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

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.

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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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Acknowledgments

We thank S. Stymne and U. Ståhl for providing the yeast strain H1246 and the corresponding parental line and C. Snyder for reviewing the manuscript. This research was supported by AVAC Ltd., the Natural Science and Engineering Research Council of Canada and the Canada Research Chairs Program.

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Correspondence to Randall J. Weselake.

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R. M. P. Siloto and M. Truksa have contributed equally to this work and should be both regarded as the first author.

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Siloto, R.M.P., Truksa, M., He, X. et al. Simple Methods to Detect Triacylglycerol Biosynthesis in a Yeast-Based Recombinant System. Lipids 44, 963 (2009). https://doi.org/10.1007/s11745-009-3336-0

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  • DOI: https://doi.org/10.1007/s11745-009-3336-0

Keywords

  • DGAT
  • Diacylglycerol acyltransferase
  • High throughput screening
  • Lipotoxicity
  • Neutral lipids
  • Nile red