Source: WASHINGTON STATE UNIVERSITY submitted to
PHYSARIA FENDLERI CROP IMPROVEMENT BY GENE EDITING AND BIOENGINEERING FOR A DOMESTIC HIGH YIELDING SOURCE OF INDUSTRIALLY VALUABLE FATTY ACIDS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1029709
Grant No.
2023-67013-39022
Cumulative Award Amt.
$650,000.00
Proposal No.
2022-08464
Multistate No.
(N/A)
Project Start Date
Feb 1, 2023
Project End Date
Jan 31, 2026
Grant Year
2023
Program Code
[A1103]- Foundational Knowledge of Plant Products
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
(N/A)
Non Technical Summary
Major plant oil crops produce triacylglycerols (TAG, e.g. oil) composed of predominantly five common fatty acids. However, the plant kingdom produces more than 450 unusual fatty acids that can be useful as chemical feed stocks in a wide range of industries. For example, castor oil (produced mostly in India) is worth over $1 billion USD per year and the hydroxy fatty acids (HFA) contained in the oil are used for pharmaceuticals, cosmetics, plastics, biodegradable polyesters, and biofuels. The US is a major importer of castor oil for the oleochemical industry. Research to engineer HFA production into a current domestic oilseed crop has had very limited success. However, Physaria fendleri is a burgeoning oilseed crop native to the US that produces HFA similar to castor. Our recent research in P. fendleri indicates it utilizes a novel TAG remodeling biosynthetic pathway to efficiently accumulate HFA and overcome the metabolic bottlenecks that are present when HFA production is engineered into other crop species. This proposal will: (1) utilize a newly developed functional genomics approach in P. fendleri to elucidate the efficient mechanisms of HFA accumulation, that could be used for engineering designer oil compositions into crop plants; (2) utilize gene editing to enhance P. fendleri as a domestic source of HFA by increasing seed oil, HFA content, and total seed yields; (3) evaluate the use of P. fendleri (and its novel TAG remodeling mechanisms) as a domestic platform crop for production of various industrially valuable fatty acids.
Animal Health Component
5%
Research Effort Categories
Basic
95%
Applied
5%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2061850104080%
2061850100010%
5111899104010%
Knowledge Area
206 - Basic Plant Biology; 511 - New and Improved Non-Food Products and Processes;

Subject Of Investigation
1850 - Lesquerella; 1899 - Oilseed and oil crops, general/other;

Field Of Science
1040 - Molecular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
Goals:Our long-term goals are: to elucidate the genetic components that control the accumulation of novel seed oil fatty acid compositions (and total amounts) that can be used for enhanced breeding or engineering of oilseed crops as feedstocks for the oleochemical industry.Within this proposal our goals are to: Identify mechanisms of efficient unusual fatty acid flux through the lipid metabolic network to enhance all oilseed crop bioengineering, characterize genes that can improve total oil and hydroxy fatty acid accumulation in Physaria fendleri through gene editing, and develop Physaria fendleri as a platform for the production of other valuable oil fatty acid compositions.OBJECTIVE 1: Functional genomics in P. fendleri to characterize the mechanisms of efficient hydroxy fatty acid flux through the lipid metabolic network into oil, and identify regulators of total oil accumulation (Years 1-3).Our newly developed VIGS based functional genomics approach in P. fendleri will be utilized to enhance our understanding of key unknowns in plant lipid metabolism and identify the molecular control of oil synthesis specifically in P. fenderli that could be targets for improving P. fendleri as a high yielding oilseed crop. The gene targets for our functional genomics approach will investigate mechanisms of fatty acid acyl editing, the selective production of phosphatidylcholine-derived diacylglycerol for triacylglycerol synthesis, and regulators of total oil synthesis and accumulation.OBJECTIVE 2. Targeted gene editing in for increased oil, HFA, and total seed yields (Years 1-2).Based on our preliminary RNA knockdown results we hypothesize a sdp1 knockout mutation will further enhance oil and HFA levels, and a srk knockout mutation will produce self-compatible flowers leading to an increase in total seed yield. Therefore, CRISPR/ cas9 technology will be used to generate mutations in SDP1 and SRK genes individually as well as together, to evaluate the individual and combined roles of each mutation on P. fendleri oil content and seed yield. Additionally, the self-compatible mutant will be a valuable background for producing homozygous transgenic P. fendleri (Objective 3), by reducing outcrossing and the manual labor required to force self-crossing during propagation of transgenic plants.?OBJECTIVE 3. Bioengineering P. fendleri as a platform crop for "castor oil" and other valuable unusual fatty acids (Years 2-3).We hypothesize that Physaria fendleri can be a highly useful platform species to engineering valuable unusual fatty acid compositions (over that of species such as camelina) due to its inherit ability to efficiently channel unusual fatty acids into seed oils. In Objective 3 we aim to produce two different seed oil compositions in P. fendleri to evaluate its value as an engineering platform crop through two phases: (1) gene editing to produce the platform backgrounds (fae1, and fae1/fah12); (2) transgene expression to produce designer oils for triacylglycerol containing three ricinoleic acids, or triacylglycerol containing two expoxy fatty acids. If the SRK gene editing in Objective 2 indicates no adverse effect on plant growth then we will cross the background lines with the srk mutant such that self-compatibility will aid in development of homozygous transgenic lines.
Project Methods
OBJECTIVE 1: Functional genomics in P. fendleri to characterize the mechanisms of efficient hydroxy fatty acid flux through the lipid metabolic network into oil, and identify regulators of total oil accumulation (Years 1-3).Candidate genes will be utilized for a virus induced gene silencing (VIGS) approach to identify genes involved in efficient trafficking of hydroxy fatty acids into seed oils and regulators total oil accumulation. Seed oil amount and fatty acid composition will be determined from silences lines through gas chromatography.OBJECTIVE 2. Targeted gene editing in for increased oil, HFA, and total seed yields (Years 1-2).CRISPR/ cas9 technology will be used to generate mutations in SDP1 and SRK genes individually as well as together, to evaluate the individual and combined roles of each mutation on P. fendleri oil content and seed yield. Seed oil amount and fatty acid composition will be determined from silences lines through gas chromatography.OBJECTIVE 3. Bioengineering P. fendleri as a platform crop for "castor oil" and other valuable unusual fatty acids (Years 2-3).CRISPR/ cas9 technology will be used to generate mutations in FAE1 and FAH12 genes, to evaluate the individual and combined roles of each mutation on P. fendleri oil content and seed yield. Additionally, transgenes will be expressed in the mutant backgrounds to attempt to produce novel seed oil fatty acid compositions first focusing on hydroxy fatty acid accumulation, second on expoxy fatty acid synthesis. Seed oil amount and fatty acid composition will be determined from silences lines through gas chromatography.

Progress 02/01/24 to 01/31/25

Outputs
Target Audience:The target audience for this project is plant breeders, the plant metabolism community, plant biochemists and scientists and engineers engaged in manipulating plant oil production for food, fuel or feed stock needs. In addition, the target audience will include graduate students and undergraduates in plant biochemistry and molecular biology who will be able to use publications from these studies to understand plant oil production. Changes/Problems:As indicated in the last report, the initial postdoc working on the project left for a job in industry. Recruitig of a new postdoc with the required skill sets took almost a year, which had slowed down and delayed the project a bit. Despite this difficulty, we have been able to maintain pushing the project along by partime work from other lab members.A new full time postdoc was hired in Sept. 2024 and our progress is increasing speed. Due to the slowdown we expect a no cost extention will be required to fully complete the main objectives. What opportunities for training and professional development has the project provided?This work has led to the training of twopostdocs and one undergraduate in research experimentation, scientific writing, and scientific presentations. Including two oral presentations by postdocs at international scientific meetings. How have the results been disseminated to communities of interest?The results generated here were disseminated to communities of interest through two peer reviewed journal articles published, one US patent published, and 3 oral presentations and one poster presentation at three international scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Six more gene candidates will be functionally evaluated for roles in seed oil accumulation through our VIGS system. Objective 2: Identification of CRISPR/Cas9 generated mutants and propagation for analysis for seed yield and oil phenotypes. Objective 3: Evaluation of P. fendleri gene based TAG remodeling to enhance the accumulation of healthy fatty acids in transgenic Camelina sativa. Identification of CRISPR/Cas9 generated mutant lines suitable for additional bioengineering of desired oil fatty acid compositions in P. fendleri.

Impacts
What was accomplished under these goals? Objective 1. Functional genomics in P. fendleri to characterize the mechanisms of efficient hydroxy fatty acid flux through the lipid metabolic network into oil, and identify regulators of total oil accumulation. (70% complete) Two journal article have been published (Parchuri et al. 2024 Nature Communications; and Azeez and Bates 2024 Plant Biotechnology Journal) and one patent application published that together characterize ten genes involved in P. fendleri lipid metabolism, self-incompatibility, and chlorophyll biosynthesis. Additionally, five genes were characterized by more in-depth functional analysis by in vitro enzyme assays, protein-protein interactions, and/or in vivo cellular localization analysis. Through this enhanced analysis we have identified a previously uncharacterized (in any plant) triacylglycerol (TAG, e.g. oil) lipase (PfeTAGL1) that is involved in the hypothesized TAG remodeling to accumulate valuable unusual hydroxylated fatty acids (HFA) in seed oil. Additionally, our enhanced biochemical analysis included the first ever (in all fields) diacylglycerol acyltransferase (DGAT) substrate assays for diacylglycerol (DAG) enantiomer selectivity (sn-1,2-DAG vs sn-2,3-DAG). Here we demonstrate that PfeDGAT1 is selective for sn-1,2-DAG whereas PfeDGAT2 is selective for sn-2,3-DAG). These results provide a biochemical understanding of how the proposed TAG remodeling pathway works. First, PfeDGAT1 adds a sn-3 HFA to sn-1,2-DAG derived from the membrane lipid phosphatidylcholine that did not contain HFA, thus generating a TAG molecule containing only sn-3 HFA. Then PfeTAGL1 which interacts with PfeDGAT1 removes the sn-1 common fatty acid generating sn-2,3-DAG containing a sn-3 HFA. Subsequently, PfeDGAT2 selectively transfers a HFA to the sn-1 position of the sn-2,3-DAG generating the final TAG containing sn-1/3 HFA. Through the TAG remodeling mechanism P. fendleri can accumulate HFA up to 66% of the oil, while limiting accumulation of HFA in membrane lipids which is detrimental to membrane structure/function. These enhanced biochemical results of the P. fendleri gene products were published in Parchuri et al. 2024 Nature Communications. The novel discovery that DGATs can be selective for different enantiomers of DAG prompted us to test DGAT1s and DGAT2s from different related Brassicaceae species (Arabidopsis thaliana and Camelina sativa), and a more distantly related species that also accumulates HFA (Ricinus communis). C. sativa DGAT1 and DGAT2 DAG enantiomer selectivity was similar to P. fendleri. However, for Arabidopsis AtDGAT1 used both enantiomers efficiently whereas DGAT2 only used sn-1,2-DAG. R. communis was distinctly different in that both RcDGAT1 and RcDGAT2 only used the sn-1,2-DAG enantiomer. Based on these results we hypothesize that other Brassicaceae species may also be able to do TAG remodeling, but R. communis cannot. Considering that both Arabidopsis and Camelina make TAG from DAG derived from the membrane lipid phosphatidylcholine (similar to P. fendleri), but R. communis synthesizes DAG for TAG production de novo, it may suggest that TAG remodeling has evolved to accumulate fatty acids in the oil that are distinct from the fatty acids within the membrane lipid substrates to oil synthesis in these species. This hypothesis will be investigated further in the future. In addition to the above results, an addtional six VIGS constructs have been generated to evaluate the function of six genes on P. fendleri lipid metabolism. P. fendleri plants will be innoculated with these constructs soon. OBJECTIVE 2. Targeted gene editing in for increased oil, HFA, and total seed yields (25% complete) Initial CRISPR/Cas9 constructs were made targeting PfeSRK to generate self-incompatible lines that could increase seed yields, PfeSDP1 to increase seed oil, and PfeFAE1 to generate 18-carbon HFA rather than 20-carbon HFA that are more suitable for the chemical industry. P. fendleri callus was generated through tissue culture and transformed. Unfortunately, issues with chimeric tissues of which only some of the cells are transformed has made screening for induced mutations difficult. Therefore, we are taking a new transformation/screening approach that will hopefully yield more efficient results throughout the remainder of the project. OBJECTIVE 3. Bioengineering P. fendleri as a platform crop for "castor oil" and other valuable unusual fatty acids (50% complete) The engineering of P. fendleri proposed was dependent on getting efficient gene editing of PfeFAE1 and PfeFAH12 in Obj. 2. While we are still working on producing these mutants, we have taken several alternative bioengineering approaches based on our Obj. 1 results to utilize P. fendleri genes to bioengineer TAG remodeling into other species to enhance production of valuable unusual fatty acids. As an initial test of bioengineering the accumulation of HFA through induced TAG remodeling in other species we took advantage of an Arabidopsis line previously engineered with RcFAH12 to produce HFA. In this prior line HFA production also leads to an ~33% reduction in total oil (from ~377 ug FA/mg seed in WT to 253 ug FA/mg seed in the RcFAH12 line). Therefore, we tested the ability of the P. fendleri genes we characterized as key to TAG remodeling in Aim 1 (PfeTAGL1, PfeDGAT1, and PfeDGAT2) for their ability to enhance both total oil and HFA accumulation within the Arabidopsis RcFAH12 lines. Each gene was overexpressed individually seed specifically and each transgenic line demonstrated increased total seed oil and HFA content (these results were published in Parchuri et al. 2024 Nature Communications). The increase in seed oil for overexpression of PfeTAGL1 alone was surprising because TAG lipases are typically thought of as involved in TAG breakdown, but this result further supports our results that the TAG lipase in volved in TAG remodeling (PfeTAGL1) distinct from other TAG lipases involved in TAG turnover (such as PfeSDP1, which we characterized previously) and that TAG remodeling is part of an anabolic process rather than a catabolic process. Next, we expressed PfeTAGL1 combined with either PfeDGAT1 or PfeDGAT2 in HFA producing Arabidopsis, or all three genes expressed together. Stacking of the three genes had a synergistic effect (likely due to recapitulation of TAG remodeling) such that the triple gene construct completely restored the transgenic Arabidopsis seed oil, even to a level higher than wild-type (~411 µg FA/mg seed), and increased HFA levels from 40 to 70 µg/mg seed. Based on these successful results in Arabidopsis we transformed the triple gene construct into the oilseed crop Camelina sativa previously engineered with RcFAH12 and PfeFAE1 (to produce 20-carbon HFA similar to P. fendleri). Again, the TAG remodeling genes increased seed oil content (from 312 to 349 µg FA/mg seed), and HFA content (from 40 to 50 µg/mg seed). Therefore, these results indicate that induced TAG remodeling utilizing P. fendleri genes can be a bioengineering approach to increase valuable unusual fatty acids in multiple plant species. To determine if bioengineered TAG remodeling can be utilized to enhance accumulation of valuable unusual fatty acids other than HFA we utilized Camelina sativa lines previously engineered to produce the healthy fatty acids EPA and DHA. We have expressed our triple gene construct of P. fendleri TAG remodeling genes seed specifically in these lines which are currently at the T2 generation. Evaluation of the effect of TAG remodeling on both total oil and EPA and DHA accumulation will occur when T3 seeds are available shortly.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Azeez A, Bates PD (2024) Self-incompatibility based functional genomics for rapid phenotypic characterization of seed metabolism genes. Plant Biotechnology Journal 22: 2688-2690
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Parchuri P, Bhandari S, Azeez A, Chen G, Johnson K, Shockey J, Smertenko A, Bates PD (2024) Identification of triacylglycerol remodeling mechanism to synthesize unusual fatty acid containing oils. Nature Communications 15: 3547
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: American Society for Biochemistry and Molecular Biology (ASBMB) Annual meeting. March 23-26, 2024. San Antonio, TX. DGAT tales: DGAT1 and DGAT2 enzymes across species differ in their specificity towards diacylglycerol enantiomers. Parchuri P, Shockey J, BATES PD. Oral presentation.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: 26th International Symposium on Plant Lipids. Lincoln, Nebraska, July 14-19, 2024. Deciphering diacylglycerol enantiomer specificities of DGAT isoforms: Insights into TAG remodeling in lipid metabolism of different species. Parchuri P, Shockey J, BATES PD. Oral presentation.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: 26th International Symposium on Plant Lipids 2024. Lincoln, Nebraska. July 14-19 2024. Triacylglycerol remodeling: Discovery and bioengineering to increase hydroxy- and polyunsaturated fatty acids in Arabidopsis and Camelina seed oils. Bates PD. Oral Presentation.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: International Camelina Conference. July 19-20, 2024. Lincoln, Nebraska. Utilization of triacylglycerol remodeling to engineer increased hydroxy- and polyunsaturated containing fatty acids in Camelina sativa seed oils. BATES PD. Poster Presentation.


Progress 02/01/23 to 01/31/24

Outputs
Target Audience:The target audience for this project is plant breeders, the plant metabolism community, plant biochemists and scientists and engineers engaged in manipulating plant oil production for food, fuel or feed stock needs. In addition, the target audience will include graduate students and undergraduates in plant biochemistry and molecular biology who will be able to use publications from these studies to understand plant oil production. Changes/Problems:The main postdoc working on this project recently took an industry job. I am currently looking for a new postdoc to complete the project, and in the meantime having other lab members work on project on the side. Depending on the time to hire a new postdoc, there may be some project delays. If that occurs I anticipate possibly needing a no cost extention. However, as there are still two years rememaining in the project it is possible once a full time replacment is hired we will be able to overcome the current delays and completment on time. What opportunities for training and professional development has the project provided?This work has led to the training of four postdocs and one undergraduate in research experimentation, scientific writing, and scientific presentations. How have the results been disseminated to communities of interest?Our target audience is scientists within academia and the biotech industry, as well as graduate and undergraduate students. We have two manuscripts that have been submitted for publication at peer reviewed journals and are currently under review. Additionally, the work was presented by the PD at three scientific meetings. Finnally,a summary of our current and past work on this topicwas published in an industry focused magazine (INFORM:: International News on Fats, Oils, and Related Materials) published by the American Oil Chemists Society. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: More gene candidates will be functionally evaluated for roles in seed oil accumulation through our VIGS system. Objective 2: Identification of CRISPR/Cas9 generated mutants and propagation for analysis for seed yield and oil phenotypes. Objective 3: Identification of CRISPR/Cas9 generated mutant lines and use for additional bioengineering of desired oil fatty acid compositions.

Impacts
What was accomplished under these goals? Objective 1. Functional genomics in P. fendleri to characterize the mechanisms of efficient hydroxy fatty acid flux through the lipid metabolic network into oil, and identify regulators of total oil accumulation. (20% complete) We have used our new VIGS methodology to functionally characterize eight genes involved in Physaria metabolism. Additionally, we have confirmed the role of three of those genes for the accumulation of hydroxylated fatty acids in developing seeds through seed specific RNAi. The genes functionally characterized in vivo within Physaria fendleri include genes responsible for self-incompatibility (and thus seed production), and the production and accumulation of hydroxylated fatty acids in seed oil. One of the VIGS targets (SRK) is further evaluated in Objective 2 by producing CRISPR/Cas9 based mutants to generate self-compatible plants for enhanced breeding and seed yields. Two of the VIGS targets (FAH12 and FAE1) confirm their role in hydroxy fatty acid production, and are targets for manipulation in Objective 3. A manuscript describing the new VIGS system and the genes characterized so far has been submitted for publication and is current awaiting review. The three gene targets confirmed by RNAi (DGAT1, DGAT2 and TAGL1) have been combined with other biochemical and cell biology results and a second manuscript has been submitted for publication. In this manuscript we describe the gene products involved in a novel pathway of oil biosynthesis coined triacylglycerol remodeling. Additionally, we demonstrate that triacylglycerol remodeling likely occurs through a metabolon residing at the endoplasmic reticulum and lipid droplet junction, and we demonstrate that each of these genes can be used for bioengineering enhanced accumulation of hydroxylated fatty acids. OBJECTIVE 2. Targeted gene editing in for increased oil, HFA, and total seed yields (20% complete). We have generated CRISPR/Cas9 constructs for producing knockout mutants of both SDP1 and SRK genes individually and together, and have produced transgenic Physaria fendleri callus containing each of the three constructs. We are currently screening callus lines for the induced mutations. OBJECTIVE 3. Bioengineering P. fendleri as a platform crop for "castor oil" and other valuable unusual fatty acids (10% complete) Our VIGS in Objective 1 has identified Physaria FAE1 as the key factor for elongation of 18 carbon hydroxy fatty acids to 20 carbon hydroxy fatty acids, and is responsible for the accumulation of the hydroxy fatty acid lesquerolic acid instead of ricinoleic acid (as what accumulates in castor). Additionally, we have identified that Physaria FAH12 is responsible for total HFA production. Therefore we have generated CRISPR/Cas9 constructs for producing knockout mutants of both FAE1 and FAH12 genes individually and together. We have generated transgenic callus for each of the three constructs and are currently screening for mutations in each of the genes. Once plants are regenerated from the callus, the fae1 mutant will be used to engineer a castor oil equivalent line, and fah12 and fah12/fae1 mutants will be used to engineer different 20 carbon and 18 carbon unusual fatty acids respectively.

Publications

  • Type: Other Status: Published Year Published: 2024 Citation: Parchuri P, Azeez A, Bates Philip D (2024) Novel oil biosynthetic pathway for hydroxy fatty acids. In INFORM: International News on Fats, Oils, and Related Materials, Vol 35. AOCS, Champain, IL USA, pp 24-26 https://www.informmagazine-digital.org/informmagazine/library/page/january_2024/24/