Type-1 Brassica napus Diacylglycerol Acyltransferases: Enzyme Characterization and Molecular Tools for Increasing Storage Lipid Production in Yeast Open Access
- Other title
- Type of item
- Degree grantor
University of Alberta
- Author or creator
Greer, Michael S
- Supervisor and department
Weselake, Randall J (Agricultural, Food and Nutritional Science)
- Examining committee member and department
Nikolau, B (Biochemistry, Biophysics and Molecular Biology - Iowa State University)
Guan, L (Agricultural, Food and Nutritional Science)
Dixon, W (Associate Vice-President/Research)
Kav, N (Agricultural, Food and Nutritional Science)
Department of Agricultural, Food, and Nutritional Science
- Date accepted
- Graduation date
Doctor of Philosophy
- Degree level
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.
- Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
- Citation for previous publication
Greer MS, Turksa M, Deng W, Lung SC, Chen G, Weselake RJ (2014a) Engineering increased triacylglycerol accumulation in Saccharomyces cerevisiae using a modified type-1 plant diacylglycerol acyltransferase. Appl. Microbiol. Biotechnol. 99: 2243-53Greer MS, Zhou T, Weselake RJ (2014b) A novel assay of DGAT activity based on high temperature GC/MS of triacylglycerol. Lipids 49: 831-8
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