Source: ARVEGENIX LLC submitted to
DEVELOPMENT OF NOVEL COVER CROP, COVERCRESS, AS A SUSTAINABLE PLANT-BASED PROTEIN SOURCE FOR FOOD APPLICATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1025904
Grant No.
2021-33530-34508
Cumulative Award Amt.
$106,489.00
Proposal No.
2021-01569
Multistate No.
(N/A)
Project Start Date
Apr 1, 2021
Project End Date
Nov 30, 2021
Grant Year
2021
Program Code
[8.2]- Plant Production and Protection-Biology
Recipient Organization
ARVEGENIX LLC
1100 CORPORATE SQUARE DR STE 254
SAINT LOUIS,MO 631322933
Performing Department
(N/A)
Non Technical Summary
The goal of this Phase I effort is to demonstrate CoverCress as a new source of protein for the plant-based protein market. CoverCress is a winter cover crop that is targeted towards corn-soy rotation farmers in the American Midwest. The unique feature of CoverCress is that it doubles as a cash crop, providing $50-70/acre in net returns to the farmer. This gives farmers a financial incentive to adopt a cover crop strategy because it more than offsets the cost of planting and harvesting the cover crop while providing environmental benefits?cover crops prevent soil erosion and nutrient runoff, reduce pesticide use and disease emergence, and sequester carbon from the atmosphere. Additionally, CoverCress is ready to harvest in late May, prior to soybean planting, and can be done using a standard soybean combine. We estimate that CoverCress can bring a potential $275/acre in economic value to the US economy from the 35 million acres of corn-soy farmland in the Midwest, about $9.6 billion overall. We expect this value to grow through improved CoverCress varieties with greater yields and climate hardiness.CoverCress was initially developed from field pennycress as a winter oilseed crop with enhanced yields, low fiber, low glucosinolates, and low erucic acid content (similar to the rapeseed-canola transition). This allows for CoverCress oil to find applications in both biofuel and culinary markets, while the meal co-product from oil extraction can be used as a source of animal feed. To improve the economic value of the meal, this Phase I effort will test the suitability of CoverCress protein in the emerging plant-based protein market. This market, estimated to be $20 billion by 2023, includes products such as dairy and meat substitutes, baking ingredients, dips, and whipped toppings. Each one of these products requires a protein input with a different functionality. For example, dairy substitutes require exceptional protein solubility to make a smooth beverage, while dips may require good protein emulsification properties. Prior encouraging results on pennycress protein revealed high solubility and emulsifiability, and we expect CoverCress protein to perform similarly.Our Phase I objectives are to analyze the functionality of CoverCress protein in order to determine which end-products can be best served by the product. We will examine meal proximate composition, protein solubility, emulsification, foaming ability, and water holding capacity. This will set the groundwork for food development testing in Phase II. Additionally, we plan to use robust genetic tools to develop CoverCress lines with the potential to alter protein functionality and target additional products in the plant-based protein market. These experimental transgenic lines will be tested for functionality and re-created using non-transgenic means (gene editing) in a subsequent Phase II effort.We expect CoverCress to yield a high protein content from the initial feedstock and perform well in at least one functionality test (solubility, emulsification, foaming ability, water holding capacity). This will enable the commercialization of CoverCress protein as competitive alternative to other plant-derived protein sources, such as soy, pea, and canola. In addition, we expect to generate at least one transgenic CoverCress line with altered protein functionalities. This should serve as a starting point for creating a functionally diverse portfolio of CoverCress protein sources, each one useful for a different plant-based protein product.Overall, we are excited to introduce CoverCress as an added value for corn-soy farmers in the American Midwest, a source of potentially new plant-based protein products, and a means to mitigate the environmental impacts of large-scale agriculture.
Animal Health Component
50%
Research Effort Categories
Basic
0%
Applied
50%
Developmental
50%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
50221401040100%
Knowledge Area
502 - New and Improved Food Products;

Subject Of Investigation
2140 - Ground covers;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
GOAL: The long-term goal of this project is to develop new plant-based source of functional protein for food applications from a novel oilseed crop based on field pennycress (Thlaspi arvense). This new crop, named CoverCress, will be used as a cover crop on up to 35M acres in US Midwest, protecting soil health and environment. It will produce over $3B of new value (from the oil and proteinaceous meal) on the land that otherwise would stay idle and undergo erosion between standard corn and soybean growing seasons.OBJECTIVES:The technical objectives of the proposal are:Demonstrate CoverCress as a viable product for the plant-based protein market.Extract CoverCress protein through standard commercial methodsBiochemically analyze the resultant product, and compare it to existing protein sources on the market and prior studies on pennycressIdentify strengths of CoverCress protein based on success criteriaObtain transgenic CoverCress lines with altered protein functionalityIdentify storage proteins in the CoverCress GenomeSilence 2S and 12S storage proteins genes in CoverCress via RNA interference
Project Methods
Efforts: This project will use established food protein characterization methods to evaluate pennycress/CoverCress protein extracts functionality. Longer term, gene editing technology will be used to create crops with improved protein composition that further enhance functionality in specific food applications.Evaluation: The following experiments will be carried out by Dr. Hojilla-Evangelista, who has already performed such work on pennycress and other oilseed and cereal crops. Seed defatting. CoverCress seeds will be defatted prior to protein extraction by the procedure outlined in Hojilla-Evangelista et al. Seeds will be screw-pressed and then solvent extracted using hexane or anhydrous ethanol until 0.5% residual oil content. The press cake will be the starting material for protein extraction.Extraction of CoverCress protein. CoverCress protein will be extracted by using conventional alkali extraction-acid precipitation or saline-based extraction methods developed for wild-type pennycress by Hojilla-Evangelista et al.Extracted sample diagnostics: Extracted samples will be analyzed by FTIR spectroscopy, soluble protein class analysis, and SDS-PAGE as a means of obtaining a coarse understanding of protein structure and composition. These diagnostics will be used to troubleshoot and optimize the sample handling and extraction protocols. For FTIR analysis, 2 mg of sample and 250 mg of KBr will be mixed and pressed into a pellet. The pressed pellet will be placed into an FTIR spectrometer set in transmission mode to obtain the IR spectra of the protein samples. For soluble protein class analysis, the four major soluble protein classes (albumin, globulin, prolamin, and glutelin) will be determined from 10 g of sample through sequential extraction with cold water, followed by 0.5 M NaCl, 70% ethanol, and finally 0.1 M NaOH. Following each solvent extraction step, the samples will be centrifuged (10,000 x g, 25oC, 20 min) and their supernatants collected, lyophilized, and analyzed for crude protein content. The amount of each soluble protein fraction will be calculated as a % of total protein. Data for CoverCress will be compared to results obtained from pennycress.Proximate composition of seed meal, press cake, and recovered protein samples: Moisture, crude protein (Dumas %N x 6.25), crude oil, and crude fiber content analyses will be done according to the American Oil Chemists' Society (AOCS) standard methods Ba 2a-38, Ba 4e-93, Am 5-04, and Ba 6-05, respectively. The ash content for each sample will be analyzed according to the Association of Official Analytical Chemists (AOAC) method 942.05.Protein solubility analysis: Protein solubilities across a range of pH will be determined according to the method of Balmaceda et al. Protein solubility will be reported as a % of total protein.Emulsification analysis: Emulsion activity index (EAI) and emulsion stability index (ESI) will be determined initially at pH 7 by following the method of Wu et al. EAI (in m2/g) and ESI will be calculated according to the equation provided in Wu et al., based on absorption values at 500 nm.Foaming analysis: Foaming capacity and foam stability of protein samples (10 mg protein/mL) will be determined initially at pH 7 according to the method described by Myers et al. Foaming capacity is the volume (mL) of foam produced in 1 min, while foam stability is the portion of foam left (%) after standing for 15 min.Water holding capacity (WHC): WHC will be determined by following the method of Balmaceda et al. for insoluble or partly soluble materials. It will then be compared with published values for wild-type pennycress protein isolate (see Background and Rationale).

Progress 04/01/21 to 11/30/21

Outputs
Target Audience:Food technology professionals, developers, innovators involved in developing plant-based protein as a newsustainable food source. Participants of the2021 AOCS Annual meeting, Biotechnology, Protein and Co-Product section. Participants of the AOCS Hot Topic Symposium on Innovative Technology, Gene Editing. Participants of two Plant Breeding Initiative international meetings. Participants of the 2022AOCS Annual meeting, Biotechnology, Protein and Co-Product and Industrial Oil Product sections. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Gordon Younkin, a Ph.D. candidate and NSF Graduate Research Fellow fromCornell University, completed his internship with CoverCress in April - August 2021. His focus and responsibility wereto identify storage proteingene targets in CoverCress genome using bioinformatics approach, design and make vectors capable of suppressing gene expression of cruciferin and albumin gene families,design and make vectors capable of gene editing individual cruciferin and albumin genes, transform both types of vectors into various elite breeding lines and identify transformants. The technical experience he received helped to significantly advance his graduate research (he is now applying the same approach to sudy function and biosynthesis of cardiac glycosides in another cruciferous plant -Erysimum).He has also received an experience and mentoring on how to succeed in fast-paced, commercially-oriented startup environment that allowed him to prepare for a career inindustry. How have the results been disseminated to communities of interest?The results of this research were presented at three conferences by the Project Director and the leading subcontractor scientist, Mila Hojilla-Evangelista from NCAUR Center, USDA-ARS, Peoria, IL. The recipients of this information were: Participants of 2021 AOCS Annual meeting, Biotechnology, Protein and Co-Product section. Participants of the AOCS Hot Topic Symposium on Innovative Technology, Gene Editing. Participants of 2022AOCS Annual meeting, Biotechnology, Protein and Co-Product and Industrial Oil Product sections. What do you plan to do during the next reporting period to accomplish the goals?Based on the promising results from Phase I, CoverCress Inc.andthe researchers from the subcontractor institution (NCAUR Center,USDA-ARS, Peoria, IL)are planning to apply for Phase II funding. Wearealso planning to prepare and submit a manuscript for a publication ina peer reviewed journal of broad interest.

Impacts
What was accomplished under these goals? 1A. Extract CoverCress protein through standard commercial methods. Comparison of CoverCress and pennycress varieties.Oil removal was achieved using either hexane or anhydrous ethanol as solvent (after a cryogenic seed grind and either with or without partial defatting step using a standard cold press). The residual oil levels in the defatted meal were ≤ 0.5% for hexane and <1% for ethanol extraction. Following oil removal, protein was extracted from fully defatted meals using the saline-based method developed for wild pennycress (Hojilla-Evangelista et al., 2015). The efficiencies of protein extraction for the black-seeded lines were generally lower (36% and 45%, for the high yielding B3 and WT pennycress, respectively), while they were generally higher for the yellow-seeded low fiber lines (49% and 50% for Y1126/TTG1 and TT8, respectively). The resulting protein samples ranged in protein purity from 71% protein (B3) to 94% (TT8-Hex) and could be classified as protein concentrates or protein isolates. Overall, TT8-Hex and TT8-Eth protein extracts had substantially more protein than those recovered from B3 and Y1126 and contained significantly (72%) less ash, indicating superior performance of the TT8 line as a source of food protein. The TT8 variety showed the best performance with respect to protein extractability, purity, and functional properties. Comparison of hexane and ethanol-based extraction methods.Another goal described in the proposal was to investigate the use of ethanol as an extraction vehicle. Ethanol has become increasingly popular as an alternative to hexane due to negative consumer perception on the use of petroleum-based solvents for food processing. Ethanol is a more polar solvent than hexane and its use in defatting may co-extract polar non-protein components that may affect the purity of the protein product. While both hexane and ethanol extracted TT8 samples demonstrated excellent proximate compositions, hexane defatting was slightly better in recovery, yield, and protein purity. While the use of ethanol for extraction produced great results, standard hexane defatting of TT8 meal (TT8-Hex) resulted in highest protein recovery, yield, and purity. 1B. Biochemically analyze the resultant product and compare it to existing protein sources on the market and prior studies on pennycress. Comparison of CoverCress and pennycress varieties.When proteins in the above extracts were resolved on SDS-PAGE gel, band patterns of B3 and Y1126 seed meals were more like those of WT pennycress, while the protein profile of TT8 line was somewhat unique and different. TT8-Hex protein isolate showed highest solubility in acidic pH, and had greater foaming capacity, foam stability, and WHC than did similarly prepared pennycress seed protein isolate. 1C. Identify strengths of CoverCress protein based on success criteria. CoverCress was found to have unique and attractive gel properties.Overall, CoverCress protein demonstrated equal or superior properties relative to pennycress. CoverCress protein extracts exhibited unique functionality that will be of significant commercial interest to companies that develop, formulate and market plant-based products. These will include plant-based meats and plant based dairy products. The formation of gels on heating will particularly benefit formulators developing plant-based cheese, yogurts, desserts, meats and other products that require texture and stability after heating. The protein extracts exhibit unique functionality that will be of significant commercial interest to companies that develop, formulate and market plant-based products. These will include plant-based meats and plant-based dairy products. CoverCress TT8 line was found to be very attractive in terms of protein yield (extraction), with equivalent to improved functionality (solubility, foaming capacity, emulsification, gel formation) versus the other varieties. The ability to form strong gels on heating will be particularly useful for developing plant-based cheese, yogurts, desserts, meats and other products that require texture and stability after heating. 2A. Identify storage protein genes in the CoverCress genome. Bioinformatics analysis of several sequenced pennycress genomes revealed that a typical pennycress/CoverCress genome has four cruciferin (12S) and six albumin (2S) storage protein genes. All four cruciferin-encoding genes had similar genomic organization, with four exons separated by 3 introns, while albumin genes were intronless. Cruciferin and albumin storage protein gene families were identified and characterized in pennycress genome. 2B. Silence 2S and 12S storage proteins genes in CoverCress via RNA interference. Production of transgenic plants. Two vectors overexpressing inverted repeats targeting conserved regions of corresponding storage protein gene families were created and RNAi cassettes were introduced into TT8 low fiber CoverCress line. Multipleindependent transformants in different germplasms were generated for each construct. The progeny of T1 plants was visually inspected for the presence of the T-DNA, and the protein profiles of individual positive seeds were analyzed using SDS-PAGE. Protein gel analysis of individual T2 seeds containing either anti-albumin or anti-cruciferin RNAi cassette revealed that storage proteins composition in selected lines was drastically different from the controls, indicating strong efficacy. Importantly, the affected seeds look normal, suggesting that the achieved suppression of the entire class of storage proteins is well tolerated by the pennycress plant opening an opportunity to significant future remodeling of the already attractive CoverCress protein composition to further tailor it to human food needs.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: "CoverCress â¿¿ a novel oilseed winter crop with canola-like composition that helps sequester carbon and prevent soil erosion", Gene Editing Technology and Advancements in Agriculture Hot Topic Symposium, American Oil Chemists Society, May 2-5, 2021.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: "Intellectual Property Rights Issues with CRISPR and Commercial Launch of CoverCress a Novel Oilseed Winter Crop with Canola like Composition", 4th Plant Breeding Initiative Roundtable, Presentation and Live Q&A Panel, June 28, 2021.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2022 Citation: "CoverCress â¿¿ a novel oilseed winter crop with canola-like composition that helps sequester carbon and prevent soil erosion", Gene Editing Technology and Advancements in Agriculture Hot Topic Symposium, American Oil Chemists Society, Atlanta, GA, May 1-4, 2022.


Progress 04/01/21 to 11/30/21

Outputs
Target Audience:Participants of 2021 AOCS Annual meeting, Biotechnology, Protein and Co-Product section. Participants of the AOCS Hot Topic Symposium on Innovative Technology, Gene Editing. Participants of two Plant Breeding Initiative international meetings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the summer of 2021, we provided training to a graduate student from Boyce Thompson Institute, Cornell University, Ithaca, NY. He desired and received the training in plant transformation, designing, building and transforming gene editing and gene suppression RNAi constructs, molecular analysis of plant trantformants, biochemistry of flavor creation in Brassica species. How have the results been disseminated to communities of interest?The preliminary results on CoverCress protein extraction and performance were reported by subcontractor scientist in the Proteins and Co-Products session of 2021 American Oil Chemists Society (AOCS) Annual meeting What do you plan to do during the next reporting period to accomplish the goals?Complete characterization of different CoverCress protein extracts and determine their relative performans versus WT pennycress protein characterized earlier. Complete transformation of gene-editing vectors into plants, propagate T1 plants for one generation, analyze for edits.

Impacts
What was accomplished under these goals? Comparison of CoverCress and pennycress varieties.Following oil removal, protein was extracted from fully defatted meals using the saline-based method developed for wild pennycress (Hojilla-Evangelista et al., 2015). The efficiencies of protein extraction for the black-seeded lines were generally lower (36% and 45%, for the high yielding B3 and WT pennycress, respectively), while they were generally higher for the yellow-seeded low fiber lines (49% and 50% for Y1126/TTG1 and TT8, respectively). The resulting protein samples ranged in protein purity from 71% protein (B3) to 94% (TT8-Hex) and could be classified as protein concentrates or protein isolates. Overall, TT8-Hex and TT8-Eth protein extracts had substantially more protein than those recovered from B3 and Y1126 and contained significantly (72%) less ash, indicating superior performance of the TT8 line as a source of food protein. 12 constructs designed to edit pennycress major protein storage genes were created and are in the porcess of being transformed into plants.

Publications