Lipids

, 44:963 | Cite as

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

  • Rodrigo M. P. Siloto
  • Martin Truksa
  • Xiaohua He
  • Thomas McKeon
  • Randall J. Weselake
Methods

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 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 

Abbreviations

BnDGAT1

Brassica napus DGAT1

DGAT

Acyl-CoA:diacylglycerol acyltransferase

FA

Fatty acids

FACS

Fluorescence-activated cell sorting

LuDGAT1

Linum usitatissimum DGAT1

NRA

Nile red assay

OD

Optical density

PDAT

Phospholipid:diacylglycerol acyltransferase

RcDGAT1

Ricinus communis DGAT1

TAG

Triacylglycerol

TLC

Thin layer chromatography

TAG-SE

TAG synthesizing enzymes

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Copyright information

© AOCS 2009

Authors and Affiliations

  • Rodrigo M. P. Siloto
    • 1
  • Martin Truksa
    • 1
  • Xiaohua He
    • 2
  • Thomas McKeon
    • 2
  • Randall J. Weselake
    • 1
  1. 1.Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional ScienceUniversity of AlbertaEdmontonCanada
  2. 2.Western Regional Research CenterUSDAAlbanyCAUSA

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