Progress 05/01/20 to 04/30/24
Outputs
Target Audience:Plant lipid biochemists Food technologists Other scientists Changes/Problems:Many initially experiments were delayed due to limited access to the lab during the COVID19 pandemic. A significant change in our approach was implemented when we shifted focus from using solely camelina to incorporating pennycress fae1 mutants for acetyl-TAG synthesis. This set of experiments leveraged transformation vectors created for camelina. Thus we thought it was a relatively low risk effort to investigate using another oil seed platform for unusual oil production. This strategic pivot allowed us to achieve ultra-high levels of acetyl-TAG (up to 98 mol%), a result that far surpassed initial expectations. Although this shift meant that some initial goals specific to camelina were not fully realized, the overall success of producing high acetyl-TAG levels validated our modified approach. This change was crucial in ultimately meeting the project's primary objectives through an alternative and more effective pathway. What opportunities for training and professional development has the project provided?Over the lifetime of the grant, the project provided training opportunities for five graduate students (4 KSU, 1 ISU): two who worked on acetyl-TAG accumulation in pennycress and camelina (including targeting lipase activity), one who optimized the of visualization of acetyl-TAG and related lipids, and two who quantified the physical properties of acetyl-TAG. In addition, the project enabled the training of two undergraduate students (KSU). Our work received multiple student competition awards highlighting the impact of our research. How have the results been disseminated to communities of interest?Over the lifetime of the grant, findings about physical property effects of acetyl-TAG were disseminated as oral presentations and posters in at least two international conferences. Two publications resulted from this particular work. Results describing the high levels of acetyl-TAG accumulation in camelina and pennycress were presented as oral presentations at four international scientific meetings and at multiple invited seminars at other universities. In addition, two manuscripts have resulted from this work (one published, one under review). Finally, we anticipate at least three additional publications in the coming year resulting from this work. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: Isolate and characterize novel promoters active in the late stages of seed development. Utilizing seed transcriptome data for Arabidopsis and camelina, we identified several genes expressed late in seed development. To confirm their expression, we quantified the transcript levels using RT-qPCR. For the characterization of these late seed promoters, segments including the upstream promoter region and portions downstream of the stop codon were cloned into a GoldenBraid2.0-compatible vector. Additionally, we employed cloned promoters from other species that are orthologous to the identified camelina late seed promoters. The napin promoter produced acetyl-TAG levels similar to the commonly used glycinin promoter, while the oleosin promoter resulted in lower levels. Attempts to combine the glycinin and oleosin promoters did not increase acetyl-TAG levels compared to using the glycinin promoter alone. Objective 2: Target lipase activity that reduces acetyl-TAG accumulation in late seed development. To suppress camelina lipase activity, RNAi hairpins targeting the expression of camelina SDP1, SDP1L, and GDSL1 were designed and cloned into plant transformation vectors, either alone or in combination with EfDAcT. Seed analysis revealed that suppression of these lipases in combination with EfDAcT expression did not enhance acetyl-TAG accumulation compared to control plants expressing EfDAcT only. This result is consistent with a model where the lipases degrade both TAG and acetyl-TAG, thus the relative ratio between the two types of storage molecules is not affected. CRISPR genome editing was also used to mutate SDP1, GDSL1, and OBL1. Analysis of mutant seed indicated changes in oil content and fatty acid composition. For example, OBL1 mutants possessed higher levels of linolenic acid (18:3) and lower levels of linoleic acid (18:2). We will continue to characterize these mutants for an anticipated publication resulting from the work. Additionally, leveraging another funding source, a combined transcriptomics and lipidomics analysis of transgenic camelina seeds was initiated, revealing high diacylglycerol (DAG) levels, a precursor to TAG and acetyl-TAG. Gene expression showed upregulation of another lipase LIP2 in transgenic seeds, identifying it as a valid target. RNAi and CRISPR will target LIP2 homeologs in camelina to assess effects on acetyl-TAG accumulation. Objective 3: Quantify the spatial accumulation of acetyl-TAG in transgenic camelina seed. We used MALDI-MS to map the localization of different lipid species, including acetyl-TAG, in transgenic seeds. In particular, we wanted to know where residual TAG was localized. Two notable findings were obtained: First, the low amounts of residual TAG were mostly confined to the embryonic axis, suggesting that DGAT1-RNAi and EfDAcT might more effectively target or compete with endogenous TAG-synthesizing enzymes in the cotyledonary tissues compared to the embryonic axis. This raises the possibility that employing tissue-specific promoters to express EfDAcT and to suppress endogenous TAG- acyltransferases within the embryonic axis could offer another strategy to eliminate the remaining residual TAG. Second, similar spatial distributions of specific TAG and acetyl-TAG molecular species, as well as their probable DAG precursors, provide additional evidence that acetyl-TAG and TAG are both synthesized from the same tissue-specific DAG pools, consistent with the fact that suppression of DGAT1 increases acetyl-TAG accumulation by reducing competition for the same substrate. Objective 4: Characterize the emulsification properties of acetyl-TAG. The goal of this objective was to evaluate the biophysical properties of various acetyl-TAG dispersions for improving the quality of dispersed food systems, such as protein foams and emulsions, and biodiverse packaging materials. We investigated the effectiveness of acetyl-TAG for stabilizing oil-in-water emulsions and protein foams. We examined two types of acetyl-TAG: one with high levels of polyunsaturated fatty acids (primarily linoleate and linolenate) at the sn-1/2 positions, and the other with higher levels of oleic acid. Minimal differences were noted in the surface tension of these different acetyl-TAG types. The stability of dispersions was further influenced by the surface elasticity of the interfacial region, evaluated through dynamic interfacial rheology measurements. The emulsification and foam-stabilizing abilities were assessed based on the rate of surface sorption and their impact on the viscoelastic properties of the surface films, particularly the storage modulus. The fatty acid compositions of acetyl-TAGs determined their attachment force and ability to modulate surface viscosity. We analyzed the interfacial rheological properties of high-oleic and wild-type acetyl-TAGs at different purities at air-water interfaces, using the pendant drop technique in an automated optical drop tensiometer. We investigated the temporal change in surface properties of oil-in-water emulsions prepared with sodium caseinate. Our results indicated that acetyl-TAG can increase packing density, providing steric repulsion and limiting Gibbs-Marangoni effects for additional stability up to a certain acetyl-TAG:protein ratio, beyond which stability decreased. We further investigated the conditions affecting the structure and stability of protein foams containing sugar formulated with acetyl-TAGs. The mechanisms involved were elucidated based on microscale interactions between interfacial protein, acetyl-TAG, and sugar content in the serum phase. Overall, the effect of acetyl-TAG on foam structure and stability was found to be specific to sugar concentration, which influenced both bulk viscosity and interfacial protein structure. On the other hand, and in line with our objectives, the addition of acetyl-TAG (up to 1 wt%) maximized foaming capacity, homogeneity, and stability compared to protein alone. This improvement was attributed to acetyl-TAG's ability to slow down the Gibbs-Marangoni mechanism and restore the thickness of thinning films. Our research demonstrated that acetyl-TAG offers great potential as a plasticizer to improve the properties of biodiverse and biodegradable packaging films derived from DDGS obtained from ethanol plants grinding sorghum. Our results showed that acetyl-TAG could replace glycerol as a plasticizer and enhance the mechanical properties of the polymer films manufactured by repurposing DDGS. Due to its unique chemical structure, FTIR analysis showed that acetyl-TAG could bind to protein moieties, providing improved elasticity. Furthermore, films prepared with acetyl-TAG exhibited superior mechanical properties compared to those made with glycerol. Optimisation of genetic background to generate ultra-high levels of acetyl-TAG: We initiated a side project to explore the production of acetyl-TAG in pennycress. Specifically, we want determine whether increasing acetyl-CoA levels in developing seeds by mutating FAE1 (a component of the fatty acid elongase machinery) would boost acetyl-TAG accumulation. Consistent with our predictions, pennycress fae1 mutants showed doubled acetyl-CoA levels in developing seeds compared to wild-type plants, whereas camelina fae1 mutants did not show significant differences. Correspondingly, EfDAcT expression in fae1 pennycress mutants led to significantly higher acetyl-TAG levels compared to wild-type pennycress, but no significant differences were observed in camelina. Further enhancement of acetyl-TAG levels was achieved by combining EfDAcT expression with suppression of DGAT1 activity, particularly in pennycress fae1 mutants, where acetyl-TAG levels reached as high as 98 mol%. Remarkably, this ultra-high production of acetyl-TAG in transgenic seeds exhibited minimal effects on seed properties, highlighting the potential for production of designer oils required for economical biofuel industries.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Nicholas Neumann, Tao Fei, Tong Wang, Timothy P. Durrett.
Defining the physical properties of blends of acetyl-triacylglycerols derived from transgenic oil seeds. (2024) J Am Oil Chem Soc 101: 197-204
https://doi.org/10.1002/aocs.12746
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Eda Ceren Kaya, Dallas Johnson, Pamela Tamura, Timothy P. Durrett, Umut Yucel.
Improving the whey protein foam structures by using novel acetylated triglycerides (acetyl-TAG): A response surface methodology (RSM) approach. (2024) J Am Oil Chem Soc 101: 397-406
https://doi.org/10.1002/aocs.12780
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Nicholas Neumann, Maxwell Harman, Andrea Kuhlman, Timothy P. Durrett
Arabidopsis diacylglycerol acyltransferase1 mutants require fatty acid desaturation for normal seed development. (2024) Plant J. https://doi.org/10.1111/tpj.16805
- Type:
Journal Articles
Status:
Under Review
Year Published:
2024
Citation:
Linah Alkotami, Dexter J. White, Kathleen M. Schuler, Maliheh Esfahanian, Brice A. Jarvis, Andrew E. Paulson, Somnath Koley, Jinling Kang, Chaofu Lu, Doug K. Allen, Young-Jin Lee, John C. Sedbrook, Timothy P. Durrett
Complete Remodeling of TAG Composition to Generate Novel Oils in Transgenic Oilseeds.
Submitted to PNAS
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2023
Citation:
Neumann, Nicholas. Harnessing biotechnology and genetics to improve oilseed biochemistry. PhD Dissertation. Kansas State University, 2023.
Available on ProQuest
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2024
Citation:
Alkotami, Linah. "Remodeling of triacylglycerol synthesis in emerging oilseed crops: genetic engineering for high-value seed oil." PhD Dissertation. Kansas State University. 2024.
Available on KRex
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Xiao, Tingyuan. "Identification of Seed Specific Promoters with A Range of Expression Strengths". Poster Presentation. International Symposium on Plant Lipids, July 2024, Lincoln, NE.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
White, Dexter. "Genetic Research on Camelina: CRISPR/Cas9-mediated gene editing to enhance seed oil in cover crops". Poster Presentation. International Symposium on Plant Lipids, July 2024, Lincoln, NE.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Durrett, Timothy. "Complete Remodeling of TAG Composition to Generate Novel Oils in Transgenic Oilseeds." Oral Presentation. International Symposium on Plant Lipids, July 2024, Lincoln, NE.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Durrett, Timothy. Identification and Characterization of Plant Membrane Bound O-acyltransferases (MBOATs) to Enable the Synthesis of Unusual Lipids .
Invited seminar. Department of Biology, Syracuse University, 13 November 2023
|
Progress 05/01/22 to 04/30/23
Outputs
Target Audience:Plant lipid biochemists Food technologists Other scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The funding has supported the work of four graduate students, two who worked on acetyl-TAG accumulation in pennycress and camelina (including targeting lipase activity), one who optimized the of visualization of acetyl-TAG and related lipids, and one who quantified the physical properties of acetyl-TAG. In addition, the project enabled the training of one undergraduate students at Kansas State. How have the results been disseminated to communities of interest?Findings about physical property effects of acetyl-TAG were disseminated as oral presentations and posters at two conferences. Results describing the high levels of acetyl-TAG accumulation in camelina and pennycress were presented as oral presentations at three international scientific meetings and at two invited seminars at other universities. In addition, two manuscripts have been submitted and are currently under review. What do you plan to do during the next reporting period to accomplish the goals?What do you plan to do during the next reporting period to accomplish the goals? Objective 1: a. Transformation of novel promoters driving EfDAcT into camelina and characterization of transgenic lines b. Submission of Goldenbraid compatible promoter and terminator parts to Addgene Objective 2: a. Characterization of transgenic lines expression EfDAcT with different lipases suppressed, with a focus on whether seed germination is affected. b. Genotyping of CRISPR lines targeting lipases. Redesign of gRNA for SDP1. c. In vitro assays on endogenous camelina lipases determine whether they are capable of hydrolyzing acetyl-TAG. Objective 3: a. Relative quantitation of the PCs, DAGs, acetyl-TAGs, and TAGs will be performed suing MALDI-MSI for new transgenic lines imbedded with low temperature techniques and using 2,5-DHAP with potassium additive as a matrix system. b. Further development of on-tissue quantification method is needed and will focus on the use of sprayed internal standards will be for on-tissue quantification of lipid classes of interest. We intend to evaluate relative ionization efficiencies of the classes and move to a single internal standard, tripentadecanoin. Though more expensive than tripalmitin, tripentadecanoin is more easily extended to other biological systems due to low natural occurrence and avoids type-II isotopic overlap. c. If successful, we will extend the on-tissue quantitation method to visualize acetyl-TAGs and other lipids in newly generated acetyl-TAG lines. Objective 4: In the remaining reporting period, we are planning to: 1) optimize the conditions for emulsions and aerated emulsions as a function of lipid content and type, and air incorporation; 2) elucidate the condition for optimization of properties and structure of thethe biodegradable DDGS films. In order to accomplish these, we will utilize a newly acquired FTIR-IR microscope module in addition to the existing array of analytical instruments to provide spatial characterization of the architecture of DDGS films formed by a biopolymer network of proteins and fibers in conjugation with acetyl-TAG.
Impacts
What was accomplished under these goals?
Objective 1: Isolate and characterize novel promoters active in the late stages of seed development. a. Expression of EfDAcT using different seed promoters. Previously, we showed that using a napin promoter produced the same amount of acetyl-TAG as using a glycinin promoter in the T2 generation. Using an oleosin promoter, predicted to be expressed later in seed development, led to lower levels of acetyl-TAG compared to the other two promoters. In addition, expressing two EfDAcT cassettes, one under the control of the glycinin promoter and one under control of the oleosin promoter, did not produce higher levels of acetyl-TAG compared to lines expressing EfDAcT only under control of the glycinin promoter. We identified homozygous lines expressing these different constructs and confirmed these results in subsequent generations. In addition, we examined EfDAcT protein accumulation at different stages of seed development. Here, seeds expressing EfDAcT under control of the oleosin promoter had much lower levels of EfDAcT compared to using glycinin or napin promoters. Expression using the glycinin promoter was earlier in seed development than with the napin promoter. The combination of both glycinin and oleosin resulted in an expression pattern that resembled napin. We are currently preparing a manuscript that describes these results. We have also isolated novel camelina promoters expressed late in seed development. Transformation vectors where these promoters drive EfDAcT are in the process of being constructed and will then be transformed into camelina. b. We continued the characterization of pennycress transgenic lines that produce ultrahigh (98 mol%) levels of acetyl-TAG. These lines were generated by expressing EfDAcT in a pennycress fae1 mutant background. In this reporting period we were able to show that these ultrahigh levels of acetyl-TAG were stable over multiple generations. Analysis of seed properties (seed size, total fatty acids, etc) revealed no significant differences between seeds with ultrahigh levels of acetyl-TAG and wild-type control seeds. However, transgenic seeds expressing both EfDAcT and DGAT1-RNAi tended to germinate slower than wild-type control seeds. We hypothesize this is due to the suppression of DGAT1 and not due to the accumulation of ultrahigh levels of acetyl-TAG, as lines expression just EfDAcT that also accumulate ultrahigh levels of acetyl-TAG germinate similar to wild-type seeds. We are currently preparing a manuscript that describes the development and characterization of these ultrahigh acetyl-TAG lines. Objective 2: Target lipase activity that reduces acetyl-TAG accumulation in late seed development. a. Suppression of camelina lipase activity. Previously, RNAi hairpins targeting the lipases SDP1, SDP1L and GDSL1 were expressed by themselves or in combination with EfDAcT in camelina. Analysis of transgenic seeds is currently in progress. We also used CRISPR genome editing to mutate SDP1, GDSL1 and OBL1. Our plan is to have three closely (~200bp) spaced target sites to obtain different small deletions in each homeolog that will facilitate easier genotyping through PCR. Vectors expressing Cas9 and the gRNAs for each target were transformed into camelina. We were able to obtain transgenic lines where GDSL1 and OBL1 are targeted, but none for SDP1 (despite repeated transformations). Genotyping to detect deletions in GDSL1 and OBL1 lines is in progress. We will try different gRNAs to obtain CRISPR lines targeting SDP1 (however, the RNAi approach we have implemented also serves as a reasonable alternative strategy to knocking out the gene). b. SDP1, GDLS1 and OBL1 have been cloned for expression in yeast. In vitro assays to determine whether these lipases can target acetyl-TAG will begin shortly. c. By leveraging another funding source, we initiated a combined transcriptomics and lipidomics analysis of transgenic camelina seeds that accumulate high levels of acetyl-TAG. Briefly, the lipidomic analysis indicated that transgenic seeds also possess high levels of diacylglycerol (DAG), which is the common precursor to both TAG and acetyl-TAG. Alternatively, DAG could also derived from the lipolytic breakdown of TAG and acetyl-TAG. Analysis of gene expression profiles indicate that another lipase LIP2 is upregulated late in transgenic seeds and thus represents an valid target. We will use both RNAi and CRISPR to target gene LIP2 homeologs in camelina and determine the effect on acetyl-TAG accumulation. Objective 3: Quantify the spatial accumulation of acetyl-TAG in transgenic camelina seed. Camelina and pennycress transgenic seeds were received and pennycress samples are currently being evaluated with MALDI-MSI for their promise for acetyl-TAG production. The 2,5-DHAP matrix protocol that we previously developed for camelina seeds was amenable to the Pennycress samples, though diffusion of TAG is still problematic. After more evaluation, minimal benefit from low temperature embedding is observed, so gelatin is now used due to ease of use and preparation. Another limitation is that isomeric or isobaric species to acetyl-TAGs are observed in the WT seeds. Both the diffusion and isomer issues make exact quantitation challenging. Regardless, general trends are observed and relative quantitation seems realistic. Importantly, our MALDI-MSI results support the same trends observed from GC-MS analysis, where ~98% acetyl-TAGs is observed in some pennycress transgenic lines. Preliminary results suggest that shorter carbon chain length triacylglycerols, regardless of having an acetyl group, are enriched in the embryonic axis. Also, though further analysis is required, there is evidence of a small degree of tissue specific (embryonic axis vs. cotyledons) accumulation of acetyl triacylglycerols in some of the transgenic lines. Though small, some of the differences are statistically significant and may provide insights into future genetic modification. Objective 4: Characterize the emulsification properties of acetyl-TAG. We continued optimizing conditions for the structure and stability of protein foams formulated with acetyl-TAGs. The related mechanisms were elucidated as a function of microscale interactions between interfacial protein and acetyl-TAG as well as sugar content in the serum phase. Overall, the effect of acetyl-TAG was on the foam structure and stability was found to be specific to sugar concentration, which affected the bulk viscosity as well interfacial protein structure. Unexpectedly, at high sugar concentrations (30 wt%), typical to conventional formulations, the addition of acetyl-TAG decreased the bulk viscosity, which probably related to partial replacement of the interfacial protein. This was also confirmed by a decrease in the zeta potential of the dispersed phase. On the other hand and in parallel with our objective, addition of acetyl-TAG (up to 1wt%) maximized the foaming capacity, homogeneity and stability in comparison to protein alone, which was explained by the ability of acetyl-TAGs to slow down the Gibbs-Marangoni mechanism and restore the thickness of thinning films. In addition, we developed protocols to manufacture emulsions and aerated-emulsions (whipped systems) with different lipid contents (10-50%) and types (hydrogenated palm stearin as solid fat and fractionated coconut oil medium chain fatty acids as the liquid lipid). The emulsions and their foams (non-aerated and aerated systems) were characterized for their foaming and emulsification capacities as a function of their physicochemical properties, such as particle size distribution, zeta potential, interfacial rheology and surface viscoelasticity. At the same time, we continued to finalize the optimization and characterization of biodegradable and renewable packaging film materials formulated from sorghum DDGS with the addition of acetyl-TAG to provide plastizing effect to increase their elasticity and improve their water resistance.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Nicholas Neumann, Tao Fei, Tong Wang, and Timothy P. Durrett.
Defining the physical properties of blends of acetyl-triacylglycerols derived from transgenic oil seeds. Journal of the American Oil Chemists Society.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Eda Ceren Kaya, Dallas Johnson, Pamela Tamura, Timothy P. Durrett, Umut Yucel.
Improving the whey protein foam structures by using novel acetylated triglycerides (acetyl-TAG): A response surface methodology (RSM) approach. Journal of the American Oil Chemists' Society.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Timothy Durrett. Targeted genome editing of industrial oilseed crops to increase synthesis of functional lipids . Oral presentation.
2022 World Congress on Oleo Science. 23 August 3 September 2022. Virtual meeting
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Linah Alkotami. Targeted genome editing of industrial oilseed crops to increase synthesis of functional lipids . Oral presentation.
2022 American Oil Chemists Society annual meeting. 1-4 May 2022. Atlanta, GA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Linah Alkotami. Modulating Seed Metabolism to Enhance Synthesis of Functional Lipids. Oral Presentation.
Plant Lipids: Structure, Metabolism and Function Gordon Research Seminar. January 2023. Galveston, TX.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Linah Alkotami. Modulating Seed Metabolism to Enhance Synthesis of Functional Lipids. Poster Presentation.
Plant Lipids: Structure, Metabolism and Function Gordon Research Conference. January 2023. Galveston, TX.
** 1st place for Student Poster Award **
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Yucel U. Sorghum DDGS as a renewable source for production of functional packaging films. ACS Spring 2022 Bonding through Chemistry. March 20-24, 2022. San Diego, CA
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Kaya E, Johnson D, Durrett T, Yucel U. Response surface methodology optimization of the use of acetyl-triacylglycerol for improving the structure of whey protein foams. 2022 AOCS Annual Meeting & Expo. May 1-4. Atlanta, GA
**1st place in Student Competition of the Analytical Division**
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Kaya E, Durrett T, Bean S, Trinetta V, Yucel U. Effect of High Oleic Acetyl Triacylglycerol (ace-TAG) on Functional Properties of Biodegradable Sorghum DDGS Packaging Films. 2022 AOCS Annual Meeting & Expo. May 1-4. Atlanta, GA
**1st place in in Student Competition of the Processing Division**
|
Progress 05/01/21 to 04/30/22
Outputs
Target Audience:Plant lipid biochemists Food technologists Other scientists Changes/Problems:New approach with fae1 pennycress: The expression of EfDAcT in pennycress was undertaken to leverage expression vectors developed in this project and determine their expression in another oilseed. In particular, we were interested to see if the higher VLCFA content of pennycress compared to camelina would result in increased acetyl-CoA availability in fae1 mutants to enhance the synthesis of acetyl-TAG. Our results (higher acetyl-CoA levels in fae1 pennycress, ~98mol% acetyl-TAG accumulation in fae1 pennycress) suggest that our hypothesis was correct. These exciting results meant that additional attention was focus on this part of the work; consequently less time was spent characterizing and suppressing camelina lipases (Objective 2). We are close to submitting a manuscript describing the pennycress fae1 results, after which we will return attention to the lipase objective. Redesign of CRISPR vectors: One challenged we have encountered with genome editing of camelina is efficiently genotyping mutant plants, particularly with three highly identical homeologous genes. While deep sequencing of amplicons spanning the target sites has been helpful in rapidly identifying which homeologs are mutated, this approach is not cost effective for genotyping many plants. In another project in the lab, we have been successful in generating short deletions in target genes by having multiple, closely-spaced gRNAs. We will extend these observations by designing at least three closely spaced target sites, and in some cases will pick sites present in only one or two of the homeologs, in order to obtain homeolog specific deletions easily genotyped by PCR. This new approach has meant redesigning and rebuilding existing vectors, but we feel this will lead to more efficient genotyping and selection of suitable lines, which will save time and cost downstream. What opportunities for training and professional development has the project provided?The funding has supported the work of three graduate students, one who identified and cloned late seed specific camelina promoters, one who optimized the of visualization of acetyl-TAG and related lipids, and one who quantified the physical properties of acetyl-TAG. In addition, the project enabled the training of two undergraduate students at Kansas State. How have the results been disseminated to communities of interest?Findings about physical property effects of acetyl-TAG were disseminated as an abstract in an international conference, invited webinar and a woman in science conference. In addition, two manuscripts were drafted and ready to be published in the next reporting period. Results describing the high levels of acetyl-TAG accumulation in pennycress were presented as part of a graduate student seminar at Kansas State University. In addition, an undergraduate student presented results describing the effects of different promoters on acetyl-TAG accumulation at an undergraduate research poster session. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: a. Grow out highest acetyl-TAG accumulating lines for each promoter type and collect developing seed for expression analysis and acetyl-TAG accumulation. b. Lipid imaging of acetyl-TAG to determine localization of expression for each seed specific promoter (Objective 3). c. Conduct additional seed property analyses (e.g. overall fatty acid content, seed size, germination efficiency, etc) on pennycress lines with ultra-high (>95 mol%) acetyl-TAG content. d. Lipid imaging determine localization of remaining endogenous TAG in ultra-high acetyl-TAG pennycress lines. Objective 2: a. Complete transformation of lipase RNAi and CRISPR constructs into camelina. Homozygous T3 seeds will be isolated based on DsRed selection and lipd content analyzed. Identify high oil lines and lines producing high levels of acetyl-TAG. b. Clone and characterize camelina lipases for expression in yeast. These work will determine whether endogenous camelina lipases are capable of hydrolyzing acetyl-TAG. Objective 3: a. Relative quantitation of the PCs, DAGs, acetyl-TAGs, and TAGs will be performed suing MALDI-MSI for new transgenic lines imbedded with low temperature techniques and using 2,5-DHAP with potassium additive as a matrix system. b. Further development of on-tissue quantification method is needed and will focus on the use of sprayed internal standards will be for on-tissue quantification of lipid classes of interest. We intend to evaluate relative ionization efficiencies of the classes and move to a single internal standard, tripentadecanoin. Though more expensive than tripalmitin, tripentadecanoin is more easily extended to other biological systems due to low natural occurrence and avoids type-II isotopic overlap. c. If successful, we will extend the on-tissue quantitation method to visualize acetyl-TAGs and other lipids in newly generated acetyl-TAG lines. Objective 4: In the next reporting period, we are planning to extent these experimental efforts to elucidate the effects of the oil properties as the dispersed phase (i.e., crystallinity and hydrophobicity) on the interfacial rheology and subsequent stability of the emulsions and foams. Crystalline fats are typically known to provide higher storage moduli than liquid lipids to limit the rate of foam drainage. Similarly other lipids, such as essential oils and oleosomes, known to show varying degree of hydrophobicities and interfacial tensions based on their composition. The acetyl-TAGs offers great potential to prepare stable flavoring preparation in the form of liquid emulsions, which will also be a subject of the next reporting period activities.
Impacts
What was accomplished under these goals?
Objective 1: Isolate and characterize novel promoters active in the late stages of seed development. a. Expression of EfDAcT using different seed promoters. Previously, we transformed EfDAcT under the control of late seed specific promoters development into camelina plants. In this funding period, we advanced these transgenic lines to the T2 generation and analysed the acetyl-TAG content of transgenic seeds. We found that on average, the napin promoter produced levels of acetyl-TAG similar to using the glycinin promoter (the promoter we have typically used to express EfDAcT). In contrast, using an oleosin promoter resulted in lower levels of acetyl-TAG. As the oleosin promoter is expressed later in seed development than glycinin, we wanted to see the effect if both were combined. However, transgenic lines expressing two EfDAcT cassettes, one under the control of the glycinin promoter and one under control of the oleosin promoter, did not produce higher levels of acetyl-TAG compared to lines expressing EfDAcT only under control of the glycinin promoter. T3 plants from these lines are currently growing to identify homozygous lines which will enable confirmation of acetyl-TAG levels using different promoters, localization of acetyl-TAG in the transgenic seed (Objective 3), and measuring EfDAcT expression levels during seed development. We also initiated an experiment to determine whether increasing acetyl-CoA levels in developing seeds would increase acetyl-TAG accumulation. We did this by using camelina and pennycress plants possessing mutations in genes encoding FATTY ACID ELONGASE1 (FAE1), a component of the fatty acid elongase complex that generates the very long chain fatty acids (VLCFA) found in Brassicaceae seeds. Pennycress contains ~52% VLCFA in its seed oil and camelina contains about ~20%; mutation of FAE1 eliminates VLCFA in both species. Fatty acid elongation uses acetyl-CoA as a substrate; we hypothesized that by eliminating fatty acid elongation more acetyl-CoA would be available for acetyl-TAG synthesis. In fae1 pennycress mutant seeds, levels of acetyl-CoA are double those in wild-type plants. In camelina, there was no significant difference in acetyl-CoA levels between wild-type and fae1 seed. Consistent with these results, expression of EfDAcT resulted in higher levels of acetyl-TAG in mutant fae1 pennycress lines (74 mol%) compared to WT penny cress lines (31 mol%) whereas the background made no significant difference in camelina (77 mol% in fae1 and 74 mol% in WT). Combining expression of EfDAcT with suppression of DGAT1 activity that produces regular TAG resulted in enhanced levels of acetyl-TAG. Similar to expression of EfDAcT alone, in camelina there were no significant differences were observed between the backgrounds (85 mol% in fae1 and 80 mol% in WT). In contrast, significantly higher levels of acetyl-TAG were achieved in pennycress fae1 mutant lines (96 mol%) compared to WT (75 mol%). Here, the best transgenic lines accumulated acetyl-TAG levels as high as 98 mol%, a particularly exciting result. Indeed, these levels of acetyl-TAG are higher than those found in plant species that naturally produce acetyl-TAG. Objective 2: Target lipase activity that reduces acetyl-TAG accumulation in late seed development. a. Suppression of camelina lipase activity. RNAi hairpins have been cloned into plant transformation vectors by themselves or in combination with EfDAcT. Transformation into camelina is underway for these different constructs. Based on success with other camelina CRISPR projects, we have redesigned our gRNA target sequences for different lipase genes so as to obtain small deletions. By having at least three closely (~200bp) spaced target sites we hope to obtain different small deletions in each homeolog. This will facilitate easier genotyping through PCR and will enable us to possibly distinguish mutations in the different homeologs. Vector construction to target these new sites is underway. Objective 3: Quantify the spatial accumulation of acetyl-TAG in transgenic camelina seed. a. In our previous report, a binary matrix of gold and 2,4,6-trihydroxyacetophenone (THAP) with a potassium additive was selected as the best sample preparation scheme for localizing lipids in camelina seed samples. However, we extended our list of potential matrices to 2,5 and 2,6-dihydroxyacetophenone (DHAP). Both of these matrices exhibited better ionization efficiencies on-tissue than the previous combinations, while the addition of gold decreased the ionization efficiencies. Although 2,5-DHAP has lower ion intensity for PCs and TAGs when compared to 2,6-DHAP, 2,5-DHAP is better suited for the ~7.5 Torr pressure of the MALDI-MS source used. Therefore, 2,5-DHAP will be used as a matrix system moving forward. b. In order to address TAG diffusion issues that were apparent in gelatin imbedding, two low temperature embedding materials were tested (carboxymethyl cellulose (CMC) and a polyvinylpyrrolidone and hydroxypropyl methylcellulose mixture (PVP&HPMC)). These techniques, especially PVP&HPMC, improved the TAG diffusion issue from the gelatin embedded samples, but some diffusion is still present. However, sputtered gold on the final sample is required for both of these embedding techniques as they do not adhere well to conductive carbon tape. c. A promising method for on-tissue quantitation by MALDI-MS is the use of tissue mimetic arrays. An attempt was made to prepare such an array from the homogenates of seeds. However, sectioning the tissue mimetics resulted in inconsistent thickness of the analyzable area, which would result in poor reproducibility in the quantitative analysis. A preliminary experiment was done using sprayed tripalmitin, a TAG not present in camelina seeds, onto the sectioned seed surface with promising results and will be the focus for further development. Further work is necessary for on tissue quantitation. We can, however, move forward with relative quantitation. Objective 4: Characterize the emulsification properties of acetyl-TAG. We focused on measuring the interfacial rheological properties of various acetyl-TAG dispersions. The dispersions were representative of foams and emulsions. The emulsification and foam-stabilizing abilities are typically determined based on the rate of surface sorption and their ability to modulate the viscoelastic properties of the surface films, mainly the storage modulus. For example, the drainage velocity of the liquid separating the air bubbles, which is inversely related to the storage modulus, determines the stability of a foam. The fatty acid compositions of acetyl TAGs determine their attachment force and ability to modulate the surface viscosity of foams. Therefore, we analyzed the interfacial rheological properties of high-oleic and wild-type acetyl-TAGs at different levels of purities at air-water interfaces in the presence of other ingredients, such as sugar and liquid lipid (i.e., fractionated coconut oil) using pendant drop technique in an automated optical drop tensiometer. In addition to the air-water interface of a foam, a modified system was used for measuring the interfacial properties of acetyl-TAG acetyl-TAGs at an oil-water interface, where oil was introduced as a dispersed droplet. In an emulsion, small molecule surfactants are often used in association with biopolymers to improve their emulsification performance, which, however, may result in competitive surface adsorption. Therefore, we also investigated the temporal change in the surface properties of an oil-in-water emulsion prepared in the presence of sodium caseinate as one of the most common proteins used to form emulsions and foams. We observed that there is a significant amount of surface displacement of the protein however above a critical concentration of the acetyl-TAG. At the end, we evaluated the stability of model emulsions and foams to correlate with their interfacial rheology.
Publications
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Stuchlik A, Alkotami L, Durrett TP. Usage of different promoters to optimize unusual lipid synthesis in transgenic plants. Poster Presentation, K-State Undergraduate Research Symposium, November 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Kaya EC, Durrett T, Yucel U. Effect of high oleic acetyl triacylglycerol (ace-TAG) on functional properties of biodegradable sorghum DDGS packaging film. Poster Presentation, IFT21 Annual Meeting, 18-21 July 2021
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Kaya EC, Yucel U. Alternative potential use of DDGS by-product from sorghum production as a biodegradable food packaging film and value-added food products. Poster Presentation, Midwest Women in Science Conference, 18-21 September 2021.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Kaya EC, Yucel U. Characterization and analysis of hydrophobicity, interfacial tension and contact angle of acetyl TAG used for various food application. Webinar Presentation, Biolin Scientific-Attension Seminar 2021, 30 November 2021
- Type:
Other
Status:
Published
Year Published:
2021
Citation:
Alkotami, L. Genetic modification of seed oil composition results in enhanced acetyl-TAG synthesis. Graduate Student Seminar, 15 November 2021
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Progress 05/01/20 to 04/30/21
Outputs
Target Audience:Plant lipid biochemists Food technologists Other scientists Changes/Problems:Many experiments were delayed due to limited access to the lab during the COVID19 pandemic. However, research operations have essentially returned to normal and we anticipate making up for lost time in the coming funding period. What opportunities for training and professional development has the project provided?The funding has supported the work of three graduate students, one who identified and cloned late seed specific camelina promoters, one who optimized the of visualization of acetyl-TAG and related lipids, and one who quantified the physical properties of acetyl-TAG. In addition, the project enabled the training of one undergraduate student. How have the results been disseminated to communities of interest?Results describing the isolation of camelina promoters were presented as part of a student seminar at Kansas State University. In addition, the project will enable two different graduate students to give a presentations at two different conferences this summer. (These will be reported in next years report as they fall outside the current reporting period) What do you plan to do during the next reporting period to accomplish the goals?Objective 1: a. Transform EfDAcT under control of different late seed promoters into camelina and isolate homozygous T3 lines. We will also express GUS under control of these promoters as an additional way to quantify the timing and localization of expression. b. Identify high acetyl-TAG lines for each promoter construct and quantify acetyl-TAG accumulation during late seed expression. Eventually, localization of expression will be determined using lipid imaging of acetyl-TAG (Objective 3). Objective 2: a. Transform lipase RNAi and CRISPR constructs into camelina. Homozygous T3 seeds will be isolated based on DsRed selection and lipd content analyzed. Identify high oil lines and lines producing high levels of acetyl-TAG. Vector construction is underway to target SDP1 and OBL1 homeolog expression. b. Clone and characterize camelina lipases in vitro. These experiments will determine whether endogenous camelina lipases are capable of hydrolyzing non-native lipid substrates such as acetyl-TAG. Objective 3: a. Use other embedding techniques to limit TAG diffusion. Gelatin, used in this work, is commonly used as an embedding material and is at ~70°C when pouring into a mold, resulting in diffusion of some TAGs. To limit the diffusion, other low temperature embedding materials will be tested including carboxymethyl cellulose or a polyvinylpyrrolidone and hydroxypropyl methylcellulose mixture, which are solutions when embedding tissues at room temperature, then frozen before sectioning. b. Develop on-tissue quantification method. The use of an internal standard will be investigated for on-tissue quantification of lipid species of interest. Internal standard method development will be performed with tripalmitin, a TAG not present in Camelina seeds. The internal standard will be sprayed on top of the tissue or under the tissue. Also, continuous ESI spray of the internal standard into the source during MS imaging analysis will be investigated. All three methods will be individually assessed for robustness of quantification. After selection of the best internal standard method, tripentadecanoin will be used as an internal standard as this compound, though more expensive, is more easily extended to other biological systems due to low natural occurrence. c. Quantitatively visualize acetyl-TAG and other lipids in existing acetyl-TAG lines. The most robust internal standard method will be used on the currently available transgenic lines to obtain quantitative spatial mapping of lipid species of interest. Objective 4: a. Evaluate the performance of acetyl-TAG on the stability of dry foam (i.e., meringue model system mainly comprised of a protein and sugar). The hypothesis is to by incorporating small amounts of acetyl-TAG the sugar requirement will decrease to have a foam with similar or better stability. The independent variables will include protein, sugar and acetyl-TAG concentrations and acetyl-TAG purity. A response surface methodology (RSM) design will be used to find the sweet spot, and elucidate the higher order interactions between acetyl-TAG concentration and purity level. The results will be disseminated as a conference abstract and a peer-reviewed journal article. b. Evaluate the performance of acetyl-TAG on the stability of whipped cream. The model system is mainly comprised of an emulsified lipid stabilized via dairy proteins (i.e., whey protein isolate). In this multi-phase complex system, the crystallinity lipid and number of lipid droplets determine the foam stability. The lipid droplet stability is determined by the protein concentration. Our hypothesis is to decrease the need of hard fat fraction and total fat content in the foam (both desirable for healthier foods) by the use of acetyl-TAG to obtain foams with similar or better stability to original formulation without acetyl-TAG. Similar to Objective 4.a, a response surface methodology (RSM) design will be used to determine the ideal conditions with improved foam stability at lower fat concentration and solid fat content. The results will be disseminated as a conference abstract and a peer-reviewed journal article.
Impacts
What was accomplished under these goals?
Objective 1: Isolate and characterize novel promoters active in the late stages of seed development. a. Identification of camelina late seed promoters - Based on publicly available seed transcriptome data for Arabidopsis, we identified 22 genes expressed late in seed development at levels higher than orthologs of glycinin (the promoter currently used for EfDAcT expression). This number of genes was reduced to six using available camelina transcript data. To confirm the expression of these genes in late camelina seed development, we extracted RNA from developing wild-type camelina seeds, focusing on later stages of seed development. Transcript levels of these candidate genes were then quantified using RT-qPCR. In this manner, we identified four genes expressed higher than the glycinin promoter late in camelina seed development. b. Characterization of late seed promoters -For each of these four genes, up to 2kb of upstream of the start codon and 500bp downstream of the stop codon has been amplified using PCR and cloned into a GoldenBraid2.0-comptable vector. We are currently in the process of inserting EfDAcT or a GUS reporter gene into these vectors to make transcriptional units (TUs) that will then be transformed into camelina. In addition, we already possessed cloned promoters from other species that were orthologous to the camelina late seed promoters identified. For example, we found that a camelina oleosin gene was expressed late in seed development and already possess a soybean oleosin promoter::terminator construct. We therefore created TUs with these existing vectors and have transformed these into camelina. Objective 2: Target lipase activity that reduces acetyl-TAG accumulation in late seed development. a. Suppression of camelina lipase activity. RNAi hairpins and CRISPR gRNAs to target the expression of camelina SDP1 and OBL1 homeologues have been designed and ordered. Vector construction is in progress. (Progress in this aim was delayed due to limited access to laboratory space due to COVID19-related restrictions). Objective 3: Quantify the spatial accumulation of acetyl-TAG in transgenic camelina seed. a. Optimization of visualization of acetyl-TAG and related lipids - Using lipid standards, different MALDI-MS matrices were evaluated for three organic matrices and an inorganic matrix, or a mixture of the inorganic and an organic matrix. Further, all matrix schemes were evaluated for performance with and without a potassium additive, totaling 14 unique sample preparation combinations. Based on these results, the three best sample preparation schemes that involved an organic matrix were tested using wild-type seed sections. In this manner, a binary matrix of gold and 2,4,6-trihydroxyacetophenone (THAP) with a potassium additive was selected as the best sample preparation scheme for localizing lipids in camelina seed samples. This method has now been used to preliminarily measure lipid (acetyl-TAG, TAG, PC and DAG) localization in transgenic seeds. Initial results of interest indicate remaining TAGs being enriched in the cotyledons. Minor issues with TAG diffusion were noted; approaches to overcome these will be attempted in the future. Higher levels of DAG in the transgenic seeds were also observed, consistent with analysis of quantification of total lipids extracted from mature seeds. Objective 4: Characterize the emulsification properties of acetyl-TAG. Two types of acetyl-TAG (i.e., wild type and high-oleic) were evaluated for their interfacial properties at an air-water interface, which relates to foam stability in various food products, such as whipped cream, confectioneries, etc. The wild-type is predominantly composed of unsaturated fatty acids (i.e., primarily linoleate and linolenate) at the sn-1/2 positions, and the high-oleic type predominantly with oleic acid. The surface tension of aqueous acetyl-TAG solutions prepared over a wide range of concentrations (0.0001 - 0.1 mg/ml) were determined by pendant drop technique in an automated optical tensiometer. Somewhat surprisingly, their sorption isotherms were similar with minimal difference in the surface tension at a given acetyl-TAG concertation. This was very likely related to similar hydrophilic-lipophilic balance (HLB) values of the two acetyl-TAG types that characteristically dominate the static measurements. However, we expect to see differences in their surface viscosity as a function of their alkyl tail packing to be evaluated through dynamic surface tension measurements. In addition, protocols were also developed to prepare different food foams (i.e., whipped cream and meringue type), and evaluate foam characteristic and stability. In a relevant application, high-oleic acetyl-TAG (i.e., related to its higher oxidative stability as compared to wild-type) was also used as a plasticizer in the development of biodegradable packaging film derived from sorghum distillers' grains. Here, acetyl-TAG served to improve mechanical properties of the films material: decreased moisture solubility and improved elongation.
Publications
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