Type-1 Brassica napus Diacylglycerol Acyltransferases: Enzyme Characterization and Molecular Tools for Increasing Storage Lipid Production in Yeast
Bibliographic record
Abstract
Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA- dependent biosynthesis of triacylglycerol (TAG). The level of DGAT activity may have a substantial effect on the flow of carbon into TAG in many organisms. In plants, yeast, and animals, two families of membrane-bound DGATs have been identified. In plants, modification of the DGAT-catalyzed step could lead to improved oil seed varieties with either increased nutritional or industrial value. Increased production of oil through manipulation of DGAT activity would be generally beneficial in both plants and yeast. In humans, alteration of DGAT activity through pharmacological intervention could lead to treatments for obesity, type-2 diabetes, and improved cardiovascular health. Four type-1 DGAT genes have been identified in the Brassica napus genome which appear to belong to two clades with representatives of each clade in the A and C genome. B. napus is of major agricultural and economic importance in Canada. Despite having highly similar amino acid sequences, the DGAT1s encoded by these genes displayed significantly different abilities to catalyze the synthesis of TAG when recombinantly produced in a strain (H1246) of Saccharomyces cerevisiae devoid of TAG synthesis. Various modifications to the N-terminal regions and/or the encoding DNA sequences of the four isoforms were shown to have a profound impact on the accumulation of recombinant enzyme polypeptide in this yeast strain. In turn, this information was used as basis for engineering increased oil accumulation in yeast. Increasing the accumulation of DGAT in yeast cells also facilitated the development of a novel gas chromatography/mass spectrometry-based in vitro DGAT assay. This assay circumvents the need for radiolabeled substrates commonly used in DGAT assays and is particularly useful in quickly evaluating substrate selectivity properties. Phylogenetic analysis of the four B. napus DGAT1 coding sequences revealed that these genes may have diverged into two separate clades relatively early in Brassicaceae history. Although all four DGAT1s could effectively use a range of molecular species of acyl-CoAs and sn-1,2-diacylglycerols, clade II DGAT1s displayed increased preference for substrates containing linoleic acid (18:29cis,12cis). In the case of acyl-CoA, α-linolenoyl (18:39cis,12cis,15cis) was the most effective acyl donor for all four DGAT1s. These differences in substrate specificity occurred despite the relatively high level of amino acid sequence identity between the two clades of DGAT1. These results suggest that the two clades of B. napus DGAT1 enzymes have slightly different functional roles in oil formation during seed development.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
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 itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".