Simple Methods to Detect Triacylglycerol Biosynthesis in a Yeast-Based Recombinant System

摘要

Standard methods to quantify the activity of triacylglycerol (TAG) synthesizing enzymes DGAT and PDAT (TAG-SE) require a sensitive but rather arduous laboratory assay based on radio-labeled substrates. Here we describe two straightforward methods to detect TAG production in baker’s yeast Saccharomyces cerevisiae. First we demonstrate that a quadruple knockout yeast strain deficient in storage lipids has a reduced growth rate in a medium supplemented with fatty acids. This phenotype is rescued by restoring TAG biosynthesis and can be thus used to select yeast cells expressing a recombinant TAG-SE. In the second method, the activity of the recombinant enzyme is measured in a fluorescent in situ assay using Nile red dye that is specific for neutral lipids. Correlation between Nile red fluorescence and enzyme activity is demonstrated with several mutants of a TAG synthesizing enzyme. This yeast live-cell-based assay is rapid, inexpensive, sensitive, and is amenable to high-throughput applications. The methods can be used for a variety of applications such as isolation of novel genes, directed evolution, gene-specific drug screening and will facilitate novel approaches in the research of TAG-SE. Keywords DGAT - Diacylglycerol acyltransferase - High throughput screening - Lipotoxicity - Neutral lipids - Nile red R. M. P. Siloto and M. Truksa have contributed equally to this work and should be both regarded as the first author.