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Simple Methods to Detect Triacylglycerol Biosynthesis in a Yeast‐Based Recombinant System
Author(s) -
Siloto Rodrigo M. P.,
Truksa Martin,
He Xiaohua,
McKeon Thomas,
Weselake Randall J.
Publication year - 2009
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-009-3336-0
Subject(s) - yeast , biochemistry , saccharomyces cerevisiae , nile red , recombinant dna , enzyme , biology , biosynthesis , high throughput screening , mutant , metabolic engineering , gene , chemistry , microbiology and biotechnology , fluorescence , physics , quantum mechanics
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.