally high TAG content. For both C. reinhardtii and N. oceanica, the process of TAG accumulation is activated by stress induction. Transcriptomic analysis of algal cells under stress conditions has been performed to understand the expression profile of TAG metabolism related genes. Moreover, forward genetics approaches have been used to identify and characterize novel genes involved in TAG accumulation. However, the mechanisms of TAG accumulation and degradation are not yet fully understood. Here, an investigation of TAG biosynthesis in stressed microalgae by analytical and biochemical approaches is presented, providing insights into algal TAG metabolism.A liquid chromatography-mass spectrometry (LC-MS) based TAG profiling approach was developed. This approach is able to identify all TAG species in microalgae - more than one hundred TAG species in both C. reinhardtii and N. oceanica. In addition, accurate and robust TAG quantification was achieved, in comparison with measurements by gas chromatography (GC). Notably, internal standards were identified for LC-MS and GC, respectively, during this work. Both internal standards have the potential to be applied in the analysis of a large number of lipid samples, enabling high throughput phenotypic screening strategies. During a series of collaborative projects, lipid analysis was performed for C. reinhardtii under N deprivation and hypoxia stress. Based on the comparison of lipid profiles, especially TAG profiles, between N deprived and hypoxia stressed C. reinhardtii, a new project was initiated, during which algal TAG accumulation under hypoxia stress was biochemically characterized. Radioactive labeling experiments demonstrated that membrane lipid remodeling is the major contributor of algal TAG biosynthesis under dark hypoxia conditions. Characterization of C. reinhardtii mutant strains with deficiencies in Phospholipid: Diacylglycerol Acyltransferase 1 (PDAT1) showed that both TAG accumulation and degradation of membrane lipids, including diacylglyceryl-N, N, N-trimethylhomoserine (DGTS) were affected in mutant strains, suggesting the PDAT1 gene is involved in algal TAG biosynthesis during hypoxia.
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