MANIPULATING OIL SEED BIOCHEMISTRY TO ENHANCE THE PRODUCTION OF ACETYL-TAGS

Master of Science === Biochemistry and Molecular Biophysics Interdepartmental Program === Timothy P. Durrett === Using vegetable oils directly as an alternative biofuel presents several problems as such oils typically possess poor fuel qualities including high viscosity, low volatility, and poor col...

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Main Author: Kornacki, Catherine
Language:en_US
Published: Kansas State University 2017
Subjects:
Online Access:http://hdl.handle.net/2097/35753
id ndltd-KSU-oai-krex.k-state.edu-2097-35753
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spelling ndltd-KSU-oai-krex.k-state.edu-2097-357532017-12-17T04:02:25Z MANIPULATING OIL SEED BIOCHEMISTRY TO ENHANCE THE PRODUCTION OF ACETYL-TAGS Kornacki, Catherine Camelina sativa Acetyl-TAGs Metabolic engineering Master of Science Biochemistry and Molecular Biophysics Interdepartmental Program Timothy P. Durrett Using vegetable oils directly as an alternative biofuel presents several problems as such oils typically possess poor fuel qualities including high viscosity, low volatility, and poor cold temperature properties. The ornamental shrub Euonymus alatus produces unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) that have an acetyl group in the sn-3 position instead of a long chain fatty acid. The presence of this sn-3 acetyl-group give acetyl-TAGs properties desirable for biofuels, such as reduced viscosity, comparted to the normal long chain triacyglycerols found in most vegetable oils. Acetyl-TAGs are synthesized by the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) and Euonymus fortunei diacylglycerol acetyltransferase (EfDAcT) enzymes. Both enzymes catalyze the transfer of an acetyl group from acetyl-CoA to diaclglycerol (DAG) to produce acetyl-TAGs. Previous work demonstrated that expression of EaDAcT combined with the suppression of a diacylglycerol aceyltransferase (DGAT1) in Camelina sativa led to seeds with 85 mol % acetyl-TAGs. Increasing acetyl-TAG levels further was explored using two strategies. Over expression of citrate lyase to increase the pool of acetyl-CoA to be used as a substrate for the acetyltransferase enzymes failed to increased levels of acetyl-TAGs. A second approach involved expressing EfDAcT in Camelina sativa. EfDAcT has demonstrated higher activity in vitro and in vivo and its expression in yeast leads to approximately 50 % higher levels of acetyl-TAGs compared to EaDAcT. The expression of EfDAcT coupled with the suppression of DGAT1 in Camelina sativa resulted in 90 mol % acetyl-TAGs in the transgenic seeds. Levels of EfDAcT protein analyzed in developing transgenic Camelina sativa seeds across a 40 day time period were highest at 15 and 20 days after flowering. Following these time points acetyl-TAG accumulation increased rapidly, coinciding with the higher enzyme expression levels. The optimization of additional promoters to ensure expression of EfDAcT in the last half of seed development could represent another way to further increase acetyl-TAGs in the future. 2017-06-30T19:37:21Z 2017-06-30T19:37:21Z 2016 December Thesis http://hdl.handle.net/2097/35753 en_US Kansas State University
collection NDLTD
language en_US
sources NDLTD
topic Camelina sativa
Acetyl-TAGs
Metabolic engineering
spellingShingle Camelina sativa
Acetyl-TAGs
Metabolic engineering
Kornacki, Catherine
MANIPULATING OIL SEED BIOCHEMISTRY TO ENHANCE THE PRODUCTION OF ACETYL-TAGS
description Master of Science === Biochemistry and Molecular Biophysics Interdepartmental Program === Timothy P. Durrett === Using vegetable oils directly as an alternative biofuel presents several problems as such oils typically possess poor fuel qualities including high viscosity, low volatility, and poor cold temperature properties. The ornamental shrub Euonymus alatus produces unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) that have an acetyl group in the sn-3 position instead of a long chain fatty acid. The presence of this sn-3 acetyl-group give acetyl-TAGs properties desirable for biofuels, such as reduced viscosity, comparted to the normal long chain triacyglycerols found in most vegetable oils. Acetyl-TAGs are synthesized by the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) and Euonymus fortunei diacylglycerol acetyltransferase (EfDAcT) enzymes. Both enzymes catalyze the transfer of an acetyl group from acetyl-CoA to diaclglycerol (DAG) to produce acetyl-TAGs. Previous work demonstrated that expression of EaDAcT combined with the suppression of a diacylglycerol aceyltransferase (DGAT1) in Camelina sativa led to seeds with 85 mol % acetyl-TAGs. Increasing acetyl-TAG levels further was explored using two strategies. Over expression of citrate lyase to increase the pool of acetyl-CoA to be used as a substrate for the acetyltransferase enzymes failed to increased levels of acetyl-TAGs. A second approach involved expressing EfDAcT in Camelina sativa. EfDAcT has demonstrated higher activity in vitro and in vivo and its expression in yeast leads to approximately 50 % higher levels of acetyl-TAGs compared to EaDAcT. The expression of EfDAcT coupled with the suppression of DGAT1 in Camelina sativa resulted in 90 mol % acetyl-TAGs in the transgenic seeds. Levels of EfDAcT protein analyzed in developing transgenic Camelina sativa seeds across a 40 day time period were highest at 15 and 20 days after flowering. Following these time points acetyl-TAG accumulation increased rapidly, coinciding with the higher enzyme expression levels. The optimization of additional promoters to ensure expression of EfDAcT in the last half of seed development could represent another way to further increase acetyl-TAGs in the future.
author Kornacki, Catherine
author_facet Kornacki, Catherine
author_sort Kornacki, Catherine
title MANIPULATING OIL SEED BIOCHEMISTRY TO ENHANCE THE PRODUCTION OF ACETYL-TAGS
title_short MANIPULATING OIL SEED BIOCHEMISTRY TO ENHANCE THE PRODUCTION OF ACETYL-TAGS
title_full MANIPULATING OIL SEED BIOCHEMISTRY TO ENHANCE THE PRODUCTION OF ACETYL-TAGS
title_fullStr MANIPULATING OIL SEED BIOCHEMISTRY TO ENHANCE THE PRODUCTION OF ACETYL-TAGS
title_full_unstemmed MANIPULATING OIL SEED BIOCHEMISTRY TO ENHANCE THE PRODUCTION OF ACETYL-TAGS
title_sort manipulating oil seed biochemistry to enhance the production of acetyl-tags
publisher Kansas State University
publishDate 2017
url http://hdl.handle.net/2097/35753
work_keys_str_mv AT kornackicatherine manipulatingoilseedbiochemistrytoenhancetheproductionofacetyltags
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