The lipid droplet (LD) is a cell organelle that has been linked to human metabolic syndromes and that can be exploited for the development of biofuels. The isolation of LDs is crucial for carrying out morphological and biochemical studies of this organelle. In the past two decades, LDs have been isolated from several organisms and investigated by microscopy, proteomics and lipidomics. However, these studies need to be extended to more model organisms, as well as to more animal tissues. Thus, a standard method that can be easily applied to these new samples with the need for minimal optimization is essential. Here we provide an LD isolation protocol that is relatively simple and suitable for a wide range of tissues and organisms. On the basis of previous studies, this 7-h protocol can yield 15-100 μg of protein-equivalent high-quality LDs that satisfy the requirements for current LD research in most organisms.

Squalene is a valuable natural substance with several biotechnological applications. In the yeast Saccharomyces cerevisiae, it is produced in the isoprenoid pathway as the first precursor dedicated to ergosterol biosynthesis. The aim of this study was to explore the potential of squalene epoxidase encoded by the ERG1 gene as the target for manipulating squalene levels in yeast. Highest squalene levels (over 1000 μg squalene per 10(9)  cells) were induced by specific point mutations in ERG1 gene that reduced activity of squalene epoxidase and caused hypersensitivity to terbinafine. This accumulation of squalene in erg1 mutants did not significantly disturb their growth. Treatment with squalene epoxidase inhibitor terbinafine revealed a limit in squalene accumulation at 700 μg squalene per 10(9)  cells which was associated with pronounced growth defects. Inhibition of squalene epoxidase activity by anaerobiosis or heme deficiency resulted in relatively low squalene levels. These levels were significantly increased by ergosterol depletion in anaerobic cells which indicated feedback inhibition of squalene production by ergosterol. Accumulation of squalene in erg1 mutants and terbinafine-treated cells were associated with increased cellular content and aggregation of lipid droplets. Our results prove that targeted genetic manipulation of the ERG1 gene is a promising tool for increasing squalene production in yeast.

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