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Acetyl-TAG (3-acetyl-1,2-diacylglycerol), unique triacylglycerols (TAG) possessing an acetate group at thesn-3 position, exhibit valuable properties, such as reduced viscosity and freezing points. Previous attempts to engineer acetyl-TAG production in oilseed crops did not achieve the high levels found in naturally producingEuonymusseeds. Here, we demonstrate the successful generation of camelina and pennycress transgenic lines accumulating nearly pure acetyl-TAG at 93 mol% and 98 mol%, respectively. These ultrahigh acetyl-TAG synthesizing lines were created using gene-editedFATTY ACID ELONGASE1(FAE1) mutant lines as an improved genetic background to increase levels of acetyl-CoA available for acetyl-TAG synthesis mediated by the expression of EfDAcT, a high-activity diacylglycerol acetyltransferase isolated fromEuonymus fortunei. Combining EfDAcT expression with suppression of the competing TAG-synthesizing enzyme DGAT1 further enhanced acetyl-TAG accumulation. These ultrahigh levels of acetyl-TAG exceed those in earlier engineered oilseeds and are equivalent or greater than those inEuonymusseeds. Imaging of lipid localization in transgenic seeds revealed that the low amounts of residual TAG were mostly confined to the embryonic axis. Similar spatial distributions of specific TAG and acetyl-TAG molecular species, as well as their probable diacylglycerol (DAG) precursors, provide additional evidence that acetyl-TAG and TAG are both synthesized from the same tissue-specific DAG pools. Remarkably, this ultrahigh production of acetyl-TAG in transgenic seeds exhibited minimal negative effects on seed properties, highlighting the potential for production of designer oils required for economical biofuel industries.
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Substrates (Acyl‐ CoA and Diacylglycerol) Entry and Products ( CoA and Triacylglycerol) Egress Pathways in DGAT1
ABSTRACT Diacylglycerol O‐acyltransferase 1 (DGAT1) is an integral membrane protein that uses acyl‐coenzyme A (acyl‐CoA) and diacylglycerol (DAG) to catalyze the formation of triacylglycerides (TAGs). The acyl transfer reaction occurs between the activated carboxylate group of the fatty acid and the free hydroxyl group on the glycerol backbone of DAG. However, how the two substrates enter DGAT1's catalytic reaction chamber and interact with DGAT1 remains elusive. This study aims to explore the structural basis of DGAT1's substrate recognition by investigating each substrate's pathway to the reaction chamber. Using a human DGAT1 cryo‐EM structure in complex with an oleoyl‐CoA substrate, we designed two different all‐atom molecular dynamics (MD) simulation systems: DGAT1away(both acyl‐CoA and DAG away from the reaction chamber) and DGAT1bound(acyl‐CoA bound in and DAG away from the reaction chamber). Our DGAT1awaysimulations reveal that acyl‐CoA approaches the reaction chamber via interactions with positively charged residues in transmembrane helix 7. DGAT1boundsimulations show DAGs entering into the reaction chamber from the cytosol leaflet. The bound acyl‐CoA's fatty acid lines up with the headgroup of DAG, which appears to be competent to TAG formation. We then converted them into TAG and coenzyme (CoA) and used adaptive biasing force (ABF) simulations to explore the egress pathways of the products. We identify their escape routes, which are aligned with their respective entry pathways. Visualization of the substrate and product pathways and their interactions with DGAT1 is expected to guide future experimental design to better understand DGAT1 structure and function.
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- Award ID(s):
- 2111728
- PAR ID:
- 10587348
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Journal of Computational Chemistry
- Volume:
- 46
- Issue:
- 11
- ISSN:
- 0192-8651
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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