Simple Methods to Detect Triacylglycerol Biosynthesis in a Yeast‐Based Recombinant System
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Bibliographic record
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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Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from it