A Synergistic Genetic Engineering Strategy Induced Nervonic Acid in Brassica juncea and Brassica napus

Nervonic acid (NA), a very-long-chain monounsaturated fatty acid, is known for its benefits in treating neurological diseases and promoting brain health. In this study, we utilized two different receptors, <i>Brassica juncea</i> (<i>B. juncea</i>, rich in erucic acid, C22:1) and <i>Brassica napus</i> (<i>B. napus</i>, high in oleic acid, C18:1), to overproduce NA through systematic metabolic engineering. Two multi-gene vector constructs, Napin-3 and Napin-5 (<i>CgKCS::SLC1-1::DGAT1</i>; <i>CgKCS::SLC1-1::BnFAE1::LdLPAAT::DGAT1</i>), are driven by seed-specific napin promoters. In <i>B. juncea</i>, Napin-3 and Napin-5 expression elevated NA levels to 48.7% and 46.3% in seed oil, respectively, compared to 2.5% in wild types. In <i>B. napus</i>, Napin-3 and Napin-5 expression achieved NA levels of 45% and 39.6%, respectively, while NA is absent in wild types. To our knowledge, this represents the highest NA production in plants to date, with stable oil content and yield, enabling cost-effective NA production. In <i>B. juncea</i>, a significant increase in NA is observed alongside a decrease in C18:1, C20:1, and C22:1 levels; in <i>B. napus</i>, the rise in NA is accompanied by a decrease in C18:1, and an increase in C20:1 and C22:1. These patterns reflect the dynamic equilibrium of fatty acids following NA conversion, influenced by the Dynamic Substrate Tugging (DST) Mechanism, in the form of either an EA-tugging mode or C18:1-tugging mode mechanism, depending on the cellular context. NA is an elongation product derived from C18:1, catalyzed by <i>CgKCS</i> with broad substrate specificity, indicating that plants with high levels of C18:1, similarly to those rich in C22:1, serve as excellent candidates for NA production. This "green factory" for NA production provides strong support for its pharmaceutical, nutraceutical, and industrial applications. The exogenous and the endogenous enzymes coordinate function remodeling of the intra-seed fatty acid elongation flux through the DST strategy, thereby systematically enhancing the synthesis and accumulation efficiency of the target fatty acid.