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Enhancing the production of acetyl-triacylglycerols through metabolic engineering of the oilseed crop Camelina sativa
Get PDFMaster of ScienceBiochemistry and Molecular Biophysics Interdepartmental ProgramTimothy P. DurrettMany Euonymus species express an acetyltransferase enzyme in their seeds which catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol (DAG) producing unusual acetyl-1,2-diacyl-sn-glycerols (acetyl-TAG). The presence of the sn-3 acetate group gives acetyl-TAG with unique physical properties over regular triacylglycerol (TAG) found in vegetable oils. The useful characteristics of acetyl-TAG oil offer advantages for its use as emulsifiers, lubricants, and 'drop-in' biofuels. One enzyme, Euonymus alatus diacylglycerol acetyltransferase (EaDAcT), responsible for acetyl-TAG synthesis in nature was previously isolated from the seeds of Euonymus alatus (burning bush) and expressed in the oilseed crop Camelina sativa. Expression of EaDAcT successfully led to production of high levels of acetyl-TAG in camelina seeds. To further increase acetyl-TAG accumulation in transgenic camelina seeds, multiple strategies were examined in this study. Expression of a new acetyltransferase enzyme (EfDAcT) isolated from the seeds of Euonymus fortunei, which was previously shown to possess higher in vitro activity and in vivo acetyl-TAG levels compared to EaDAcT, increased acetyl-TAG accumulation by 20 mol%. Suppression of the endogenous competing enzyme DGAT1 further enhanced acetyl-TAG accumulation to 90 mol% in selected transgenic line. Studying the regulation of EfDAcT transcript, protein, and acetyl-TAG levels during seed development further provided new insights on the factors limiting acetyl-TAG accumulation
Remodeling of triacylglycerol synthesis in emerging oilseed crops: genetic engineering for high-value seed oil
No full textDoctor of PhilosophyBiochemistry and Molecular Biophysics Interdepartmental ProgramTimothy P. DurrettThe utilization of vegetable oils, which predominantly consist of triacylglycerols (TAG), for diverse industrial purposes is hindered by many challenges due to their unfavorable physical properties. To overcome this constraint, metabolic engineering studies have focused on modifying plant seeds to produce oil with improved physical properties in non-food crops. However, altering oil composition to generate novel TAG molecules has proven to be challenging due to pathway bottlenecks and their potential impact on plant growth and seed viability. One unique class of TAG, known as acetyl-1,2-diacyl-sn-glycerols (acetyl-TAG), naturally constitutes more than 90% of the seed oil in Euonymus species. Acetyl-TAG is characterized by a distinct structure in which an sn-3 acetate replaces the long acyl group found in regular TAG. Consequently, acetyl-TAG oil exhibits lower viscosity, making it advantageous for applications as an emulsifiers, lubricants, or "drop-in" biofuel. Previous attempts to produce acetyl-TAG in oilseed crops such as soybean (Glycine max), Camelina sativa, and pennycress (Thlaspi arvense) have only achieved an average of 45-80% acetyl-TAG. The primary objective of this study is to engineer acetyl-TAG production in oilseed crops to match the highest naturally occurring levels in Euonymus seeds. Furthermore, the study aims to investigate the impact of acetyl-TAG on plant growth, seed properties, seed lipids, and global gene expression. The non-food crops camelina and pennycress were chosen due to their ease of transformation using an Agrobacterium-mediated floral-dip method. To achieve the desired outcome, multiple strategies were employed. Initially, an acetyltransferase enzyme (EfDAcT) from Euonymus fortunei seeds, known for its higher activity compared to the previously used Euonymus alatus enzyme (EaDAcT), was introduced. Subsequently, the endogenous TAG-synthesizing enzyme DGAT1, which competes with EfDAcT for the diacylglycerol (DAG) substrate, was inhibited. Lastly, EfDAcT was expressed in FATTY ACID ELONGASE1 (FAE1) mutant lines to enhance acetyl-CoA availability for acetyl-TAG synthesis while eliminating very long-chain fatty acids (VLCFA), which are unfavorable substrates for EfDAcT. Using these strategies, transgenic plants capable of producing 98% acetyl-TAG in pennycress seeds and 93% acetyl-TAG in camelina seeds were generated. Compared to wild-type seeds, transgenic seeds exhibited higher accumulation of TAG and polar lipids, while maintaining comparable or slightly lower levels of total fatty acid content. Seeds that produced high levels of acetyl-TAG showed a slight delay in germination with no significant impact on plant growth. Global transcript levels in camelina transgenic seeds indicated changes in gene expression, including those involved in flavonoid biosynthesis and hormone response. Overall, this study demonstrates the feasibility of achieving nearly complete alteration of the type of oil synthesized by a plant and offer valuable insights into the genetic and metabolic mechanisms underlying lipid biosynthesis
Targeted engineering of camelina and pennycress seeds for ultrahigh accumulation of acetyl-TAG
Get PDFAcetyl-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.This article is published as Alkotami, Linah, Dexter J. White, Kathleen M. Schuler, Maliheh Esfahanian, Brice A. Jarvis, Andrew E. Paulson, Somnath Koley et al. "Targeted engineering of camelina and pennycress seeds for ultrahigh accumulation of acetyl-TAG." Proceedings of the National Academy of Sciences 121, no. 47 (2024): e2412542121. doi: https://doi.org/10.1073/pnas.241254212
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Increasing Oil Production in Camelina sativa Engineered to Synthesize Unusual Lipids
Get PDFDifferent Euonymus species naturally produce acetyl-triacylglycerol (acTAG) in their seed oil. AcTAG are unusual structures of the triacylglycerols (TAG) typically found in vegetable oil, and have lower freezing points and viscosity compared to regular vegetable oil. Seed oil containing AcTAG can be used as biodiesel, plasticizers, and other alternatives to petroleum-based products. AcTAG can be synthesized in Camelina sativa by expressing key enzymes from Euonymus and by using RNA interference (RNAi) to suppress competing camelina biochemical pathways. Previous observations of high acetyl-TAG camelina lines have shown an increase in overall TAG molecules, but lower fatty acid levels compared to wild-type seeds. It is hypothesized the decrease in overall fatty acid content is due to a limited amount of glycerol-3-phosphate (G3P) backbone. To increase G3P, C. sativa seeds were transformed to overexpress the gene that encodes glycerol-3-phosphate dehydrogenase (GPD1), which converts dihydroxyacetone phosphate (DHAP) to G3P. By increasing G3P synthesis, the fatty acid content is expected to increase to wild-type content or higher. C. sativa seeds were collected from third-generation homozygous transgenic lines for lipid analysis. These lines were analyzed in three or more replicates to measure the amount of AcTAG, TAG, and overall fatty acid per seed. The resulting data is expected to show increased fatty acid content closer to wild-type seeds. Higher fatty acid content using acTAG and TAG molecules can be used to decrease the number of plants needed to produce C. sativa oils and save agricultural space for farmers.
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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