INFORM September 2024

28 • inform September 2024, Vol. 35 (8)

produced nearly pure protein isolates (90 percent–97 percent crude protein content). The WTP protein isolates had nutritional quality (amino acid profile) that was comparable to those of soybean and canola protein products. They also showed excellent func tional properties, such high solubility across a broad pH range, remarkable foaming and emulsifying abilities, and high water-holding capacities, which are desirable traits for an alternative protein source to be used in dairy substitutes, emulsions, foamed products, and baked goods. The paper goes on to describe how advances in agronomic breeding practices and genomic breeding tools, such as molec ular markers, association mapping, and gene editing have allowed plant breeding researchers to domesticate pennycress in less than a decade. In the process, they have produced new pennycress varieties with improved agronomic traits, as well as specialty traits, such as low erucic acid and glucosinolate con tents, low fiber, and higher oil/protein. Based on these efforts, in 2022, CoverCress Inc., a com pany based in St. Louis, Missouri, developed and commercially launched the covercress™ family of pennycress-derived seed varieties. The seeds have a yellow color (instead of the usual black in WTP seeds) because of a key mutation in a transcrip tion factor from the transparent testa (TTG1 or TT8) family. Hojilla-Evangelista and her team evaluated the varietal effects on the extractability, composition, and functional prop erties of covercress™ TT8 and Y1126 (TTG1 mutant) seed pro teins. They found that the TT8 and Y1126 hexane defatted seed meals had 20 percent–28 percent greater protein recovery and their saline-extracted protein isolates generally had superior functional properties compared with those of similarly prepared WTP. TT8 performance surpassed Y1126 based on protein yield, purity (94 percent vs. 75 percent for Y1126), very high solubil ity in acidic pH (like whey protein), foaming, and gelation. They also found that protein extracted from another new experimen tal pennycress line with zero erucic acid ( T. arvense fae1-1) had structure and functional properties like those of WTP. OPTIMIZING EXTRACTION The research team considered whether covercress™ varieties were suitable for producing “clean” label protein products that align with current consumer preferences. As such, they stud ied the possibility of using non-hexane solvent alternatives for oilseed extraction. Ethanol, which can be recycled during oil extraction and comes from a biorenewable resource, is among those that have generated interest for many decades. In the 1930s, anhydrous ethanol used to extract soybean oil on a commercial scale also removed sugars, saponins, and phosphatides. As a result, the quality of the defatted meal was improved, determined by a whiter color, no beany/bit ter flavor, and no flatus-causing sugars. However, the limita tion of ethanol, and other alcohols, for defatting is that they should remain anhydrous and be used near their boiling point. Hojilla-Evangelista and her team reported an 84 percent oil recovery from flaked corn using 95 percent ethanol at 40 °C. This amount was only 9 percent less than at the boiling point

of 75 °C. They did find that using aqueous ethanol at higher extraction temperatures led to greater protein loss because of co-extraction with oil. Cold pressing of oilseeds involves milder thermal condi tions (no seed cooking step) that may benefit oil quality and PC proteins. Cold-pressed rapeseed cake was found to have less glucosinolate than commercial rapeseed protein isolates but more phytate than those in commercial soy protein prod ucts. Pennycress cold-pressing gave oil yields of 70 percent and crude oil with low amounts of phospholipids and sulfur-con taining compounds. Cold-pressed pennycress cake had 62 per cent more albumins and 20 percent more globulins than did cake that underwent seed cooking and the protein was more soluble when pH was above five. The teams most recent work applied elements of these physical and aqueous approaches to TT8 and Y1126 PCs, the typical product of oil processing and likely starting materials for producing protein isolates. The goals of their work were to: (1) develop/evaluate the combination of cold-pressing, non-hexane defatting, and saline-based extraction to pro duce proteins from new pennycress varieties TT8 and Y1126; (2) determine the process impact on yields and chemical and functional properties of PC proteins; and (3) identify potential uses of resulting protein isolates from TT8 and Y1126 PCs. GAINING EXEMPTION Before the researchers reached this point of fine-tuning extraction methods for the best chance at commercial success, they had to first apply for regulatory exemption. The team first had to request a Regulatory Status Review (RSR) for pennycress developed using genetic engineering. This letter to the USDA described how pennycress was modified to impart lowered eru cic acid in seeds via reduction or loss of fatty acid elongation 1 (FAE1) and to confer lowered fiber in seeds via reduction or loss of transparent testa 8 (TT8). A year later, once the review was complete, they filed a requested to confirm the exemption of covercress™ from regulation pursuant to 7 CFR § 340.1(b)(1). The letter described the intended single genetic modifi cation leading to a loss of function of a gene resulting from cellular repair of a targeted DNA break in the absence of an externally provided repair template. And explained how the researchers insert exogenous DNA into the plant’s genome to generate the modification and how they eliminate it in the final modified plant. It also described methods they planned to use to verify they made only the intended modification, and that no exogenous DNA remained in the plant. Consistent with 7 CFR 340.4, APHIS reviewed the modified pennycress to determine whether it is subject to the regulations in 7 CFR part 340. Specifically, APHIS reviewed the modified pennycress to determine whether there is a plausible pathway by which the pennycress would pose an increased plant pest risk relative to the plant pest risk posed by an appropriate pen nycress comparator. Based on information provided, publicly available resources, and APHIS’ familiarity with pennycress and knowledge of the trait, phenotype, and mechanism of action, APHIS considered the (1) biology of nonmodified pennycress