Related Experiment Video
Updated: Jun 20, 2026
10:57
Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae
Published on: February 16, 2015
Simple methods to detect triacylglycerol biosynthesis in a yeast-based recombinant system
Rodrigo M P Siloto1, Martin Truksa, Xiaohua He
1Agricultural Lipid Biotechnology Program, Department of Agricultural, Food and Nutritional Science, University of Alberta, 4-10 Ag/For Centre, Edmonton, AB, T6G 2P5, Canada.
Lipids
|September 19, 2009
Summary
We developed two simple yeast-based methods to measure triacylglycerol (TAG) production. These assays detect TAG synthesis by engineered yeast, offering a faster, cheaper alternative to traditional radio-labeled assays for TAG-synthesizing enzymes.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- Standard assays for triacylglycerol (TAG) synthesizing enzymes (TAG-SE), such as DGAT and PDAT, rely on radio-labeled substrates, which are sensitive but laborious.
- Developing efficient and high-throughput methods to quantify TAG production is crucial for understanding lipid metabolism and for applications in biotechnology.
Purpose of the Study:
- To establish straightforward, yeast-based methods for detecting and quantifying TAG production.
- To provide rapid, inexpensive, and sensitive alternatives to traditional assays for TAG-SE activity.
- To facilitate high-throughput screening for novel TAG-SE genes and drug discovery.
Main Methods:
- Utilizing a quadruple knockout yeast strain deficient in storage lipids; its growth defect in fatty acid-supplemented media is rescued by restoring TAG biosynthesis, enabling selection of yeast expressing recombinant TAG-SE.
- Employing a fluorescent in situ assay with Nile red dye, which specifically stains neutral lipids, to measure TAG-SE activity in live yeast cells.
- Correlating Nile red fluorescence intensity with TAG-SE activity using various enzyme mutants.
Main Results:
- A quadruple knockout yeast strain's growth phenotype accurately reflects TAG biosynthesis capacity.
- Nile red fluorescence provides a reliable measure of TAG-SE activity in live yeast cells.
- The developed methods demonstrate a strong correlation between fluorescence and enzyme activity across different mutants.
Conclusions:
- Two novel, yeast-based assays for TAG production have been successfully developed.
- These methods offer rapid, cost-effective, and sensitive alternatives for studying TAG-SE.
- The assays are suitable for high-throughput applications, including gene discovery and drug screening in lipid research.
