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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPMENT OF A DOMESTIC SOURCE FOR PRODUCTION OF RICINOLEATE AND OTHER INDUSTRIAL-USE FATTY ACIDS Title: Simple method to detect triacylglycerol biosynthesis in a yeast-based recombinant system

Authors
item Siloto, Rodrigo -
item Truksa, Martin -
item He, Xiaohua
item McKeon, Thomas
item Weselake, Randall -

Submitted to: Lipids
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 7, 2009
Publication Date: September 10, 2009
Citation: Siloto, R.M., Truksa, M., He, X., McKeon, T.A., Weselake, R. 2009. Simple method to detect triacylglycerol biosynthesis in a yeast-based recombinant system. Lipids. 44:963-973.

Interpretive Summary: Vegetable oil is a valuable commodity for food uses and industrial applications like soap and biodiesel production. However, there is limited understanding of how plants regulate their oil production. The final step in oil biosynthesis, the diacylglycerol acyltransferase (DGAT), is one of the enzymes thought to be involved in controlling oil content and composition. This manuscript reports on the development of mutant strains of yeast that can be used to identify and evaluate genes encoding DGAT activity. This technology can be used to screen genes that have been modified in order to detect greater activity or specificity. The result of such research could lead to development of oilseeds with higher oil content or altered fatty acid composition.

Technical Abstract: 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 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 synthase. 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.

Last Modified: 10/23/2014