INFORM January 2024

26 • inform January 2024, Vol. 35 (1)

HFA can be detrimental to membrane lipid structure func tion, and the inherent differences in the TAG assembly path ways are likely limiting HFA accumulation in transgenic crops such as Camelina . Hence, it is vital for scientists to understand the molecular factors that control acyl flux through the lipid metabolic network to engineer high levels of industrially useful fatty acids in plants (https://doi.org/10.1093/jxb/erab533 ). SOLUTIONS Two approaches to produce a domestic source of HFA are: (1) to develop new crops from non-toxic HFA-producing plants native to the US; and (2) to identify and engineer TAG assem bly pathways that overcome the HFA flux limitations through membrane lipid intermediates as found in Arabidopsis and Camelina . P. fendleri is both a potential new crop species and a source of novel metabolic enzymes for bioengineering HFA accumulation in oilseed crops. Multiple species in the Brassicaceae family accumulate HFA, are non-toxic, and are adapted to grow in the US. P. fen dleri (previously Lesquerella fendleri , commonly known as Fendler’s bladderpod) has been considered the prime can didate for domestication, and research toward that end has proceeded over the past several decades. P. fendleri seeds accumulate (~60%) the industrially valuable HFA, lesquerolic acid, the plants have a high tolerance to abiotic stresses, and need low water making them suitable for marginal lands. Due to similar chemical properties between ricinoleic acid and lesquerolic acid, the P. fendleri seed oil can be used for many of the same applications as castor oil. The addition of P. fendleri seed oil to diesel at as low as 0.25% shows high per formance as a lubricant in fuel-injected diesel engines as com pared to traditional castor and soybean oil. In addition to the oil, P. fendleri has other useful agro nomic features indicating it could be a valuable crop. It con tains a natural gum that can be used as a food and industrial thickener; and 30% of the defatted seed meal is protein that could be used for livestock feed. P. fendleri will not compete with food crops as it can be grown as a winter annual in rota tion with existing crops, acting as an off-season cover crop. The current seed yields are approximately 2000 kg/ha but could reach 2500-3000 kg/ha. Therefore, the continued develop ment of P. fendleri can provide a domestic source of HFA and a valuable alternative crop. IMPROVING HFA CONTENT IN P. FENDLERI P. fendleri is transformable. Researchers have demon strated its potential for oil and HFA content improvement using multiple engineering approaches. Unlike castor, which accumulates ricinoleic acid at all three positions of the TAG backbone, lesquerolic acid accumulates at just at the sn -1 and sn -3 positions. To produce a castor equivalent TAG in P. fendleri , a research team expressed castor’s lysophosphatidic acid acyl transferase gene. The result was accumulation of HFA at sn -2, yet total HFA content was not increased, indicating more work is needed to understand HFA limiting bottlenecks (https://doi. org/10.3390/plants10061093). Our group recently demon

strated a seed-specific knockdown of the HFA-selective TAG lipase, known as sugar-dependent 1, increased P. fendleri seed weight, seed oil content, and increased the amount of indus trially valuable HFA without an effect on seed germination (https://doi.org/10.3389/fpls.2022.931310). Thus, enhancing P. fendleri oil production by gene editing to knockout specific enzymes may be a valuable target for future engineering and breeding. NOVEL METABOLISM AS A TARGET Recent research indicates that P. fendleri uses multiple, uncon ventional pathways to channel carbon into lipids (https://doi. org/10.1093/jxb/erad343 ). In addition, it uses a novel path way to assemble TAG containing lesquerolic acid distinctly at the sn -1 and sn -3 positions (https://doi.org/10.1093/plphys/ kiab294). The novel oil biosynthesis, coined triacylglycerol remodel ing, overcomes the barrier of moving HFA through membrane lipid intermediates common to HFA biosynthesis engineered into Brassicaceae seed oils. P. fendleri HFA metabolism starts like castor, where ricinoleic acid is produced on the membrane lipid PC and transferred to the acyl-CoA pool, then in P. fen dleri it is elongated to lesquerolic acid. Unlike castor, lesque royl-CoA is not used to produce de novo DAG containing HFA. Instead, the DAG used to synthesize TAG is derived from PC (not containing HFA) similar to other Brassicaceae species. TAG is initially synthesized from PC-derived DAG by add ing the sn -3 lesquerolic acid, producing a TAG with only a sin gle HFA. Subsequently, the sn -1 common fatty acid is removed and a second lesquerolic acid is incorporated at sn -1 (Figure 2). Thus, the initially synthesized TAG containing one HFA is remodeled to contain two HFA. By not first incorporating the sn -1 through de novo DAG biosynthesis, P. fendleri over comes the constraint of pushing HFA-containing DAG through the membrane as in the PC-derived DAG pathway found in many crop species. We identified the TAG remodeling pathway through isotopic tracing of lipid metabolism, but not the gene products responsible for it yet, indicating the need for further research into P. fendleri lipid metabolism. For a domestic non-toxic source of HFA, using the iden tified novel metabolism to target breeding in P. fendleri may be important for crop development. In addition, enhancing oil and HFA content through bioengineering and producing a cas tor equivalent TAG species will elevate the usefulness of P. fen dleri seed oil. And the development of herbicide-resistant lines will make P. fendleri suitable for modern agriculture. Finally, as researchers identify TAG remodeling genes the bioengineering P. fendleri like metabolism into an industrial fatty acid platform crop such as Camelina sativa or pennycress ( Thlapsi arvense ) may be a novel approach for creating the HFA source of the future. Prasad Parchuri, a postdoctoral research associate, Abdul Azeez, a research assistant professor, and Philip D. Bates, an associate professor, of the Institute of Biological Chemistry at Washington State University, Pullman, USA, contributed to this story. Bates can be contacted at phil_bates@wsu.edu.