Progress 01/15/24 to 01/14/25
Outputs
Target Audience:The audience for the this research is biochemists, molecular biologists, geneticists, and biotechnologists who study the underlying metabolism and genes that control the biosynthesis of high-value carotenoids and who aim to use this knowledge to develop crops with increased value for human nutrition and aquaculture feedstocks. Findings from this research are also of interest to oilseed processors, aquaculture feedstock manufacturers, and aquaculture producers who seek high value traits and alternative, cost-competitive, and sustainable sources of natural pigments for food production and aquaculture feed. Ultimately, the audience for the research outcomes of this project are US soybean and other oilseed producers who desire enhanced germplasm with new traits for high-value markets to sustain and increase the profitability of farm production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The research was primarily conducted by a postdoctoral associate Dr. Hyojin Kim who gained experience in oral presentation of her research through three presentations at international conferences in 2024. Most notably, she presented her research in a poster presentation at the 26th International Symposium on Plant Lipids in July, 2024. She also gained experience in intellectual property protection by contributing to a non-provisional US patent application, filed in October 2024, on research from this project. How have the results been disseminated to communities of interest?Project findings were shared with the plant science and plant lipid research communities through both oral and poster presentations at local and international conferences. These included an oral presentation by project postdoctoral associate Dr. Hyojin Kim: Hyojin Kim, Kiyoul Park, Edgar B. Cahoon. Oilseed Metabolic Engineering for High-Value Aquaculture Traits. Oral presentation at the Center for Plant Science Innovation Research Group Meeting, Lincoln, Nebraska, USA. March 18, 2024. Hyojin Kim, Kiyoul Park, Edgar B. Cahoon. Nature-Guided Strategies to Maximize Astaxanthin Production and Purity in Camelina Oil for Aquaculture Feed and High-Value Food Applications. Poster presentation at the International Symposium on Plant Lipids, Lincoln, Nebraska, USA. July 14-19, 2024. Edgar B. Cahoon,Metabolic Redesign of Oil Metabolism for Enhanced Food, Feed, and Biofuel Traits. Oral presenation at theFirst International Summit on Plant Resilience, East Lansing, Michigan, USA, May 16, 2024. What do you plan to do during the next reporting period to accomplish the goals?Aim 1: Optimize Astaxanthin Production and Purity in Camelina and Soybean Seeds Plan 1a: Validate genotype-to-phenotype consistency in advanced camelina and soybean lines Transgene analysis: We will continue evaluating transgenic camelina and soybean lines for astaxanthin content and purity in seeds and extracted oil. Expression analysis of the introduced genes will also be performed. HPLC and TLC analyses: High-Performance Liquid Chromatography (HPLC) and Thin-Layer Chromatography (TLC) will be used to quantify total ketocarotenoids and determine the ratio of free versus esterified astaxanthin. Plan 1b: Investigate the evolutionary origin of astaxanthin biosynthesis in Adonis To inform future engineering strategies, we will continue investigating the evolutionary context of astaxanthin biosynthesis in the Adonis genus, including red-petaled species (A. aestivalis) and yellow-petaled relatives, to better understand the genetic and biochemical mechanisms underlying astaxanthin production. Aim 2: Define Metabolic and Transcriptomic Effects of Astaxanthin Production in Camelina and Soybean Seeds Plan 2a: Integrative analysis of metabolic flux and transcriptomic profiles Systems biology analysis: We will analyze previously collected transcriptome and metabolomic data to identify metabolic shifts and gene expression changes associated with astaxanthin biosynthesis. Bottleneck identification and optimization: Key metabolic bottlenecks limiting astaxanthin accumulation will be identified. Gene editing tools will be applied to overcome these constraints and enhance pathway efficiency and seed performance. Aim 3: Optimize Compositional Quality and Agronomic Performance of Astaxanthin-Producing Camelina and Soybean Seeds Plan 3a: Expanded field trials of transgenic lines We will conduct field trials of T4 generation transgenic camelina and soybean lines at multiple locations. Key agronomic traits--such as seed yield, seed size, oil content, and astaxanthin quality--will be evaluated to assess environmental robustness and scalability. Plan 3b: Commercial-scale evaluation and industry engagement We will perform detailed analyses of oil composition and functional co-products in engineered seeds to assess the broader nutritional and industrial value of astaxanthin accumulation. Efforts will also be made to establish collaborations with partners in food science, nutraceuticals, or cosmetics to explore commercial applications of astaxanthin-enriched seeds and their co-products.
Impacts
What was accomplished under these goals?
Aim 1. Optimize Astaxanthin Production and Purity in Camelina Seeds During the current reporting period, we advanced metabolic engineering strategies to enhance astaxanthin biosynthesis in Camelina sativa seeds. Our efforts focused on comparative analyses between prototype I (pASTA), which contains a single copy of CBFD and HBFD, and the ASXx2 construct, which includes two copies of each gene under seed-specific promoters. Result 1a. Gene stacking enhances astaxanthin production and purity In 2024, we progressed to the T4 generation of ASXx2 camelina and assessed astaxanthin accumulation and composition in greenhouse-grown seeds. Visual inspection of T4 seeds indicated consistent pigmentation associated with ketocarotenoid accumulation. Biochemical analyses confirmed stable transgene expression and metabolite profiles. Astaxanthin comprised approximately 30% and 80% of total ketocarotenoids in prototype I and ASXx2 camelina, respectively. HPLC analysis showed average astaxanthin content of 63 µg/g and total ketocarotenoids of 76 µg/g in ASXx2 seeds, compared to 37 µg/g and 124 µg/g in prototype I. Result 1b. Agronomic performance of engineered camelina Greenhouse-based agronomic evaluations of ASXx2 lines were conducted to assess potential trade-offs associated with astaxanthin production. Key traits included seed mass, oil content, and oil composition. Both prototype I and ASXx2 camelina lines showed normal growth. Prototype I seeds had an average 100-seed mass of 85 mg and oil content ranging from 24% to 26%. In ASXx2, 100-seed mass averaged 96 mg, and oil content ranged from 27% to 32%. Result 1c. Evaluation of astaxanthin esterifying enzyme function To improve astaxanthin stability, we evaluated the function of two esterifying enzymes--AaPES (from Adonis) and SlXES (from tomato)--in the ASXx2 background. Both ASXx2-AaPES and ASXx2-SlXES lines were advanced to the T4 generation and analyzed under greenhouse and field conditions. In greenhouse-grown T4 seeds, HPLC analysis showed increased esterified astaxanthin levels. ASXx2-AaPES seeds accumulated 109 µg/g astaxanthin and 132 µg/g total ketocarotenoids; ASXx2-SlXES seeds accumulated 55 µg/g and 69 µg/g, respectively. In both lines, astaxanthin comprised over 80% of total ketocarotenoids. Astaxanthin levels in ASXx2-AaPES seeds were 1.7-fold higher than in ASXx2 alone. Field-grown seeds exhibited similar or slightly elevated astaxanthin levels, indicating stable gene expression across environments. ASXx2 field-grown seeds accumulated 88 µg/g astaxanthin and 94 µg/g total ketocarotenoids. ASXx2-AaPES and ASXx2-SlXES seeds reached 114 µg/g and 95 µg/g astaxanthin, and 122 µg/g and 102 µg/g total ketocarotenoids, respectively. These results confirm the effectiveness of esterifying enzymes in enhancing astaxanthin yield and stability, particularly under field conditions. Result 1d. Progress in soybean transformation and early characterization We continued advancing our soybean transformation pipeline with constructs encoding astaxanthin biosynthetic and esterification genes. T2 ASX-SlXES soybean plants were grown and analyzed in the greenhouse, and T1 ASX-AaPES seeds were obtained, containing two copies of CBFD and HBFD along with the Adonis esterifying gene. We also generated ASXx2 constructs for soybean and obtained T0 plants. T2 ASX-SlXES soybean seeds displayed consistent pigmentation, indicating ketocarotenoid accumulation. Preliminary HPLC analysis showed up to 66 µg/g astaxanthin and 131 µg/g total ketocarotenoids. No growth defects were observed, and T3 seed production is underway. Red/orange T1 ASX-AaPES seeds are being grown for further analysis. Aim 2. Define Metabolic Effects of Astaxanthin Production in Camelina Seeds Result 2a. Metabolite profiling of developing camelina seeds We extended our metabolic flux analyses in astaxanthin-producing camelina lines. Developing seeds from prototype I, ASXx2, ASXx2-SlXES, and ASXx2-AaPES grown in the greenhouse were collected and analyzed. Metabolomic profiling revealed that developmental stage, especially after 20 days after flowering (DAF), was the primary driver of metabolic shifts. PCA showed that all transgenic lines had distinct metabolite profiles from wild-type (WT), especially at 35 DAF, likely reflecting metabolic reprogramming due to astaxanthin biosynthesis. ANOVA identified 61 metabolites with significant genotype × DAF interaction effects, suggesting temporal regulation of metabolic impacts. Compared to WT, transgenic lines accumulated higher levels of several primary metabolites at maturity, including organic acids (pyruvate, malate, glycerate, threonate), sugars and derivatives (fructose, tagatose, glucose-6-phosphate, galactinol), sugar alcohols (myo-inositol), amino acids (histidine), and fatty acids (linoleic acid), indicating a broad upregulation of central carbon metabolism. Prototype I exhibited a unique temporal signature, with slower metabolic progression between 20-30 DAF. Metabolites such as phosphate, threonate, citric acid, fructose, dehydroascorbate, and glucose-6-phosphate were elevated during this period. Notably, threonic acid levels remained high at all measured stages, suggesting possible redox imbalance or perturbations in ascorbate metabolism. This may relate to the delayed germination phenotype observed in prototype I. Aim 3. Optimize Compositional Quality and Agronomic Performance of Astaxanthin-Producing Camelina Result 3a. Agronomic evaluation under greenhouse and field conditions Extensive agronomic evaluation of prototype I and improved astaxanthin-producing lines (ASXx2, ASXx2-AaPES, ASXx2-SlXES) was conducted under greenhouse and field conditions. Assessment included seed yield, size, oil content, and composition. Due to dry conditions during early field growth, germination and development were impaired, and yield data are not included in this report. Seed mass and yield: In the greenhouse, improved lines showed comparable performance to WT (seed mass: ~96 mg/100 seeds; seed yield: 8-13 g/plant). Prototype I showed slightly reduced performance (85 mg/100 seeds; 6-11 g/plant), suggesting improved lines did not compromise yield potential. Oil content and composition: ASXx2 lines exhibited oil contents (28%-30%) within WT ranges. Fatty acid profiles were unchanged across lines, with consistent levels of oleic, linoleic, and α-linolenic acids, supporting the compatibility of carotenoid engineering with camelina's oilseed quality. In 2024, we leveraged this funding in part toward a successful NSF Global Centers grant to UNL "FoodID". As a part of this grant, Camelina and soybeans engineered for ketocarotenoids will be evaluated for food applications.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Hyojin Kim, Kiyoul Park, Edgar B. Cahoon. Nature-Guided Strategies to Maximize Astaxanthin Production and Purity in Camelina Oil for Aquaculture Feed and High-Value Food Applications. Poster presentation at the International Symposium on Plant Lipids, Lincoln, Nebraska, USA. July 1419, 2024.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2024
Citation:
Edgar B. Cahoon, Metabolic Redesign of Oil Metabolism for Enhanced Food, Feed, and Biofuel Traits. Oral presenation at the First International Summit on Plant Resilience, East Lansing, Michigan, USA, May 16, 2024.
|
Progress 01/15/23 to 01/14/24
Outputs
Target Audience:The audience for the this research is biochemists, molecular biologists, geneticists, and biotechnologists who study the underlying metabolism and genes that control the biosynthesis of high-value carotenoids and who aim to use this knowledge to develop crops with increased value for human nutrition and aquaculture feedstocks. Findings from this research are also of interest to oilseed processors, aquaculture feedstock manufacturers, and aquaculture producers who seek high value traits and alternative, cost-competitive, and sustainable sources of natural pigments for food production and aquaculture feed. Ultimately, the audience for the research outcomes of this project are US soybean and other oilseed producers who desire enhanced germplasm with new traits for high-value markets to sustain and increase the profitability of farm production Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The research was primarily conducted by a postdoctoral associate Hyojin Kim who gained experience in oral presentation of her research through three presentations at international conferences in 2023. She also gained experience in mentorship of undergraduate students by serving as a mentor to student Bernadette Traina from Juniata College during the summer of 2023. Bernadette was part of the 2023 cohort of students in the USDA-NIFA funded Research and Extension Experiences for Undergraduates (REEU) grant "Expanding Opportunities in Agricultural Sciences: Crop-to-Food Innovation" that Dr. Cahoon serves as PI . Through participation in the REEU program and in this grant, Bernadette gained laboratory and research training as well as experience in science communication by presentation of her results at the University of Nebraska-Lincoln Undergraduate Summer Research Symposium on August 2, 2023. How have the results been disseminated to communities of interest?The project findings were disseminated to the plant science and plant lipid research communities through three oral presentations at international conferences and two poster presentations at local conferences. These presentations included oral presentations by project postdoctoral associate Dr. Hyojin Kim: 1. Hyojin Kim, Kiyoul Park, Hae Jin Kim, Tara J Nazarenus, Tam Nguyen, Rebecca E Cahoon, Ozan Ciftci, Johnathan A Napier, Edgar B Cahoon.Interrogating Plant Metabolic Diversity for High-value Carotenoid Production in Oilseeds. 2023 Plant Lipids: Structure, Metabolism and Function Gordon Research Conference. Grand Galvez, Texas, USA. Jan. 29-Feb. 03, 2023. 2. Hyojin Kim, Truyen Quach, Shirley J Sato, Hanh T Nguyen, Kiyoul Park, Thomas E Clemente, Edgar B Cahoon. Development of EPA- and Astaxanthin-Enriched Soybean Germplasm for Aquaculture Feedstocks. SOY 2023, Biennial Cellular and Molecular Biology of the Soybean Conference. Lincoln, Nebraska, USA. Aug. 10-13, 2023. 3. Hyojin Kim, Kiyoul Park, Edgar B. Cahoon. Learning from Nature for Strategies to Maximize Production and Purity of the High Value Carotenoid Astaxanthin in Oilseeds. The 9th Asian-Oceanian Symposium on Plant Lipids. Seoul, Republic of Korea. Oct. 10-13, 2023. Project findings were also disseminated through poster presentations by undergraduate researcher Bernadette Traina and Dr. Hyojin Kim: 1. Bernadette Traina, Hyojin Kim, Edgar B. Cahoon. Combinatorial Genetic Strategies for Enhanced Astaxanthin Production in Camelina. University of Nebraska-Lincoln Summer Undergraduate Research Symposium, Lincoln, NE, August 2, 2023. 2. Hyojin Kim, Kiyoul Park, Hae Jin Kim, Tara J Nazarenus, Tam Nguyen, Rebecca E. Cahoon, Ozan Ciftci, Johnathan A. Napier, and Edgar B. Cahoon. Interrogating plant metabolic diversity for high-value carotenoid production in oilseeds. NEBRASKA PLANT SCIENCE SYMPOSIUM. Lincoln, Nebraska, USA. Apr. 25, 2023. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Optimize astaxanthin production and purity in camelina and soybean seeds. Plan 1a. Stacking of genes that promote enhanced astaxanthin production and purity in camelina and soybean seed. We will analyze astaxanthin/astaxanthin esters in red/orange seed of transgenic camelina and soybean. The quantity and quality of ketocarotenoids will be analyzed using TLC and HPLC. Also we will test agronomic performance in the lab scale. Plan 1b. Understand evolutionary position of astaxanthin biosynthesis in Adonis genus. Adonis plant with yellow petal (e.g. Adonis dentata or Adonis vernalis) and with red petal (e.g., Adonis aestivalis) will be grown and we will submit transcriptome analysis using RNA extracted from Adonis petal for comparison analysis for astaxanthin biosynthesis. Aim 2. Define metabolic and transcriptomic effects of astaxanthin production in camelina and soybean seeds. We will initiate these studies using developing soybean and camelina seeds from our current generation of astaxanthin-producing lines. These analyses will be extended to our next-generation of our engineered camelina and soybean lines, as seeds become available. We expect that these studies will identify metabolic bottlenecks that limit ketocarotenoid and astaxanthin accumulation (e.g., catabolic processes; Goal 1) as well as genes whose altered expression affects seed agronomic performance (Goal 3). Working with collaborators at Rothamsted Researcher, our top performing transgene combinations will be transformed into high EPA/DHA omega-3 polyunsaturated fatty acid (PUFA) lines for a complete aquaculture oil feedstock. Aim 3. Optimize compositional quality and agronomic performance of astaxanthin-producing camelina and soybean seeds through application of systems biology-guided approaches. Extensive agronomic evaluation will be conducted on our improved astaxanthin camelina lines in 2024 field trial studies. The lines also will be tested agronomic performance and sufficient amounts of oil from 2024 field will be obtained for functionality studies.
Impacts
What was accomplished under these goals?
Aim 1. Optimize astaxanthin production and purity in camelina seeds. Result 1a. Stacking of genes that promote enhanced astaxanthin production and purity. In the previous reporting period, we described the use of transcriptomics to identify genes for key enzymes for asxtaxanthin biosynthesis4-hydroxy-β-ring 4-dehydrogenase (HBFD) and carotenoid β-ring 4-dehydrogenase (CBFD) from the astaxanthin-rich flower petals ofAdonis aestivalis (CBFD1, CBFD2, HBFD1, HBFD2). We also identified a petal-specific gene for a phytyl ester synthase-like enzyme that we demonstrated is involved in making astaxanthin fatty acid esters, the primary astaxanthin storage form in Adonis flower petals. In this reporting period, we introduced these genes under seed-specific promoters in camelina and soybean.We obtained total thirty T1 plants for ASXx2 (two copies of each of CBFD and HBFD genes and maize phytoene synthase or ZmPSY) transgenic camelina, we selected four lead lines accumulating high level of astaxanthin in seeds. Even we assembled an herbicide resistance cassette (Basta) into the final binary vector to select transformed ASXx2 camelina plant by the herbicide spraying method, ASXx2 T2 seeds were visually sorted out based on the deep red/brown color in their seeds due to ketocarotenoid accumulation. Similarly, with TLC analysis using extracts from T1 seeds which were previously characterized in 2022 in our lab, we found that nearly all of the ketocarotenoid was ketocarotenoid esters in T2 seeds of ASXx2 lines. Using high-performance liquid chromatography (HPLC) analysis, we detected that average value for astaxanthin and total ketocarotenoids was 145 μg/g and 165 μg/g, respectively, in T2 seeds of four lead ASXx2 lines. This was ≥87% of ketocarotenoids present as astaxanthin, rather than ketocarotenoids intermediates, as found in our previous studes. To obtain homozygous line of ASXx2 camelina line, T2 plants were grown in the greenhouse and their T3 seeds were harvested. There were no differences in growth of T2 ASXx2 camelina plants. Based on the red/brown seed color, we obtained the homozygous line for ASXx2 camelina. Using HPLC and thin layer chromatography (TLC) analysis, we found that nearly all of the ketocarotenoid accumulated in the T3 seeds was ketocarotenoid esters in ASXx2 camelina lines. The average value for astaxanthin and total ketocarotenoids after saponification,was 135 μg/g and 153 μg/g, respectively, in the T3 seeds of ASXx2 camelina. According to the previous results in our lab, prototype ASTA camelina, which contains CBFD2, HBFD1 and ZmPSY genes, accumulated astaxanthin, but its content was only 20% of total ketocarotenoids in seed. Therefore, our result in this period indicated the expression of two copies of each of CBFD and HBFD genes are sufficient to produce high quality of astaxanthin in camelina seed. Result 1b. Stacking of genes that promote enhanced seed agronomic performance. The agronomic traits such as seed mass, germination rate, oil content, and oil composition in T3 seeds of ASXx2 camelina were analyzed. As a control, we analyzed prototype ASTA camelina. Prototype ASTA camelina had a reduced seed mass, delayed seed germination and reduced seed oil content. Interestingly, T3 seed mass from our improved astaxanthin producing camelina, ASXx2 camelina, was no big differences from wild type (0.92 mg for ASXx2 vs. 0.90 mg for wild type). To test germination rate of T3 seeds, sterilized seeds were monitored for both germination rate and percentage under sterilized and moisturized filter paper. The germination rate (percentage) was over 85% in T3 seeds of ASXx2 camelina after 24 hours after sowing, which is similar observation of wild type, while our prototype ASTA camelina showed 10% of germination rate at the same time point. In addition, similar with wild type level, the seed oil content in T3 seed of ASXx2 camelina was ~21 to ~ 22 % of seed weight (wild type: 21 % of seed weight). These results suggested that the ASXx2 camelina has been improved the quality and quantity of astaxanthin in their seeds and has normal agronomic traits compared to our prototype ASTA camelina. Result 1c. Test of astaxanthin biosynthetic enzymes and its esterifying enzyme in camelina. According to the previous report, one candidate for astaxanthin esterifying enzyme from Adonis (transcript 25425; AaAES) and one candidate from tomato (SlXES) were investigated their activity for astaxanthin esterification in N. benthamiana leaves and ASXx2-AaPES transgenic camelina was generated. In this period, we also obtained ASXx2-SlXES transgenic camelina. To test quality and quantity of astaxanthin in transgenic camelina lines, we harvested T2 seeds from each of ASXx2-AaPES and ASXx2-SlXES camelina. Using HPLC analysis, we quantified ketocarotenoids in T2 seed of ASXx2-AaPES and ASXx2-SlXES camelina. After saponification, the average value of astaxanthin and total ketocarotneoids was 189 μg/g and 217 μg/g, respectively, in ASXx2-AaPES camelina, and 164 μg/g and 196 μg/g, respectively, in ASXx2-SlXES camelina. And the rate of free astaxanthin level without saponification over free astaxanthin level with saponification processing was less than 8% in both transgenic camelina expressing astaxanthin esterifying gene (ASXx2-AaAES and ASXx2-SlXES). As a control, we calculated the rate of free astaxanthin level without saponification over free astaxanthin level with saponification processing in ASXx2 camelina, which was over than 11%. In addition, the astaxanthin esterifying gene was well expressed in seeds of each of ASXx2-AaAES and ASXx2-SlXES camelina. Using TLC and HPLC analysis in T3 seed of ASXx2-AaPES and ASXx2-SlXES camelina, we detected ~220 μg/g of astaxanthin level and ~250 μg/g of total ketocarotenoid level in seed of ASXx2-AaPES and ~176 μg/g of astaxanthin level and ~205 μg/g of total ketocarotenoid level in seed of ASXx2-SlXES. Interestingly, the astaxanthin level from transgenic camelina expressing astaxanthin esterifying gene was 120 to 150% higher than that from ASXx2 camelina. These results indicated transgenic camelina expressing astaxanthin esterifying gene is one of key steps to confer astaxanthin accumulation in the ester form in seeds. Aim 2: Generation of astaxanthin producing soybean Result 2a. Generation of transgenic soybean producing astaxanthin and astaxanthin esters. Our lab attempted to generate astaxanthin producing camelina and soybean and found the metabolic flux for astaxanthin biosynthesis in seed was distinguishable between camelina and soybean. To define this differences in metabolic and transcriptomic effects of astaxanthin production in camelina and soybean seeds, we also designed binary vectors harboring astaxanthin biosynthetic genes, CBFD1/2 and HBFD1/2, and/or astaxanthin esterifying genes, AaPES or SlXES for the soybean transformation. We constructed binary vector with all genes expression driven by seed-specific promoters and assembled an herbicide resistance cassette into final binary vector. Now, soybean transformation is in progress and we obtained one event (1443-4) for ASX-SlXES soybean. We found orange/red T1 seed of ASX-SlXES soybean and twenty-five T1 plants are growing in the greenhouse to get next generation. Aim 3. Optimize compositional quality and agronomic performance of astaxanthin-producing camelina and soybean seeds through application of systems biology-guided approaches. For agronomic evaluation of our improved astaxanthin camelina, we obtained homozygous lines for each of ASXx2, ASXx2-AaAES, and ASXx2-SlXES camelina. We selected two lead plants for each of ASXx2, ASXx2-AaAES, and ASXx2-SlXES camelina and prepared seed for a 2024 field trial.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Konda AR, Gelli M, Pedersen C, Cahoon RE, Zhang C, Obata T, Cahoon EB (2023) Vitamin E biofortification: Maximizing oilseed tocotrienol and total vitamin E tocochromanol production by use of metabolic bypass combinations. Metabolic Engineering 79: 66-77.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Hyojin Kim, Kiyoul Park, Hae Jin Kim, Tara J Nazarenus, Tam Nguyen, Rebecca E. Cahoon, Ozan Ciftci, Johnathan A. Napier, and Edgar B. Cahoon. Interrogating plant metabolic diversity for high-value carotenoid production in oilseeds. NEBRASKA PLANT SCIENCE SYMPOSIUM. Lincoln, Nebraska, USA. Apr. 25, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Bernadette Traina, Hyojin Kim, Edgar B. Cahoon. Combinatorial Genetic Strategies for Enhanced Astaxanthin Production in Camelina. University of Nebraska-Lincoln Summer Undergraduate Research Symposium, Lincoln, NE, August 2, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Hyojin Kim, Kiyoul Park, Hae Jin Kim, Tara J Nazarenus, Tam Nguyen, Rebecca E Cahoon, Ozan Ciftci, Johnathan A Napier, Edgar B Cahoon. Interrogating Plant Metabolic Diversity for High-value Carotenoid Production in Oilseeds. 2023 Plant Lipids: Structure, Metabolism and Function Gordon Research Conference. Grand Galvez, Texas, USA. Jan. 29-Feb. 03, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Hyojin Kim, Truyen Quach, Shirley J Sato, Hanh T Nguyen, Kiyoul Park, Thomas E Clemente, Edgar B Cahoon. Development of EPA- and Astaxanthin-Enriched Soybean Germplasm for Aquaculture Feedstocks. SOY 2023, Biennial Cellular and Molecular Biology of the Soybean Conference. Lincoln, Nebraska, USA. Aug. 10-13, 2023.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Hyojin Kim, Kiyoul Park, Edgar B. Cahoon. Learning from Nature for Strategies to Maximize Production and Purity of the High Value Carotenoid Astaxanthin in Oilseeds. The 9th Asian-Oceanian Symposium on Plant Lipids. Seoul, Republic of Korea. Oct. 10-13, 2023.
|
Progress 01/15/22 to 01/14/23
Outputs
Target Audience:The audience for the this research is biochemists, molecular biologists, geneticists, and biotechnologists who study the underlying metabolism and genes that control the biosynthesis of high-value carotenoids and who aim to use this knowledge to develop crops with increased value for human nutrition and aquaculture feedstocks. Findings from this research are also of interest to oilseed processors, aquaculture feedstock manufacturers, and aquaculture producers who seek high value traits and alternative, cost-competitive, and sustainable sources of natural pigments for food production and aquaculture feed. Ultimately, the audience for the research outcomes of this project are US soybean and other oilseed producers who desire enhanced germplasm with new traits for high-value markets to sustain and increase the profitability of farm production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The research was primarily conducted by a postdoctoral associate Hyojin Kim who has gained experience in oral presentation of her research through two presentations at international conferences. She also gained experience in mentorship of undergraduate students by serving as a mentor to student Gannon Cole from West Virginia State University during the summer of 2022. In January, 2022, PD Cahoon was awarded a USDA- NIFA funding Research and Extension Experiences for Undergraduates (REEU) grant "Expanding Opportunities in Agricultural Sciences: Crop-to-Food Innovation" that has a recruitment focus on underrepresented minority students from 1890 Land Grant Universities (LGU) and other HBCUs. During the summer of 2022, undergraduate student Gannon Cole, recruited from the HBCU/1890 LGU West Virginia State University, conducted research as part of this grant. Through participation in this grant, he gained laboratory and research training and experience in science communication by presentation of his results at the University of Nebraska-Lincoln Undergraduate Summer Research Symposium on August 5, 2022. How have the results been disseminated to communities of interest?Dissemination to farmers/producers: PDCahoon described the value of oilseed engineering research to US farmers during an interviewon the September 17, 2022 US Farm Report, aired on RFD TV. Link: https://www.youtube.com/watch?v=cAy5NecWmLA. Dissemination to public researchers: PD Cahoon presented project findings in a virtual oral presentation at the 2nd World Congress on Oleo Science (WCOS 2022), "Development of oilseed aquaculture feedstocks: synthetic biology approaches for productions of oils with high value carotenoids", authors: Edgar Cahoon, Hyojin Kim, Truyen Quach, Kiyoul Park, Tom Elmo Clemente, September 2, 2022. Postdoctoral researcher Hyojin Kim presented project findings in an oral presentation at the 2022 Annual Meeting of the Phytochemical Society of North America, "Metabolic engineering of oilseeds for sustainable astaxanthin production", July 26, 2022, Blacksburg, VA. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Optimize astaxanthin production and purity in camelina and soybean seeds. Aim 1a. Stacking of genes that promote enhanced astaxanthin production and purity. We will analyze astaxanthin/astaxanthin esters in red/orange seed of transgenic camelina. The quantity and quality of ketocarotenoids will be analyzed using TLC and HPLC. Plan 1b. Understand evolutionary position of astaxanthin biosynthesis in Adonis genus. Adonis plant with yellow petal (e.g. Adonis dentata or Adonis vernalis) and with red petal (e.g. Adonis aestivalis) will be grown and we will submit transcriptome analysis using RNA extracted from Adonis petal for comparison analysis for astaxanthin biosynthesis. Aim 2. Generation of astaxanthin producing camelina or soybean. Define metabolic and transcriptomic effects of astaxanthin production in camelina and soybean seeds. We will initiate these studies using developing soybean and camelina seeds from our current generation of astaxanthin-producing lines. These analyses will be extended to our next-generation of our engineered camelina and soybean lines, as seeds become available. We expect that these studies will identify metabolic bottlenecks that limit ketocarotenoid and astaxanthin accumulation (e.g., catabolic processes; Goal 1) as well as genes whose altered expression affects seed agronomic performance (Goal 3). Working with collaborators at Rothamsted Researcher, our top performing transgene combinations will be transformed into high EPA/DHA omega-3 polyunsaturated fatty acid (PUFA) lines for a complete aquaculture oil feedstock. Aim 3. Optimize compositional quality and agronomic performance of astaxanthin-producing camelina and soybean seeds through application of systems biology-guided approaches. Extensive agronomic evaluation will be conducted on our improved astaxanthin camelina lines in greenhouse studies. The lines will also be bulked in the greenhouse for a 2024 field trial of these lines and to generation sufficient amounts of oil for functionality studies.
Impacts
What was accomplished under these goals?
Aim 1: Optimize astaxanthin production and purity in plant. Result Ia. Stacking of genes that promote enhanced astaxanthin production and purity. Adonis aestivalisflower petals accumulate 1% to 2% dry weight of astaxanthin, the highest known concentration of astaxanthin in plants. We tapped the biochemistry and genetics ofAdonis aestivalisto identify genes that will enhance asxtaxanthin production and accumulation in engineered camelina and soybean seeds. Given thatAdonis aestivalispetals accumulate astaxanthin in a form that contains two fatty acid esterified to its ionone ring hydroxyl groups, we focused not only on biosynthetic genes but also plastid localized fatty acid acyltransferases.To quantify gene expression in Adonis aestivalis petal, we submitted PacBio Isoform Sequencing and Illumina RNA Sequencing. Carotenoid biosynthetic pathway genes were identified and their gene expression quantifiedusing transcriptome data. Based on KEGG pathway (ath00906 carotenoid biosynthesis), the paralogue genes of Arabidopsis genes were explored in BLAST server of A. aestivalis petal transcriptome data. In addition, we also explored astaxanthin biosynthetic genes based on astaxanthin biosynthetic pathway, which was previous reported in A.aestivalis. Since A. aestivalis is a polyploid, two paralog genes corresponding to biosynthetic genes on the carotenoid pathway was detected. The transcript level of CBFD1/2 gene for 3-hydroxylation of β-carotene which is the first branch to produce astaxanthin was enriched compared to transcript level for BCH1/2 which is responsible for 4-hydroxylation of β-carotene to zeaxanthin synthesis. To produce astaxanthin in plant host, we designed binary vectors containing CBFD1/2 and HBFD1/2 genes from Adonis plant or/and phytoene synthase (ZmPSY) gene from maize using Golden Braid stacking methodology. For the transient expression assay in Nicotiana benthamiana leaves, CaMV 35S promoter was used to drive the expression of CBFD1/2 or/and HBFD1/2 genes. For the generation of transgenic camelina and soybean, seed-specific glycinin promoter was used to drive the expression of CBFD1/2, HBFD1/2 and ZmPSY genes. Result 1b. Binary vector construction by applying a 2A self-cleaving peptides system to avoid possibility of gene silencing of transgenes by repetitive use of same promoter in plant host. We designed transient expression vector containing astaxanthin biosynthetic genes linked each other using P2A or T2A linkers. Astaxanthin was successfully accumulated when CBFD1-P2A-HBFD1-T2A-ZmPSY expression cassette was introduced in N. benthamiana leaves. In addition, we constructed two gene expression cassettes under the control of seed-specific glycinin promoter to produce transgenic camelina that produces astaxanthin in seed. One expression cassette was designed so that CBFD1, HBFD1, and ZmPSY genes were interconnected using P2A and T2A. Other expression cassette was designed so that CBFD2 and HBFD2 genes were connected to each other using P2A. These two expression cassettes and basta resistance cassette were assembled into the final binary vector using the Golden Braid method. We found that T1 camelina seed showed red/orange color in their seeds. To quantify and qualify the astaxanthin, T1 plants are growing in the greenhouse. Result 1c. Test of astaxanthin esterifying enzyme candidates. Based on the previous reports, the genes for xanthophyll esterase and GDSL-lipase as candidates for an astaxanthin esterifying enzyme were blasted in our Adonis transcriptome. For the paralogousgenes of xanthophyll esterase (XES), we selected a transcript 5819/25015 as well as transcript 6566/25425 which was characterized at previous year 2022 in our lab. In addition, transcript 23871 as a paraloggene of GDSL-lipase was isolated. To investigate the activity for astaxanthin esterification of candidates, transient expression vector was designed and constructed using CaMV35S promoter. The ketocarotenoid esters were accumulated in N. benthamiana leaves when transcript 5819/25015 was expressed along with astaxanthin synthetic genes as well as transcript 6566/25425 or XES from tomato (SlXES) did before, but not transcript 23871. Among the products detected from transient expression were astaxanthin fatty acid esters, which were absent in transient expression assays that lacked the acyltransferase transgenes. This result suggested that the Adonis and tomato genes encode enzymes withastaxanthin acyltransferase activity. Aim 2: Generation of astaxanthin producing camelina or soybean Result 2a. Gerneration of transgenic camelina producing astaxanthin and astaxanthin esters. To improve the purity and quantity in engineered camelina and soybean seeds, we transformed the binary vector (mentioned above) containing two copies of each of CBFD and HBFD genes and ZmPSY into camelina (ASXx2). Two additionalexpression cassettes containing astaxanthin esterifying genes, transcript 6566/25425 (ASXx2-AaePES) or SlXES (ASXx2-SlXES), were assembled with astaxanthin biosynthesis cassette into new binary vectors vectors and used for camelina transformation. As a control, we also transformed camelina with only the astaxanthin biosynthetic genes lacking the acyltransferase.Using thin-layer chromatography (TLC), we found that nearly all of the ketocarotenoid accumulated in the T1 seeds was ketocarotenoid esters in camelina lines engineered with the acyltransferase transgenes. Following saponification, we observed that nearly all of the ketocarotenoids were present as astaxanthin.In contrast, the majority of ketocarotenoids in seeds from camelina transformed with only astaxanthin biosyntheticgenes were ketocarotenoid intermediates of astaxanthin. This result indicatest that the acyltransferases yield much higher astaxanthin purity than use of only the biosynthetic transgenes. Using high-performance liquid chromatography (HPLC)analysis, we found that astaxanthin content ranged from 27 μg/g to 71 μg/g in T2 seeds of ASXx2 transgenic camelina, while it ranged from 43 μg/g to 98 μg/g in T2 seeds of ASXx2-AaePES transgenic camelina. This is ~two-times more astaxanthin than in our previous lines lacking acyltransferase transgenes.We are very excited about these findings! These constructs are now in queue for soybean transformation. Aim 3. Optimize compositional quality and agronomic performance of astaxanthin-producing camelina and soybean seeds through application of systems biology-guided approaches. Our initial results from camelina engineered with biosynthetic and acyltransferase transgenes indicate improved germination and seedling stand, comparable of wild-type/non-egineered seeds. This will be examined in additional detail in the next year.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Kim H, Park K, Cahoon EB (2022) Metabolic engineering of oilseeds for sustainable
astaxanthin production. 2022 Annual Meeting of the Phytochemical Society of North America, Blacksburg, VA, July 26, 2022. (oral presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Cahoon EB, Kim H, Quach T, Park K, Clemente T (2022) Development of oilseed aquaculture feedstocks: synthetic biology approaches for productions of oils with high value carotenoids. 2nd World Congress on Oleo Science (WCOS 2022), September 2, 2022. (virtual oral presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Cole G, Kim H, Cahoon EB (2022) Synthetic Biology of EPA and Ketocarotenoids Production in Soybean to Provide Sustainable Aquacultural Feedstocks. University of Nebraska-Lincoln Summer Undergraduate Research Symposium, Lincoln, NE, August 5, 2022 (Poster presentation)
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Kim H, Park K, Clemente TE, Cahoon EB (2023) Interrogating plant metabolic diversity for high-value carotenoid production in oilseeds. Gordon Research Conference Plant Lipids: Structure, Metabolism and Function, Gallveston, TX, February 1, 2023. (Oral presentation)
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Progress 01/15/21 to 01/14/22
Outputs
Target Audience:The audience for the this research is biochemists, molecular biologists, geneticists, and biotechnologists who study the underlying metabolism and genes that control the biosynthesis of high-value carotenoids and who aim to use this knowledge to develop crops with increased value for human nutrition and aquaculture feedstocks. Findings from this research are also of interest to oilseed processors, aquaculture feedstock manufacturers, and aquaculture producers who seek high value traits and alternative, cost-competitive, and sustainable sources of natural pigments for food production and aquaculture feed. Ultimately, the audience for the research outcomes of this project are US soybean and other oilseed producers who desire enchanced germplasm with new traits for high-value markets to sustain and increase the profitability of farm production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The researchis primarily conducted by a postdoctoral associate who has gained experience in preparation and writing of journal articles and progress reports through participation in this project. She also has developed her presentation skills through periodic lab meeting oral reporting of her research. She also has access to career development workshops through participation in Collective Research Organization of Plant Scientists ("CROPS"), an early career researcher professional organization in the Center for Plant Science Innovation at the University of Nebraska-Lincoln. It is also planned that the postdoctoral associate will report project findings at an international conference in 2022. The project has also had participation of undergraduate students who have gained lab research experience to better prepare them for STEM careers and/or graduate school training. To advance participation of underrepresented minority students in STEM, we also hosted a summer research experiences for undergraduate students in 2021. Two students from 1890 Land Grant HBCUs (North Carolina A&T University and Alabama A&M) participated in the program and conducted research related to this project on the characterization of oilseed quality traits. We leveraged the experience from this pilot program to successfully obtain USDA-NIFA funding for a Research and Externsion Experiences for Undergraduates (REEU) program (Expanding Opportunities in Agricultural Sciences: Crop-to-Food Innovation) that will focus recruitment efforts on underrepresented minority students from 1890 Land Grant Universities and other HBCUs. How have the results been disseminated to communities of interest?During this reporting period, project results were reported by publication in the peer-reviewed journal aBIOTECH and through oral presentations at the 2021 Annual Meeting of the American Oil Chemists Society (virtual) and at the first annual Soybean Research Forum and Think Tank (August 25-27, 2021; Indianapolis, IN). What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Optimize astaxanthin production and purity in camelina and soybean seeds. Towards this goal, we will stack or express combinations of genes identified from our Adonispetal transcriptomic studies in camelina and soybean seeds. We will use GoldenBraid modular gene assembly method to generate multigene vector constructs for astaxanthin-producing engineered camelina and soybean lines using four astaxanthin biosynthetic genes (CBFD1/2 and HBFD1/2) and the astaxanthin esterification gene. The 2A self-cleaving peptides system will be applied to construct binary vector to avoid possibility of gene silencing of transgenes by repetitive use of promoters in plant system. We will also compare the in planta activities of the newly identified Adonis esterification enzyme withother known astaxanthin esterifying enzyme candidates, including the Arabidopsis PES1 or tomato PYP1 that encode a phytyl ester synthase. These studies will be conducted using transient expression of genes for these enzymes inNicotianabenthamiana leaves.These studies will inform our camelina and soybean metabolic engineering efforts. Goal 2. Define metabolic and transcriptomic effects of astaxanthin production in camelina and soybean seeds. We will initiate these studies using developing soybean and camelina seeds from our current generation of astaxanthin-producing lines. These analyses will be extended to our next-generation of our engineered camelina and soybean lines, as seeds become available. We expect that these studies will identify metabolic bottlenecks that limit ketocarotenoid and astaxanthin accumulation (e.g., catabolic processes; Goal 1)as well as genes whose altered expression affects seed agronomic performance (Goal 3).
Impacts
What was accomplished under these goals?
Research during the past year focused primarily on Goal 1: optimize astaxanthin production and purity in camelina and soybean seeds. Field studies were conducted for astaxanthin producing camelina and soybean lines were initiated in May 2021 using APHIS-regulated biotech plotsat the Eastern Nebraska Research and Extension Center (ENREC) near Mead, NE. Seeds from these lines accumulated ketocarotenoids to amounts of up to 137 µg/g seed weight. Of these amounts, ~25 to 40% of the total ketocarotenoids were in the form of astaxanthin. The remainder were biosynthetic intermediates, including adonixanthin. TAnd we have observed that total ketocarotenoid content ranged from 12 to 13.7% and 1.3 to 4.0 % of seed weight, respectively. Interestingly, astaxanthin producing camelina and soybean lines have been observed to accumulate not only astaxanthin but also intermediate metabolites (e.g., adonixanthin) in biosynthetic pathways. These lines were generated by seed-specific expression of theAdonis aestivalisCBFD2 and HBFD1 genes. We explored the possibility that additional or alternative genes from Adonis may increase total ketocarotenoid and astaxanthin production. To pursue this, we generated a PacBio transcriptome of RNA extracted from astaxanthin-richAdonispetals. Notably, we determined through multiple analytical methodsthat >95% of the total ketocarotenoids in Adonis petals occursas a diester form with fatty acids linked to the hydroxyl groups of the rings of astaxanthin. Total amounts of astaxanthin in these petals is in the range1.3% to 2.2% of petal dry weight.Though BLAST gene homology analyses, we identifed transcript 29266 for CBFD1 and transcript 28613 for it homologCBFD2 as well as transcript 22383 for HBFD1and transcript 20101 for its homlogHBFD2.CBFD1andHBFD2are "new" paralogs ofCBFD2andHBFD1. Functional characterization of full-length transcripts for these genes was conducted by transient expression in Nicotianabenthamianaleaves and thin layer chromatography (TLC) anlysis of products. Expression ofCBFD2 and HBFD1 (gene set from our previous studies) resulted in the accumulation of astaxanthin to ~50% of total ketocarotenioids along with intermediates, such as adonixanthin. When all four astaxanthin biosynthetic genes (CBFD1/2 and HBFD1/2) were expressed together, the accumulation of intermediate metabolites was significantly reduced and astaxanthin purity increased to ~90% of total ketocarotenoids. Based on previous studies thatreported several genes involved in astaxanthin esterification, four xanthophyll esterase transcripts and three acyltransferase genes involved in oil synthesis were identifed in the Adonis petal transcriptome by BLAST analysis. Among these candidates, the transcript 6566 wasselected for further study, since its expression level is relatively higher in Adonis petals compared to leaves. Nicotiana benthamiana infiltration system was used to co-express the gene for the astaxanthin-ester forming candidate enzyme eith thefour astaxanthin biosynthetic genes with the transcript 6566. Results from these studies showed the production of astaxanthin esters, whichwere not detectable by expression of the four astaxanthin biosynthetic genes alone. The findings confirmed that Adonis transcript 6566 encodes an astaxanthin esterifying enzyme. Overall, transcriptome mining revealed that Adoniscan provide an extended genetic source for enhancing total ketocarotenoid production and astaxanthin purity in oilseeds.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Sun, T., Zhu, Q., Wei, Z., Owens, L. A., Fish, T., Kim, H., Thannhauser, T. W., Cahoon, E. B. & Li, L. (2021) Multi-strategy engineering greatly enhances provitamin A carotenoid accumulation and stability in Arabidopsis seeds, aBIOTECH. 2, 191-214 [10.1007/s42994-021-00046-1].
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cahoon, Edgar, Kiyoul Park, Hyojin Kim, and Tom Clemente. (2021) "Synthetic Biology Application for Development of High-value Oil Traits." Annual Conference of the American Oil Chemists Society. vol. 98, pp. 41-42. May 6, 2021 (virtual).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Cahoon, E.B. (2021) Moving Soybean Beyond Commodity Markets. Soybean Research Forum and Think Tank. August 25-27, 2021, Indianapolis, IN.
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