Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system.
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.
Siloto
Rodrigo M P
RM
Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Ag/For Centre, Edmonton, AB, T6G 2P5, Canada.
Truksa
Martin
M
He
Xiaohua
X
McKeon
Thomas
T
Weselake
Randall J
RJ
D002626:Chemistry, Pharmaceutical / Q000379:methods*; D051048:Diacylglycerol O-Acyltransferase / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis*; D002785:Sterol O-Acyltransferase / Q000096:biosynthesis / Q000235:genetics; D014280:Triglycerides / Q000096:biosynthesis*
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014280:Triglycerides; D051048:Diacylglycerol O-Acyltransferase; D002785:Sterol O-Acyltransferase