Targeted engineering of camelina and pennycress seeds for ultrahigh accumulation of acetyl-TAG

Alkotami, Linah; White, Dexter J.; Schuler, Kathleen M.; Esfahanian, Maliheh; Jarvis, Brice A.; Paulson, Andrew E.; Koley, Somnath; Kang, Jinling; Lu, Chaofu; Allen, Doug K.; Lee, Young-Jin; Sedbrook, John C.; Durrett, Timothy P.

doi:10.1073/pnas.2412542121

Targeted engineering of camelina and pennycress seeds for ultrahigh accumulation of acetyl-TAG

Journal Article · Mon Nov 11 00:00:00 EST 2024 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.2412542121· OSTI ID:2476880

^[1]; ^[1]; ^[2]; ^[3]; ^[3]; Paulson, Andrew E. ^[4]; ^[5]; Kang, Jinling ^[6]; ^[6]; ^[7]; ^[4]; ^[3]; ^[1]

Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS 66506
Division of Biology, Kansas State University, Manhattan, KS 66506
School of Biological Sciences, Illinois State University, Normal, IL 61790
Department of Chemistry, Iowa State University, Ames, IA 50011
Donald Danforth Plant Science Center, St. Louis, MO 63132
Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717
Donald Danforth Plant Science Center, St. Louis, MO 63132, United States Department of Agriculture, Agricultural Research Service, St. Louis, MO 63132

Acetyl-TAG (3-acetyl-1,2-diacylglycerol), unique triacylglycerols (TAG) possessing an acetate group at the sn -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 producing Euonymus seeds. 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-edited FATTY 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 from Euonymus 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 in Euonymus seeds. 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.

View Journal Article

Research Organization:: Kansas State University, Manhattan, KS (United States)

Sponsoring Organization:: National Science Foundation (NSF); USDOE; USDOE Office of Science (SC), Biological and Environmental Research (BER); United States Department of Agriculture–National Institute of Food and Agriculture

Grant/Contract Number:: SC0023142

OSTI ID:: 2476880

Alternate ID(s):: OSTI ID: 2477973

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 47 Vol. 121; ISSN 0027-8424

Publisher:: Proceedings of the National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: English

References (38)

Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method Livak, Kenneth J.; Schmittgen, Thomas D. Methods, Vol. 25, Issue 4 https://doi.org/10.1006/meth.2001.1262	journal	December 2001
Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants Ma, Fangfang; Jazmin, Lara J.; Young, Jamey D. Methods in Molecular Biology, p. 167-194 https://doi.org/10.1007/978-1-4939-7225-8_12	book	August 2017
(S)-1,2-diacyl-3-acetins: Optically active triglycerides fromEuonymus verrucosus seed oil Kleiman, R.; Miller, R. W.; Earle, F. R. Lipids, Vol. 2, Issue 6 https://doi.org/10.1007/BF02533174	journal	November 1967
Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation Lu, Chaofu; Kang, Jinling Plant Cell Reports, Vol. 27, Issue 2 https://doi.org/10.1007/s00299-007-0454-0	journal	September 2007
Camelina sativa, an oilseed at the nexus between model system and commercial crop Malik, Meghna R.; Tang, Jihong; Sharma, Nirmala Plant Cell Reports, Vol. 37, Issue 10 https://doi.org/10.1007/s00299-018-2308-3	journal	June 2018
A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa Snapp, Anna R.; Kang, Jinling; Qi, Xiaoli Planta, Vol. 240, Issue 3 https://doi.org/10.1007/s00425-014-2122-2	journal	July 2014
Towards the production of high levels of eicosapentaenoic acid in transgenic plants: the effects of different host species, genes and promoters Cheng, Bifang; Wu, Guohai; Vrinten, Patricia Transgenic Research, Vol. 19, Issue 2 https://doi.org/10.1007/s11248-009-9302-z	journal	July 2009
Rapid Quantification of Low-Viscosity Acetyl-Triacylglycerols Using Electrospray Ionization Mass Spectrometry Bansal, Sunil; Durrett, Timothy P. Lipids, Vol. 51, Issue 9 https://doi.org/10.1007/s11745-016-4179-0	journal	August 2016
Content and Fatty Acid Composition of Neutral Acylglycerols in Euonymus Fruits Sidorov, Roman A.; Zhukov, Anatoly V.; Pchelkin, Vasily P. Journal of the American Oil Chemists' Society, Vol. 91, Issue 5 https://doi.org/10.1007/s11746-014-2425-2	journal	February 2014
Seed dormancy and germination Koornneef, Maarten; Bentsink, Leónie; Hilhorst, Henk Current Opinion in Plant Biology, Vol. 5, Issue 1 https://doi.org/10.1016/S1369-5266(01)00219-9	journal	February 2002
Using lipidomics to reveal details of lipid accumulation in developing seeds from oilseed rape (Brassica napus L.) Woodfield, Helen K.; Cazenave-Gassiot, Amaury; Haslam, Richard P. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, Vol. 1863, Issue 3 https://doi.org/10.1016/j.bbalip.2017.12.010	journal	March 2018
Camelina sativa: An ideal platform for the metabolic engineering and field production of industrial lipids Bansal, Sunil; Durrett, Timothy P. Biochimie, Vol. 120 https://doi.org/10.1016/j.biochi.2015.06.009	journal	January 2016
Field production, purification and analysis of high-oleic acetyl-triacylglycerols from transgenic Camelina sativa Liu, Jinjie; Tjellström, Henrik; McGlew, Kathleen Industrial Crops and Products, Vol. 65 https://doi.org/10.1016/j.indcrop.2014.11.019	journal	March 2015
Oil content of Arabidopsis seeds: The influence of seed anatomy, light and plant-to-plant variation Li, Yonghua; Beisson, Fred; Pollard, Mike Phytochemistry, Vol. 67, Issue 9 https://doi.org/10.1016/j.phytochem.2006.02.015	journal	May 2006
Mutagenesis of the FAE1 genes significantly changes fatty acid composition in seeds of Camelina sativa Ozseyhan, Mehmet E.; Kang, Jinling; Mu, Xiaopeng Plant Physiology and Biochemistry, Vol. 123 https://doi.org/10.1016/j.plaphy.2017.11.021	journal	February 2018
Reconstitution of EPA and DHA biosynthesis in Arabidopsis: Iterative metabolic engineering for the synthesis of n−3 LC-PUFAs in transgenic plants Ruiz-Lopez, Noemi; Haslam, Richard P.; Usher, Sarah L. Metabolic Engineering, Vol. 17 https://doi.org/10.1016/j.ymben.2013.03.001	journal	May 2013
Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana Rossak, Maren; Smith, Mark; Kunst, Ljerka Plant Molecular Biology, Vol. 46, Issue 6, p. 717-725 https://doi.org/10.1023/A:1011603923889	journal	August 2001
Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid Walsh, Terence A.; Bevan, Scott A.; Gachotte, Daniel J. Nature Biotechnology, Vol. 34, Issue 8 https://doi.org/10.1038/nbt.3585	journal	July 2016
Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase Bansal, Sunil; Durrett, Timothy P. Bioscience Reports, Vol. 36, Issue 6 https://doi.org/10.1042/BSR20160277	journal	November 2016
dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics Neff, Michael M.; Neff, Joseph D.; Chory, Joanne The Plant Journal, Vol. 14, Issue 3 https://doi.org/10.1046/j.1365-313X.1998.00124.x	journal	May 1998
A distinct DGAT with sn-3 acetyltransferase activity that synthesizes unusual, reduced-viscosity oils in Euonymus and transgenic seeds Durrett, T. P.; McClosky, D. D.; Tumaney, A. W. Proceedings of the National Academy of Sciences, Vol. 107, Issue 20, p. 9464-9469 https://doi.org/10.1073/pnas.1001707107	journal	May 2010
Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus Andre, C.; Haslam, R. P.; Shanklin, J. Proceedings of the National Academy of Sciences, Vol. 109, Issue 25 https://doi.org/10.1073/pnas.1204604109	journal	June 2012
Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly Bates, P. D.; Johnson, S. R.; Cao, X. Proceedings of the National Academy of Sciences, Vol. 111, Issue 3 https://doi.org/10.1073/pnas.1318511111	journal	January 2014
An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids Koley, Somnath; Chu, Kevin L.; Gill, Saba S. Journal of Experimental Botany, Vol. 73, Issue 9 https://doi.org/10.1093/jxb/erac062	journal	February 2021
A simple and rapid method for the preparation of plant genomic DNA for PCR analysis Edwards, K.; Johnstone, C.; Thompson, C. Nucleic Acids Research, Vol. 19, Issue 6 https://doi.org/10.1093/nar/19.6.1349	journal	January 1991
Simultaneous Targeting of Multiple Gene Homeologs to Alter Seed Oil Production in Camelina sativa Aznar-Moreno, J. A.; Durrett, T. P. Plant and Cell Physiology, Vol. 58, Issue 7 https://doi.org/10.1093/pcp/pcx058	journal	April 2017
Alteration of Seed Fatty Acid Composition by an Ethyl Methanesulfonate-Induced Mutation in Arabidopsis thaliana Affecting Diacylglycerol Acyltransferase Activity Katavic, V.; Reed, D. W.; Taylor, D. C. Plant Physiology, Vol. 108, Issue 1 https://doi.org/10.1104/pp.108.1.399	journal	May 1995
Development Defects of Hydroxy-Fatty Acid-Accumulating Seeds Are Reduced by Castor Acyltransferases Lunn, Daniel; Smith, Gracen A.; Wallis, James G. Plant Physiology, Vol. 177, Issue 2 https://doi.org/10.1104/pp.17.01805	journal	April 2018
Spatial Mapping of Lipids at Cellular Resolution in Embryos of Cotton Horn, Patrick J.; Korte, Andrew R.; Neogi, Purnima B. The Plant Cell, Vol. 24, Issue 2 https://doi.org/10.1105/tpc.111.094581	journal	February 2012
Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity, freezing point and calorific value Liu, Jinjie; Rice, Adam; McGlew, Kathleen Plant Biotechnology Journal, Vol. 13, Issue 6 https://doi.org/10.1111/pbi.12325	journal	March 2015
Molecular tools enabling pennycress ( Thlaspi arvense ) as a model plant and oilseed cash cover crop McGinn, Michaela; Phippen, Winthrop B.; Chopra, Ratan Plant Biotechnology Journal, Vol. 17, Issue 4 https://doi.org/10.1111/pbi.13014	journal	October 2018
Enhancing the accumulation of eicosapentaenoic acid and docosahexaenoic acid in transgenic Camelina through the CRISPR‐Cas9 inactivation of the competing FAE1 pathway Han, Lihua; Haslam, Richard P.; Silvestre, Susana Plant Biotechnology Journal, Vol. 20, Issue 8 https://doi.org/10.1111/pbi.13876	journal	July 2022
Membrane topology and identification of key residues of Ea DAcT, a plant MBOAT with unusual substrate specificity Tran, Tam N. T.; Shelton, Jennifer; Brown, Susan The Plant Journal, Vol. 92, Issue 1 https://doi.org/10.1111/tpj.13636	journal	August 2017
Expression of a high‐activity diacylglycerol acetyltransferase results in enhanced synthesis of acetyl‐TAG in camelina seed oil Alkotami, Linah; Kornacki, Catherine; Campbell, Shahna The Plant Journal, Vol. 106, Issue 4 https://doi.org/10.1111/tpj.15210	journal	March 2021
Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes Hutcheon, Carolyn; Ditt, Renata F.; Beilstein, Mark BMC Plant Biology, Vol. 10, Issue 1 https://doi.org/10.1186/1471-2229-10-233	journal	January 2010
Rapid analysis of seed size in Arabidopsis for mutant and QTL discovery Herridge, Rowan P.; Day, Robert C.; Baldwin, Samantha Plant Methods, Vol. 7, Issue 1 https://doi.org/10.1186/1746-4811-7-3	journal	January 2011
Generating Pennycress (Thlaspi arvense) Seed Triacylglycerols and Acetyl-Triacylglycerols Containing Medium-Chain Fatty Acids Esfahanian, Maliheh; Nazarenus, Tara J.; Freund, Meghan M. Frontiers in Energy Research, Vol. 9 https://doi.org/10.3389/fenrg.2021.620118	journal	January 2021
Heterogeneous Distribution of Erucic Acid in Brassica napus Seeds Lu, Shaoping; Aziz, Mina; Sturtevant, Drew Frontiers in Plant Science, Vol. 10 https://doi.org/10.3389/fpls.2019.01744	journal	January 2020

Similar Records

Molecular tools enabling pennycress (Thlaspi arvense) as a model plant and oilseed cash cover crop

Journal Article · Tue Sep 18 20:00:00 EDT 2018 · Plant Biotechnology Journal · OSTI ID:1611817

Related Subjects

09 BIOMASS FUELS
biofuels
oil seeds
synthetic biology
triacylglycerols

Targeted engineering of camelina and pennycress seeds for ultrahigh accumulation of acetyl-TAG

Citation Formats

References (38)

Similar Records

Related Subjects