INFORM October 2025

20 • inform October 2025, Vol. 36 (9)

increase in acetyl-TAG levels compared to EaDAcT. The high est-performing lines expressing EfDAcT reached 81 mol per cent acetyl-TAG. We also transformed pennycress with EfDAcT and observed acetyl-TAG levels up to 68 mol percent in the best-performing lines. These results identified EfDAcT as our With improved enzymes and reduced pathway competition, we looked at another critical limiting factor: the supply of ace tyl-CoA. Acetyl-CoA serves as the two-carbon donor for ace tyl-TAG synthesis via DAcTs, but in oilseeds, it is also used in other pathways such as fatty acid elongation. In members of the Brassicaceae family, including camelina and pennycress, the fatty acid elongase (FAE) complex uses acetyl-CoA to con vert oleic acid (18:1) into very-long-chain fatty acids (VLCFA), such as eicosenoic acid (20:1) and erucic acid (22:1). Camelina seeds contain more than 20 mol percent VLCFA, with eicose O O O O O O O O O O O O sn-1 sn-2 sn-3 sn-1 sn-2 sn-3 FAE1 18:1-CoA FAE1 20:1-CoA 22:1-CoA FA elongation Acyl-CoA pool TAG RNAi DAG Acetyl-TAG DGAT1 EfDAcT 2 3 1 Acetyl-CoA pool Euonymus alatus Euonymus fortunei Celastrus scandens TAG Acetyl-TAG top candidate gene for acetyl-TAG synthesis. ELIMINATING THE COMPETITION In native and transgenic plants that produce acetyl-TAG, DGAT1 uses the same DAG pool as DAcTs to synthesize regu lar TAGs. To shift the balance toward acetyl-TAGs synthesis, we needed to reduce this internal competition. Our lab designed a seed-specific RNA interference (RNAi) construct targeting DGAT1, with the aim of suppressing its expression during oil biosynthesis. By downregulating DGAT1, we hoped to reduce conventional TAG formation and redirect more DAG toward acetyl-TAGs production. We introduced this DGAT1-RNAi construct alongside EfDAcT into both camelina and pennycress. In the best came lina lines, acetyl-TAGs reached 87 mol percent, while pen nycress lines achieved 85 mol percent. These levels were a significant improvement over previous results using EfDAcT alone and demonstrated that reducing native pathway com petition can lead to enhanced product synthesis in our engi neered biosynthetic pathway. INCREASING PRECURSOR SUPPLY

noic acid making up about 14 mol percent. In contrast, penny cress seeds have a much higher VLCFA content, exceeding 55 mol percent, with erucic acid accounting for roughly 43 mol percent. We hypothesized that eliminating fatty acid elonga tion in camelina and pennycress could significantly increase the acetyl-CoA supply available to acetyl-TAG production. To test this, we measured VLCFA and acetyl-CoA lev els in fae1 knockout mutants of camelina and pennycress. Our collaborators at Montana State University and Illinois State University generated these mutants using CRISPR-Cas9 genome editing in camelina and pennycress, respectively. The fae1 mutant lines produced almost no VLCFA, less than 2 mol percent in both crops. More importantly, we observed a corresponding increase in acetyl-CoA levels, par ticularly in pennycress. We then introduced our best-perform ing construct, EfDAcT + DGAT1-RNAi, into the fae1 mutant backgrounds. In camelina, acetyl-TAG content increased to 93 mol percent, the highest level we had observed in this crop. Pennycress exceeded that, reaching 98 mol percent ace tyl-TAG, essentially replacing conventional long-chain TAGs with our desired low-viscosity acetyl-TAG. As a result of this final modification, we reached our goal in achieving levels that are equivalent to or greater than those naturally produced in Euonymus species. WHAT COMES NEXT: FROM LAB TO FIELD The successful generation of native-like acetyl-TAG levels in transgenic camelina and pennycress seeds provides a foun dation for further research and evaluation of these lines. Field evaluations of high acetyl-TAG lines will be essential to examine how acetyl-TAG production is affected by agronomic factors outside of controlled environments. Key questions include: Will acetyl-TAG levels remain stable under common biotic and abiotic stresses? Can engineered camelina and pen nycress match wild-type seed yield and oil content? Do mod ifications impact seed germination, flowering time, or overall plant performance over multiple growing seasons? While acetyl-TAGs look promising in terms of structure, they must also meet performance expectations across indus trial applications. Specific industries will be need to examine their oxidative stability, flow at low temperatures, and func tional properties for a given end use such as biodegradable lubricants, plasticizers, and biofuels. Growing genetically modified oilseed crops in the United States requires navigating a complex regulatory process. This process can be demanding in terms of time and resources. Despite these challenges, acetyl-TAGs represent a promising opportunity to introduce tailored plant oils into real-world applications. Linah Alkotami earned her PhD in Biochemistry from Kansas State University working in the Durrett lab. Timothy Durrett is a Professor in the Department of Biochemistry and Molecular Biophysics at Kansas State University. He can be contacted at tdurrett@ksu.edu.

EfDAcT 1

EfDAcT DGAT1-RNAi 2

EfDAcT DGAT1-RNAi fae1 background 3

WT

Pennycress

0%

68%

85%

98%

0%

81%

87%

93%

Camelina

Stepwise enhancement of acetyl-TAG content in engineered oil seeds. Oil droplet sizes represent the highest acetyl-TAG levels achieved in camelina and pennycress under different metabolic engineering strategies. Source: Durrett Lab