Progress 10/01/10 to 09/30/15
Outputs
Target Audience: The project provides outcomes of relevance to (1) U.S. farmers for higher value oilseed products, (2)oilseed processors for vegetable oils with expanded functionality for bio-based lubricant and biodiesel markets, (3) lubricant manufacturers for sustainable and biodegradable lubricant products, (4) biochemists, geneticists and metabolic engineers for basic knowledge of the regulation and metabolic plasticity of oil biosynthetic pathways in camelina, (5) plant breeders who require genetic information for camelina crop improvement, and (6) chemical and agricultural engineers and chemists for the design of new vegetable oil formulations for bio-based lubricants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has involved undergraduate student, graduate student, and postdoctoral researchers. They have received scientific and career mentorship and multidisciplinary training in biochemistry, lipid analytical chemistry, functional genomics, and plant biotechnology. How have the results been disseminated to communities of interest?The results have been disseminated by publication in international, peer-reviewed journals. Portions of the research were disseminated through oral and poster presentations at the following meetings and seminars: (1) Gordon Research Conference on Plant Lipids, Galveston, TX, February 2-6, 2015; (2) European Symposium on Plant Lipids, Rothamsted, UK, July 5 2015; (3) UNL Department of Agronomy & Horticulture, Lincoln, NE, May 10, 2015; (4)Huazhong Agricultural University, Wuhan, China, May 31, 2015; (5) Asian Symposium on Plant Lipids, Singapore, December 2, 2015; and (6)Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, China, December 11, 2015. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The research was directed at developing the emerging oilseed crop camelina as a platform for producing novel or improved vegetable oils for industrial uses including biofuels and bio-based lubricants through the application of biotechnology. Camelina is especially attractive for these applications because it is has limited use in the US as a food crop and can be easily and quickly engineered with existing biotechnological methods. During the past year, research addressed at Aim 1 mined transcriptomes of seeds of Cuphea species for genes associated with short- and medium-chain-length fatty acids for use in metabolic engineering of camelina.Seeds of members of the genus Cuphea accumulate medium-chain fatty acids (MCFAs; 8:0-14:0). MCFA- and palmitic acid- (16:0) rich vegetable oils have received attention for jet fuel production, given their similarity in chain length to Jet A fuel hydrocarbons. Studies were conducted to test genes, including those from Cuphea, for their ability to confer jet fuel-type fatty acid accumulation in seed oil of the emerging biofuel crop Camelina sativa. Transcriptomes from Cuphea viscosissima and Cuphea pulcherrima developing seeds that accumulate >90% of C8 and C10 fatty acids revealed three FatB thioesterase cDNAs (CpuFatB3, CvFatB1, and CpuFatB4) expressed predominantly in seeds and structurally divergent from typical FatB thioesterases that release 16:0 from acyl carrier protein (ACP). Expression of CpuFatB3 and CvFatB1 resulted in Camelina oil with capric acid (10:0), and CpuFatB4 expression conferred myristic acid (14:0) production and increased 16:0. Co-expression of combinations of previously characterized Cuphea and California bay FatBs produced Camelina oils with mixtures of C8-C16 fatty acids, but amounts of each fatty acid were less than obtained by expression of individual FatB cDNAs. Increases in lauric acid (12:0) and 14:0, but not 10:0, in Camelina oil and at the sn-2 position of triacylglycerols resulted from inclusion of a coconut lysophosphatidic acid acyltransferase specialized for MCFAs. RNA interference (RNAi) suppression of Camelina β-ketoacyl-ACP synthase II, however, reduced 12:0 in seeds expressing a 12:0-ACP-specific FatB. Camelina lines presented here provide platforms for additional metabolic engineering targeting fatty acid synthase and specialized acyltransferases for achieving oils with high levels of jet fuel-type fatty acids.Seed-specific acyltransferases with in vivo selectivity for saturated (10:0) fatty acids were also identified in Cuphea seed transcriptomes. These acylytransferases included lysophosphatidic acid acyltransferases(LPATs) and diacyglycerol acyltransferases (DGATs). Expression of these LPATs and DGATs together with a 10:0-producing FatB in camelina seeds resulted in the accumulation of seed oils with up to 40% 10:0 in the sn-2 position oftriacylglycerols (TAGs). By comparison, 10:0 was excluded from the sn-2 position of TAGs in wild-type seeds. These acylytransferasesprovide tools for understanding the structural basis for LPAT and DGAT substrate specificity and should facilitate the engineering of different oil functionalities. In support of Aim 3, high oleic acid camelina lines planted in field sites in Mead, NE and Bozeman, MT had seeds oils with up to 74% oleic acid and approximately 35% oil. Oleic acid levels in these lines have been stable in thetwo field sites in the 2013, 2014, and 2015 growing seasons. The high oleic camelina oil with low linolenic acid content (<3% linolenic acid) from the engineered lines had a measured oxidative stability index of 29 hours versus 3.9 hours of convential camelina oil with ~30% linolenic acid.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Kim HJ, Silva JE, Vu HS, Mockaitis K, Nam JW, Cahoon EB (2015) Toward production of jet fuel functionality in oilseeds: identification of FatB acyl-acyl carrier protein thioesterases and evaluation of combinatorial expression strategies in Camelina seeds. Journal of Experimental Botany 66: 4251-4265.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Shockey J, Mason C, Gilbert M, Cao H, Li X, Cahoon E, Dyer J (2015) Development and analysis of a highly flexible multi-gene expression system for metabolic engineering in Arabidopsis seeds and other plant tissues. Plant Molecular Biology 89: 113-126.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Huai D, Zhang Y, Zhang C, Cahoon EB, Zhou Y (2015) Combinatorial effects of fatty acid elongase enzymes on nervonic acid production in Camelina sativa. PLoS One 10: e0131755.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Liu J, Tjellstr�m H, McGlew K, Shaw V, Rice A, Simpson J, Kosma D, Ma W, Yang W, Strawsine M, Cahoon E, Durrett TP, Ohlrogge J (2014) Field production, purification and analysis of high-oleic acetyl-triacylglycerols from transgenic Camelina sativa. Industrial Crops and Products 65:259-268.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Belayneh HD, Wehling RL, Cahoon E, Ciftci ON (2015) Extraction of omega-3-rich oil from Camelina sativa seed using supercritical carbon dioxide. Journal of Supercritical Fluids 104: 153-159.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2015
Citation:
Kim HJ, Silva JE, Iskandarov U, Andersson M, Cahoon RE, Mockaitis K, Cahoon EB (2015) Structurally divergent lysophosphatidic acid acyltransferases with high selectivity for saturated medium chain fatty acids from Cuphea seeds. Plant Journal In Press.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The project provides outcomes of relevance to (1) U.S. farmers for higher value oilseed products, (2)oilseed processors for vegetable oils with expanded functionality for bio-based lubricant and biodiesel markets, (3) lubricant manufacturers for sustainable and biodegradable lubricant products, (4) biochemists, geneticists and metabolic engineers for basic knowledge of the regulation and metabolic plasticity of oil biosynthetic pathways in camelina, (5) plant breeders who require genetic information for camelina crop improvement, and (6) chemical and agricultural engineers and chemists for the design of new vegetable oil formulations for bio-based lubricants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has involved undergraduate student, graduate student, and postdoctoral researchers. They have received scientific and career mentorship and multidisciplinary training in biochemistry, lipid analytical chemistry, functional genomics, and plant biotechnology. Aspects of the project have also been presented in outreach instruction to high school students as a component of the University of Nebraska-Lincoln Annual Women in Science meeting. How have the results been disseminated to communities of interest? The results have been disseminated by publication in international, peer-reviewed journals. Camelina transcriptomic data has also been disseminated through searchable databases on the project website www.camelinagenome.org. Furthermore, portions of the research were disseminated through oral and poster presentations at the following meetings: (1) 53rd Annual Meeting of the Phytochemical Society of North America, Raleigh, NC, "Camelina: A Designer Oilseed Crop for Metabolic Engineering of Advanced Biofuels and Bio-Based Lubricants", August 12, 2014; (2) Society for Industrial Microbiology & Biotechnology Annual Meeting, Saint Louis, MO, "Development of Camelina as Oilseed Platform for Advanced Metabolic Engineering and Synthetic Biology", July 22, 2014; (3) 21st International Symposium on Plant Lipids, Guelph, Ontario, Canada, "Metabolic Engineering of Vitamin E synthesis for Enhance Seed Oil Oxidative Stability", July 9, 2014, (4) 4th Pan-American Congress on Plants and BioEnergy, Guelph, Ontario, Canada, "Biotechnological Development of Vegetable Oil-Based Advanced Biofuels, June 5, 2014. What do you plan to do during the next reporting period to accomplish the goals? In support of Specific Aim 1, studies will be conducted to evaluate the oxidative stability and other functional properties of oils and biodiesel generated from high oleic acid and high omega-7 camelina lines. This aim of these studies is to demonstrate the enhanced value of engineered camelina lines for bio-based lubricants and biodiesel. Research will also be initiated to enhance the oxidative stability of camelina oil by using metabolic engineering strategies that overcome bottlenecks for enhanced vitamin E antioxidant production. In support of Specific Aim 3, field evaluation of new high oleic acid camelina lines will be conducted in biotech crop-dedicated field sites in Mead, NE and Bozeman, MT to determine whether the strategies used in the generation of these lines affect agronomic properties and yield and to determine whether oleic acid levels in seeds of these lines are stable under field conditions in two differing climatic locations.
Impacts
What was accomplished under these goals?
The research was directed at developing the emerging oilseed crop camelina as a platform for producing novel or improved vegetable oils for industrial uses including biofuels and bio-based lubricants through the application of biotechnology. Camelina is especially attractive for these applications because it is has limited use in the US as a food crop and can be easily and quickly engineered with existing biotechnological methods. During the past year, a complex metabolic pathway was introduced into camelina for expression of genes that confer the production of a novel oil that has improved superior oxidation properties for biodiesel and can also be used as a feedstock for producing polyurethane. In addition, a pathway was engineered into camelina to sustainably generate biodegradable plastics. As a further component of the project, camelina varieties engineered for producing improved vegetable oils for biodiesel were found to have yields comparable to those of non-engineered camelina in field tests conducted in Nebraska and Montana. These findings demonstrate the utility of camelina as a dedicated oilseed crop for producing bio-based industrial traits without concerns of compromising food and animal feed value-chains. Progress for Aim 1, the engineering of fatty acid composition of camelina seeds for improved lubricant functionaliites, included the successful complex metabolic engineering of omega-7 fatty acid biosynthetic pathway in camelina seeds. Seed oils enriched in omega-7 monounsaturated fatty acids, including palmitoleic acid (16:1?9) and cis-vaccenic acid (18:1?11), have nutraceutical and industrial value for polyethylene production, lubricants, and biofuels. Existing oilseed crops accumulate only small amounts (<2%) of these novel fatty acids in their seed oils. A strategy was developed and demonstrated for enhancing production of omega-7 monounsaturated fatty acids in camelina that is dependent on redirection of metabolic flux from the typical ?9 desaturation of stearoyl (18:0)-acyl carrier protein (ACP) to ?9 desaturation of palmitoyl (16:0)-acyl carrier protein (ACP) and coenzyme A (CoA). This was achieved by seed-specific co-expression of a mutant ?9-acyl-ACP and an acyl-CoA desaturase with high specificity for 16:0-ACP and CoA substrates, respectively. Seed oils with ~17% omega-7 monounsaturated fatty acids were obtained. Further increases in omega-7 fatty acid accumulation to 60-65% of the total fatty acids in camelina seeds were achieved by inclusion of seed-specific suppression of 3-keto-acyl-ACP synthase II and the FatB 16:0-ACP thioesterase genes to increase substrate pool sizes of 16:0-ACP for the ?9-acyl-ACP desaturase and by blocking C18 fatty acid elongation. Seeds from these lines also had total saturated fatty acids reduced to ~5% of the seed oil versus ~12% in seeds of nontransformed plants. Consistent with accumulation of triacylglycerol species with shorter fatty acid chain lengths and increased monounsaturation, seed oils from engineered lines had marked shifts in thermotropic properties that may be of value for biofuel applications. Also in support of Aim 1, a metabolic engineering strategy was developed and used for procuction of the biodegradable plastic poly-3-hydroxybutyrate (PHB) production in plastids camelina seeds. This study compared levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed-specific promoters for expression of transgenes. Three bacterial genes for PHB biosynthesis and containing added plastid targeting sequences were used for camelina transformation. Camelina lines with up to 15% polymer in mature T2 seeds. High molecular weight polymer was produced with weight-averaged molecular weights varying between 600000 and 1500000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T4 seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed-based platform for PHB production. Research for Aim 2 was concluded in 2013. Aim 3 involves evaluation of the agromomic performance of camelina lines egineered for altered oil composition. For these studies, two camelina lines engineered for seed oils with oleic acid content >65% of seed weight were field tested in Mead, NE and Bozeman, MT in 2014. These lines, designated Oleic2 and Oleic3, had polyunsaturated fatty acid content reduced from ~55% in seeds of non-engeinned plants to ~17% in seeds of the high oleic acid lines. In addition, the linolenic acid content of seed oils from the Oleic3 line was ~4% compared to ~35% in seed oils from non-engineered lines. In the NE and MT field sites, oleic acid content of seeds from Oleic2 and Oleic3 lines was consistently around 70% of the total fatty acids, and oil content of seeds from these lines were not significantly different than those from non-engineered lines. This was the third year of field testing of Oleic 2 and the second year of field testing for Oleic 3. Notably, the oil content and quality of these engineered lines appears to be stable over multiple generations in the field and in the greenhouse. Results to date, indicate that high oleic acid traits in camelina are suitable for commercial production to generate oils with enhanced oxidative stability for lubricants and biodiesel. Research for Aim 4 was completed in 2013. Transcriptomic information from these studies is publically available at the project website: www.camelinagenome.org. These data are avaliable to facilitate the genetic improvement of camelina.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Iskandarov U, Kim HJ, Cahoon EB (2014) Chapter 8: Camelina: An emerging oilseed platform for improved biofuels and bio-based materials. In Plants and BioEnergy, Advances in Plant Biology 4, M. C. McCann, N.C. Carpita M.S. Buckeridge, eds. (New York: Springer), pp. 131-140.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Nguyen HT, Park H, Koster KL, Cahoon RE, Shanklin J, Clemente TE, Cahoon EB (2014) Redirection of metabolic flux for high levels of omega-7 monounsaturated fatty acid accumulation in camelina seeds. Plant Biotechnology Journal. Published online July 26, 2014.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Malik MR, Yang W, Patterson N, Tang J, Wellinghoff RL Preuss ML, Burkitt C, Sharma N, Ji Y, Jez JM, Peoples OP, Jaworski JG, Cahoon EB, Snell KD (2014) Production of high levels of poly-3-hydroxybutyrate in plastids of Camelina sativa seeds. Plant Biotechnology Journal. Published online November 21, 2014.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Napier JA, Haslam RP, Beaudoin F, Cahoon EB (2014) Understanding and manipulating plant lipid composition: Metabolic engineering leads the way. Current Opinion in Plant Biology 19: 68-75.
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Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The project provides outcomes of relevance to (1) U.S. farmers for higher value oilseed products, (2)oilseed processors for vegetable oils with expanded functionality for bio-based lubricant and biodiesel markets, (3) lubricant manufacturers for sustainable and biodegradable lubricant products, (4) biochemists, geneticists and metabolic engineers for basic knowledge of the regulation and metabolic plasticity of oil biosynthetic pathways in camelina, (5) plant breeders who require genetic information for camelina crop improvement, and (6) chemical and agricultural engineers and chemists for the design of new vegetable oil formulations for bio-based lubricants. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has involved undergraduate student, graduate student, and postdoctoral researchers. They have received scientific and career mentorship and multidisciplinary training in biochemistry, lipid analytical chemistry, functional genomics, and plant biotechnology. Aspects of the project have also been presented in outreach instruction to high school students as a component of the University of Nebraska-Lincoln Annual Women in Science meeting. How have the results been disseminated to communities of interest? The results have been dissminated by publication in international, peer-reviewed journals. Camelina transcriptomic data has also been disseminated through searchable databases on the project website www.camelinagenome.org. Furthermore, portions of the research were disseminated through a poster presentation at the 2013 Gordon Research Conferernce on Plant Lipids. What do you plan to do during the next reporting period to accomplish the goals? We will intiate studies to generate high-value protein co-products in camelina seeds, with the goal of increasing the overall value of the seed. This is needed to improve the economic viability of camelina as an oilseed crop platform for complex metabolic engineering of industrial oil traits. In addition, metabolic engineering studies will continue that are aimed at enhancing the content of vitamin E antioxidants in camelina seeds for oxidative stabilization of oil traits. Furthermore, functionality studies will be conducted on seed oil extracted from field grown high oleic acid camelina lines to determine the efficacy of this trait for high temperature lubricant and biofuel uses.
Impacts
What was accomplished under these goals?
A transcriptome reference was built from 2047 Sanger ESTs and more than 2million 454-derived sequence reads, representing genes expressed in developing camelina seeds. The transcriptome of approximately 60K transcripts from 22597 putative genes includes camelina homologues of nearly all known seed-expressed genes, suggesting a high level of completeness and usefulness of the reference. These sequences included candidates for 12S (cruciferins) and 2S (napins) seed storage proteins (SSPs) and nearly all known lipid genes, which have been compiled into an accessible database. To demonstrate the utility of the transcriptome for seed quality modification, seed-specific RNAi lines deficient in napins were generated by targeting 2S SSP genes, and high oleic acid oil lines were obtained by targeting FATTY ACID DESATURASE 2 (FAD2) and FATTY ACID ELONGASE 1 (FAE1). The high sequence identity between Arabidopsis thaliana and camelina genes was also exploited to engineer high oleic lines by RNAi with Arabidopsis FAD2 and FAE1 sequences. It is expected that these transcriptomic data will be useful for breeding and engineering of additional camelina seed traits and for translating findings from the model Arabidopsis to an oilseed crop. Engineering compositional changes in oilseeds is typically accomplished by introducing new enzymatic step(s) and/or by blocking or enhancing an existing enzymatic step(s) in a seed-specific manner. However, in practice, the amounts of lipid species that accumulate in seeds are often different from what one would predict from enzyme expression levels, and these incongruences may be rooted in an incomplete understanding of the regulation of seed lipid metabolism at the cellular/tissue level. We showed by mass spectrometry imaging approaches that triacylglycerols and their phospholipid precursors are distributed differently within cotyledons and the hypocotyl/radicle axis in embryos of camelina, indicating tissue-specific heterogeneity in triacylglycerol metabolism. Phosphatidylcholines and triacylglycerols enriched in linoleic acid (C18:2) were preferentially localized to the axis tissues, whereas lipid classes enriched in gadoleic acid (C20:1) were preferentially localized to the cotyledons. Manipulation of seed lipid compositions by heterologous over-expression of an acyl-acyl carrier protein thioesterase, or by suppression of fatty acid desaturases and elongases, resulted in new overall seed storage lipid compositions with altered patterns of distribution of phospholipid and triacylglycerol in transgenic embryos. Our results reveal previously unknown differences in acyl lipid distribution in camelina embryos, and suggest that this spatial heterogeneity may or may not be able to be changed effectively in transgenic seeds depending upon the targeted enzyme(s)/pathway(s). Further, these studies point to the importance of resolving the location of metabolites in addition to their quantities within plant tissues.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Nguyen HT, Silva JE, Podicheti R, Macrander J, Yang W, Nazarenus TJ, Nam JW, Jaworski JG, Lu C, Scheffler BE, Mockaitis K, Cahoon EB (2013) Camelina seed transcriptome: a tool for meal and oil improvement and translational research. Plant Biotechnology J 11: 759-769.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Horn PJ, Silva JE, Anderson D, Fuchs J, Borisjuk L, Nazarenus TJ, Shulaev V, Cahoon EB, Chapman KD (2013) Imaging heterogeneity of membrane and storage lipids in transgenic Camelina sativa seeds with altered fatty acid profiles. Plant Journal 76: 138-150.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kim S, Yamaoka Y, Ono H, Kim H, Shim D, Maeshima M, Martinoia E, Cahoon EB, Nishida I, Lee Y (2013) AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum. Proc. Natl. Acad. Sci. USA 110:773-778
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Progress was made toward the development of camelina that produces high value wax ester seed oils. Wax esters consist of a fatty acid bound to a fatty alcohol and have superior properties over conventional vegetable oils as high temperature lubricants. Camelina lines were generated that accumulate 20% to 30% of their seed oils in the form of wax esters by introduction of genes for wax synthase (WS), fatty acyl-CoA reductase (FAR), and fatty acid elongase 1(FAE1) from jojoba under control of seed-specific promoters. The wax esters produced contained carbon chain lengths of primarily C46 and C48. In these experiments, the jojoba FAE1 displayed broad substrate specificity resulting in the production of a mixture of wax esters containing saturated, monounsaturated, and polyunsaturated fatty acids and fatty alcohols. A more uniform wax ester composition with a preponderance of monounsaturated C22 and C24 fatty acids and fatty alcohols was obtained by replacement of the jojoba FAE1 with a FAE1 from Lunaria alba, which has greater specificity for elongation of oleic acid (18:1) to 22:1 and 24:1. Using this background, camelina lines with seed wax esters highly enriched in monounsaturated fatty acids and fatty alcohols were generate by seed-specific silencing of the FAD2 gene, which is responsible for conversion of 18:1 to the polyunsaturated linoleic acid (18:2). These lines contained total wax ester content of 30% to 35% of the seed oil. Results from these studies were disseminated in a poster presentation at 2012 International Plant Lipid Symposium in Seville, Spain and in an oral presentation to the European Commission Framework Programme 7 ICON General Assembly meeting in Seville, Spain. PARTICIPANTS: The research on during the reporting period was conducted primarily by Tara Nazarenus (University of Nebraska-Lincoln), a research technician in the UNL Department of Biochemistry. Also participating in this research was Dongxin Hua, a visiting graduate student from the Huazhong Agricultural University in Wuhan, China. The project was conducted collaboratively with the European Commission Framework Programme 7 project: ICON: Industrial Crops Producing Added Value Oils for Novel Chemicals, led by Professor Sten Stymne, Swedish Agricultural University. TARGET AUDIENCES: The project provides outcomes of relevance to (1) U.S. farmers for higher value oilseed products, (2) oilseed processors for vegetable oils with expanded functionality for bio-based lubricant and biodiesel markets, (3) lubricant manufacturers for sustainable and biodegradable lubricant products, (4) biochemists, geneticists and metabolic engineers for basic knowledge of the regulation and metabolic plasticity of oil biosynthetic pathways in camelina and (5) chemical and agricultural engineers and chemists for the design of new vegetable oil formulations for bio-based lubricants. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The wax ester-type oil generated in camelina is a novel type of seed oil that is valued for its stability as a high temperature lubricant. The primary source of wax esters for industrial applications was historically sperm whales, but global bans on whale hunting have virtually eliminated this source of wax esters. Our camelina lines therefore represent an advance toward generating renewable sources of wax esters in a temperate oilseed crop (camelina) that has only limited food use in North America. Our studies demonstrate the power of systematic metabolic engineering and biotechnology to generate wax esters enriched in monounsaturated C22 and C24 fatty acids and fatty alcohols that are not typically found in nature. It is expected that through functionality studies, the camelina oils generated will find uses in bio-based lubricant formulations. The project outputs have been produced by technicians, graduate students, and post-doctoral associates applying techniques of molecular biology and biotechnology. Access to state of the art growth chambers, greenhouses, and biotech field facilities also enabled the production of camelina with novel oil traits.
Publications
- Zhang C, Cahoon RE, Hunter SC, Chen M, Han J & Cahoon EB (2012) Genetic and bochemical basis for alternative routes of tocotrienol biosynthesis for enhanced vitamin E antioxidant production. Plant Journal In press.
- Eiamsa-Ard P, Kanjana-Opas A, Cahoon EB, Chodok P & Kaewsuwan S (2012) Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization. Appl Microbiol Biotechnol, In press
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: A major goal of this project is to develop camelina oil with improved fatty acid and oil compositions for biodiesel and bio-based lubricant applications through the use of metabolic engineering. To this end, we have succeeded in generating a number of novel oil compositional traits. These traits include: (1) a high oleic acid phenotype (65% to 70% oleic acid) achieved by seed-specific RNA interference (RNAi) suppression of FAD2 delta12 desaturase and FAE1 fatty acid elongase genes, (2) a high myristic acid oil(25% 14:0)obtained by seed-specific expression of the cuphea Thio14 FatB thioesterase, (3) a high lauric acid oil (25% to 30% 12:0)achieved by seed-specific co-expression of a California bay FatB thioesterase and a coconut lysophosphatidic acid acyltransferase, and (4)a high omega-7 monounsaturated oil (60% total of palmitoleic and vaccenic acids) obtained by seed-specific expression of a mutant stearoyl-ACP desaturase, C. elegans FAT5 desaturase and RNAi suppression of beta-ketoacyl-acyl carrier protein synthase II (KASII) and FAE1 fatty acid elongase. In addition, a seed transcriptome of developing camelina seeds was completed from which most of the known fatty acid biosynthetic and metabolic genes were identified. Results from oil metabolic engineering studes will be disseminated by publication in scientific journals and by presentations in scientific meetings. Camelina seed transcriptomic data will be made available on a publicly accessible website and by deposition of sequence information in GenBank. PARTICIPANTS: Dr. Tam Nguyen, Anji Reddy Konda, and Tara Nazarenus (University of Nebraska-Lincoln) have conducted metabolic engineering studies to develop novel fatty acid profiles and oil compositions in camelina. The project provides post-doctoral training to Dr. Tam Nguyen and graduate student training to Anji Reddy Konda. The development of bio-based lubricants involves collaboration with the European Commission Framework Programme 7 project: ICON: Industrial Crops Producing Added Value Oils for Novel Chemicals, led by Dr. Sten Stymne, Swedish Agricultural University. Next-generation DNA sequencing is conducted in collaboration with Dr. Keithanne Mockaitis, Director of Sequencing Operations, Indiana University Center for Genomics and Bioinformatics. TARGET AUDIENCES: The project provides outcomes of relevance to (1) U.S. farmers for higher value oilseed products, (2) oilseed processors for vegetable oils with expanded functionality for bio-based lubricant and biodiesel markets, (3) lubricant manufacturers for sustainable and biodegradable lubricant products, (4) biochemists, geneticists and metabolic engineers for basic knowledge of the regulation and metabolic plasticity of oil biosynthetic pathways in camelina and (5) chemical and agricultural engineers and chemists for the design of new vegetable oil formulations for bio-based lubricants and biodiesel. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The novel oils that have been generated in camelina will be subjected to functionality testing upon collection of sufficient amounts of engineered seeds for oil extraction. It is expected that the high oleic acid and high omega-7 monounsaturated acid camelina oils will have superior properties for biodiesel and bio-based lubricant applications compared to conventional camelina oil, which is rich in linolenic acid. Most notably, the high content of monounsaturated fatty acids will yield improved oxidative stability of the oil for lubricant applications and enhanced pour point properties and ignition quality for biodiesel. The high myristic and lauric acid oils may contribute to jet fuel-type functionalities. In addition, the camelina 454 transcriptomic data will likely be useful for efforts to improve seed quality traits by conventional breeding and biotechnological approaches. The project outputs have been produced by technicians, graduate students, and post-doctoral associates applying techniques of molecular biology and biotechnology. Access to state of the art growth chambers and greenhouses also enabled the production of camelina with improved oil traits.
Publications
- Lu C, Napier JA, Clemente TE, Cahoon EB (2011) New frontiers in oilseed biotechnology: meeting the growing global demand for vegetable oils for food, feed, biofuel, and industrial uses. Current Opinion in Biotechnology 22: 252-259.
- Collins-Silva J, Lu C, Cahoon EB (2011) Camelina: A designer biotech oilseed crop. Inform 22: 610-613.
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