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




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Saccharomyces cerevisiae, Lipid Metabolism, Triacylglycerol, Diacylglycerol Acyltransferase, DGAT, Brassia napus, Canola

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: Department of Agricultural, Food, and Nutritional Science

Specialization: Plant Science

Date accepted: 2015-06-29T14:19:46Z

Graduation date: 2015-11

Degree: Doctor of Philosophy

Degree level: Doctoral

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 18:2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.

Language: English

DOI: doi:10.7939-R36M3386S

Rights: 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.





Author: Greer, Michael S

Source: https://era.library.ualberta.ca/


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Type-1 Brassica napus Diacylglycerol Acyltransferases: Enzyme Characterization and Molecular Tools for Increasing Storage Lipid Production in Yeast by Michael Scott Greer A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Plant Science Department of Agriculture, Food, and Nutritional Science University of Alberta © Michael Scott Greer, 2015 ABSTRACT Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoAdependent 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 Nterminal 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. ii Increas...





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