Type-1 Brassica napus Diacylglycerol Acyltransferases: Enzyme Characterization and Molecular Tools for Increasing Storage Lipid Production in Yeast

  • Author / Creator
    Greer, Michael S
  • 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 (18:29cis,12cis). In the case of acyl-CoA, α-linolenoyl (18:39cis,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.

  • Subjects / Keywords
  • Graduation date
    Fall 2015
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.